Modular FLIM Systems for Zeiss
LSM 710 / 780 / 880 Family
Laser Scanning Microscopes
With Appendix for LSM 510 FLIM Systems
Becker & Hickl GmbH
Nunsdorfer Ring 7-9
Tel. +49 / 30 / 212 800 20
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7th Edition, November 2017
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The bh FLIM systems are based on a multi-dimensional time-correlated single photon counting (TCSPC) technique introduced by Becker & Hickl in 1993 . Since then, the bh TCSPC systems have become faster in terms of acquisition speed, got larger and larger memories, and more dimensions have been added to the recording process. Fluorescence lifetime imaging started in 1996 with applications in ophthalmology . Parameter-tag recording was added in 1996. The first FLIM module for laser scanning microscopes was introduced in 1998. bh FLIM systems for the Zeiss LSM laser scanning microscopes are available since 2000 . Since then, several new generations of LSM family laser scanning microscopes, and several generations of bh FLIM modules have been introduced. As a result, a wide variety of bh FLIM systems and of FLIM system configurations are in use [55, 66]. The excitation light source can be the Ti:Sapphire laser of a multiphoton microscope, a picosecond diode laser attached to or integrated in the microscope, or a visible-range tuneable solid state laser. The fluorescence light may be detected via a confocal port of the scan head or via a non-descanned port of a multiphoton microscope. Signals may be detected by one detector, simultaneously by two, three, or four detectors, or by the 16 channels of a bh multi-wavelength detector. Since 2008, bh FLIM systems are using highly efficient GaAsP hybrid detectors. By combining extremely high efficiency with large active area, high counting speed, high time-resolution, and low background, these detectors have triggered a breakthrough in FLIM recording. Another step was made by the introduction of 64-bit data acquisition software. FLIM data are now recorded at unprecedented pixel numbers, high dynamic range, short acquisition time, and minimum exposure of the sample. New hardware and software functions have resulted in advanced FLIM functions, like time-series FLIM, Z stack FLIM, spatial and temporal Mosaic FLIM, combined fluorescence and phosphorescence lifetime imaging (FLIM/PLIM), and fluorescence lifetime-transient scanning (FLITS).
Fig. 1: LSM 710 Multiphoton NDD FLIM systems
This handbook describes the bh TCSPC FLIM systems for the Zeiss LSM 710/780/880 family microscopes (LSM 710, LSM 710 NLO, LSM 7 MP, LSM 780, LSM 780 NLO, LSM 880 and LSM 880 NLO). It describes the basic FLIM modes of the system, advanced FLIM techniques and procedures, the technical background of FLIM, and typical FLIM applications in biology. It ends with a list of more than 500 references related to the bh FLIM systems. FLIM systems for the LSM 510, LSM 510 NLO, and LSM 510 Meta are described in an appendix, see page 297. As supplementary literature we recommend the bh TCSPC Handbook , which presents general information about multi-dimensional TCSPC, photon counting detectors, TCSPC devices, operation modes, and TCSPC applications.
The bh FLIM systems use a combination of bh’s multidimensional time-correlated single-photon counting process with confocal or multiphoton laser scanning. The sample is repetitively scanned by a high-repetition rate pulsed laser beam, single photons of the fluorescence signal are detected, and each photon is characterised by its time in the laser pulse period and the coordinates of the laser spot in the scanning area in the moment its detection. The recording process builds up a photon distribution over these parameters, see Fig. 2. The photon distribution can be interpreted as an array of pixels, each containing a full fluorescence decay curve in a large number of time channels.
Fig. 2: Principle of TCSPC FLIM
The recording process delivers a near-ideal photon efficiency, excellent time resolution, and is independent of the scan rate of the microscope. The signal-to-noise ratio depends only on the total acquisition time and the photon rate available from the sample.
The technique can be extended by including additional parameters in the photon distribution. These can be the depth of the focus in the sample, the wavelength of the photons, the time after a stimulation of the sample, or the time within the period of an additional modulation of the laser. These techniques are used to record Z stacks or lateral mosaics of FLIM images, multi-wavelength FLIM images, images of physiological effects occurring in the sample, or to record simultaneously fluorescence and phosphorescence lifetime images.
Due to the wide variety of microscopes, lasers, excitation principles, optical interfaces, FLIM detectors, and TCSPC modules various optical configurations are possible. All have in common that the sample is excited by a pulsed laser of high repetition rate, and that the fluorescence light is detected by one or several fast photon counting detectors via a suitable optical interface. Typical optical FLIM configurations for the LSM 710, LSM 780 and LSM 880 microscopes are shown in Fig. 3 and Fig. 4.
Fig. 3 shows FLIM configurations for inverted microscopes. The configuration on the left uses multiphoton excitation by a femtosecond titanium-sapphire laser for excitation. The fluorescence light is detected via a non-descanned detection (NDD) beam path. Typically, the light is split in two spectral components by a Zeiss 'T Adapter', and detected by two parallel detectors and TCSPC channels.
The configuration shown on the right uses picosecond diode lasers or the tuneable ‘Intune’ laser. The sample is excited by one-photon excitation. The fluorescence light is detected back through the confocal beam path of the scanner and sent out of the scan head via an optical port. It is split into two spectral channels by a bh beamsplitter module and fed into two FLIM detectors. The signals from the detectors are recorded by two separate TCSPC channels of the FLIM system.
Fig. 3: LSM 710/780 family FLIM systems, inverted microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon FLIM with confocal detection.
Fig. 3 shows FLIM at an LSM 710/780 in the upright version. The configuration on the left uses multiphoton excitation and non-descanned detection, the configuration on the right one-photon excitation and confocal detection.
Fig. 4: LSM 710/780/880 family FLIM systems, upright microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon excitation FLIM with confocal detection.
The FLIM configurations for the LSM 880 are shown in Fig. 5. The multiphoton NDD system is the same as for the LSM 710/80, see Fig. 5, left. The one-photon (confocal system) is shown in Fig. 5, right. The LSM 880 uses a special 'Airy Scan' detector at the confocal output of the scan head. To provide an optical output to the FLIM detectors Zeiss deliver a beam switch between the scan head and the Airy Scan detector. Upright LSM-880s are similar to the inverted versions, except for the fact that the microscope body is different.
Fig. 5: LSM 880 FLIM systems, inverted microscopes. Left: Multiphoton-excitation FLIM with non-descanned detection. Right: One-photon excitation FLIM with confocal detection.
Standard bh FLIM systems for the LSM 710, 780 and 880 use the bh HPM‑100-40 hybrid detectors [33, 60]. However, the bh systems work also with the Zeiss BIG 2 detector, with NIR versions of the HPM‑100 detectors, with new ultra-fast HPM-100 detectors, and with the bh MW-FLIM GaAsP multi-spectral FLIM detectors. The optical configuration for multiphoton multi-wavelength FLIM is shown in Fig. 6. The light is collected from an NDD port by a fibre bundle. The light is dispersed spectrally, and detected by a bh PML-16 GaAsP (16-channel) PMT module. Similarly, the multi-wavelength FLIM setup can be attached to the confocal port of the scan head.
Fig. 6: Multi-wavelength FLIM
The FLIM systems for the Zeiss LSM 710/780/880 family use the SPCM data acquisition software. Since 2013 the SPCM software is available in a 64-bit version. SPCM 64 bit exploits the full capability of Windows 64 bit, resulting in faster data processing, capability of recording images of extremely large pixel numbers, and availability of additional multi-dimensional FLIM modes [36, 66, 457]. Although running on different computers, the bh SPCM software and the Zeiss ZEN software can be operated from the same keyboard and the same mouse.
The main panel of the SPCM data acquisition software is configurable by the user . Four configurations for FLIM systems are shown in Fig. 7. During the acquisition the SPCM software displays intermediate results in predefined intervals, usually every few seconds. The acquisition can be stopped after a defined acquisition time or by a user commend when the desired signal-to-noise ratio has been reached .
Fig. 7: SPCM software panel. Top left to bottom right: Dual-channel FLIM, multi-spectral FLIM, online FLIM, temporal mosaic FLIM, simultaneous FLIM/PLIM, fluorescence decay and FCS.
Frequently used operation modes and user interface configuration can be selected from a panel of predefined setups. Switching between Preview modes, FLIM acquisition, different pixel and time-channel numbers, time-series recording, Z-stack recording, FCS, or any other conceivable recording procedure is a matter of a single mouse click, see Fig. 8. The predefined setups are the key to the efficient use using the bh FLIM systems for the Zeiss LSMs.
Fig. 8: Switching the instrument configuration via the ‘Predefined Setup’ panel
When FLIM is applied to live samples the time and exposure needed for sample positioning, focusing, laser power adjustment, and region-of-interest selection has to minimised. Therefore, the FLIM systems have a fast preview function. The preview function displays images in intervals on the order of 1 second and less, see Fig. 9.
Fig. 9: SPCM software in fast preview mode. 1 second per image.
Whatever you change in the microscope: The position of the samples, the scan area, the zoom factor, the focal plane, pinhole size or the laser power - the result becomes immediately visible in the preview images, see Fig. 10.
Fig. 10: When the scan area definition in the Zeiss ZEN software is changed the result is shown in the images immediately.
The bh HPM‑100‑40 GaAsP hybrid detectors combine SPAD-like sensitivity with the large active area of a PMT [33, 60]. The large area allows light to be efficiently collected from non-descanned ports or through large pinholes. In contrast to SPADs, there is no ‘diffusion tail’ in the temporal response. Moreover, the hybrid detectors are free of afterpulsing. The absence of afterpulsing results in improved contrast, higher dynamic range of the decay curves recorded, and in the capability to obtain FCS data from a single detector.
bh FLIM systems can be equipped with ultra-fast HPM-100-06 or -07 hybrid detectors . In combination with Ti:Sa laser systems and bh SPC-150N or SPC-160N FLIM modules the FLIM systems achieve instruments response widths of less than 20 ps fwhm (full width at half maximum). This is world record in FLIM time resolution!
Possible applications are metabolic FLIM via the bound and unbound components of NAD(P)H, and quantitative FRET experiments with resolution of the bound and unbound donor components.
The bh FLIM systems also work with the Zeiss GaAsP BIG-2 detector. The BIG-2 has two channels for different wavelength. The two outputs are connected the inputs of the two TCSPC channels of a bh dual-channel FLIM system.
The bh FLIM systems are perfectly compatible with the fast beam scanning used in the Zeiss LSM 710/780/880 family microscopes. Frame times can be from about 30 ms to a few seconds, with pixel dwell times down to one microsecond. The multi-dimensional TCSPC process used in the bh FLIM systems delivers identical results for different scan rates, provided the total acquisition time is the same. FLIM can be acquired at short acquisition time. Fig. 11 shows lifetime images of a BPAE cell recorded within 5 seconds acquisition time.
Fig. 11: FLIM acquired within 5 seconds of acquisition time. Left 485 to 560 nm, right 560 to 650 nm. BPAE cell stained with Alexa 488 and Mito Tracker Red.
Acquisition at high scan rate is also the basis of the fast preview mode (see above) and of the fast FLIM time-series recording. With the bh FLIM systems time-series can be recorded as fast as two images per second. Fig. 12 shows time-series FLIM of an amoeba. The images are 0.4-second snapshots taken very one second.
Fig. 12: Moving amoeba. Autofluorescence, acquisition time 0.5 s, image rate 1 image per second.
Starting from Version 9.72 SPCM software the FLIM systems for the Zeiss LSM 710 / 780 / 880 family are able to display lifetime images online, both during the accumulation of FLIM data and for the individual steps of a fast image sequence. An example is shown in Fig. 13.
Fig. 13: Intensity image (left) and online lifetime image (right) calculated online by SPCM software
The calculation of the lifetime images is based on the first moment of the decay data in the pixels of the images . The first-moment technique combines short calculation times with near-ideal photon efficiency. It does not require to reduce the time resolution (time channels per pixel) to obtain high image rates. Even if the fast online lifetime function is used during the FLIM acquisition the data can later be processed by precision SPCImage multi-exponential data analysis.
With Version 9.73 SPCM Software, the bh TCSPC / FLIM systems support the bidirectional scanning function of the Zeiss LSMs. As usual, data recording is synchronised with the scanning by frame clock, line clock, and pixel clock pulses from the scanner. Each first line clock pulse indicates the beginning of a forward scan, each second one the beginning of a backward scan. The recording procedure automatically reverses the data from the backward scan and compensates for the line shift caused by the dynamic behaviour of the scanner. The FLIM data structure is the same as for unidirectional scanning. Thus, standard online intensity and lifetime display functions of the SPCM software are available, and data can be analysed by SPCImage as usual.
Fig. 14: FLIM of Convallaria sample (left, 512x512 pixels) and BPAE Cell sample (right, 1024x1024 pixels), recorded with bidirectional scanning. Images created by online-lifetime function of SPCM software.
Until recently lifetime images have been recorded with 256 x 256 or 512 x 512 pixels and 256 time channels. With bh’s megapixel technology, pixel numbers can be increased up to 2048 x 2048 while maintaining a temporal resolution of 256 time channels. Or, the temporal resolution can be increased to, for example, 1024 time channels, while still having 1024x1024 pixels available. Thus, the useful pixel resolution is rather limited by the optical resolution and the maximum field of view of the microscope lens than by the capabilities of the bh FLIM system. By using the ‘Mosaic FLIM’ option, the useful pixel number can be increased even beyond the capabilities of the microscope lens, see Fig. 31 page 18. Alternatively, the number of time channels can be increased up to 1024 for images of 1024x1024 pixels, and up to 4096 for images of 512x512 pixels or less.
Fig. 15 shows a FLIM image of a BPAE sample recorded at a resolution of 1024 x 1024 pixels. The image on the left shows the entire area of the scan. The image on the right have been selected from the full scan by the zoom function of the SPCImage data analysis software.
Fig. 15: Left: Image recorded with 1024 x 1024 pixels. Right: Digital zoom into the data of Fig. 15, showing the two cells on the upper left. Effective resolution 256x256 pixels. LSM 710 Intune system, excitation 535 nm, emission from 550 nm up. BPAE cell stained with Alexa 488 and Mito Tracker Red.
Standard bh FLIM systems record in two wavelength intervals simultaneously. The signals are detected by separate detectors and processed by separate TCSPC modules . There is no intensity or lifetime crosstalk. Even if one channel overloads the other channel is still able to produce correct data.
Fig. 16: Dual-channel detection. BPAE cells stained with Alexa 488 phalloidin and Mito Tracker Red. Left: 460 nm to 550 nm. Right: 550 nm to 650 nm.
The number of (parallel) FLIM channels can be increased up to four by adding additional beamsplitters and detectors to the NDD or confocal beam path.
The bh FLIM system can synchronise two ps diode lasers or a ps diode laser and the Zeiss Intune laser for wavelength-multiplexed FLIM by pulse-interleaved excitation (PIE). An example is shown in Fig. 17.
Fig. 17: FLIM by PIE, Zeiss LSM 710 Intune laser (‘green laser’) and 405 nm ps diode laser (‘blue laser’). Images recorded by the two parallel SPC‑150 TCSPC modules of the FLIM system.
The bh Multiphoton FLIM systems use the non-descanned detection (NDD) path of the LSM 710/780/880 NLO microscopes. With non-descanned detection, fluorescence photons scattered on the way out of the sample are detected far more efficiently than in a confocal system. The result is that clear images are obtained from deep tissue layers. An example is shown in Fig. 18. The images show a pig skin sample exited by two-photon excitation at 800 nm. The left image shows the wavelength channel below 480 nm. This channel contains both fluorescence and SHG signals. The SHG fraction of the signal has been extracted from the FLIM data and displayed by colour. The right image is from the channel >480 nm. It contains only fluorescence, the colour corresponds to the amplitude-weighted mean lifetime of the multi-exponential decay functions.
Fig. 18: Two-photon FLIM of pig skin. LSM 710 NLO, excitation 800nm, HPM‑100‑40, NDD. Left: Wavelength channel <480nm, colour shows percentage of SHG in the recorded signal. Right: Wavelength channel >480nm, colour shows amplitude-weighted mean lifetime.
In 2017 bh introduced ultra-fast hybrid detectors with a timing jitter of less than 9 ps rms. The instrument-response function of a multiphoton FLIM system with these detectors has a full-width at half maximum (FWHM) of less than 20 ps [42, 66]. The fast response greatly improves the accuracy at which fast decay components can be extracted from a multi-exponential decay. Applications are mainly in the field of metabolic FLIM, which requires separation of the decay components bound and unbound NADH, and in the field of FRET, where interacting and non-interacting donor decay components need to be separated. An NADH FLIM image recorded with an ultra-fast FLIM system on a Zeiss LSM 880 is shown in Fig. 18. Images of the images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH) are shown in Fig. 20.
Fig. 19: Left: NADH Lifetime image, amplitude-weighted lifetime of double-exponential fit. Right: Decay curve in selected spot, 9x9 pixel area. FLIM data format 512x512 pixels, 1024 time channels. The IRF width is 19 ps, the time-channel width 10ps.
Fig. 20: Left to right: Images of the amplitude ratio, a1/a2 (unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay component (t2, bound NADH). FLIM data format 512x512 pixels, 1024 time channels. Time-channel width 10ps.
The LSM 710/780/880 confocal microscopes are available with fully integrated FLIM lasers based on bh / Lasos BDL‑SMC picosecond diode lasers. Together with the superior efficiency of the bh hybrid detectors and of the Zeiss LSM 710, LSM 780 or LSM 880 scan head FLIM is performed at excellent sensitivity, see Fig. 21.
Fig. 21: Confocal FLIM with diode-laser excitation. Left: Plant tissue, autofluorescence. Right: HEK cell, interacting proteins, FRET from GFP into RFP.
Tuneable-excitation FLIM uses the ‘Intune’ laser of the Zeiss LSM 710/780/880 systems. With the Intune laser FLIM images of the same sample can be obtained for different excitation wavelength, see Fig. 23 and Fig. 23.
Fig. 22:Confocal FLIM with tuneable ‘Intune’ laser. BPAE cells stained with Alexa 488 phalloidin and Mito tracker red. Amplitude weighted lifetime of double-exponential model. Excitation at 490 and 520 nm, all images 1024 x 1024 pixels.
Fig. 23: Confocal FLIM with tuneable ‘Intune’ laser. BPAE cells stained with Alexa 488 phalloidin and Mito tracker red. Amplitude weighted lifetime of double-exponential model. Excitation at 535, 556 nm, all images 1024 x 1024 pixels.
By tuning the excitation wavelength to exactly the absorption maximum of the fluorophores maximum excitation efficiency is obtained at minimum sample exposure. High laser power, excellent spectral purity of the laser, high efficiency of the confocal detection path, and high efficiency of the GaAsP hybrid detectors result in FLIM data of excellent special and temporal resolution.
Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they bind to. NIR FLIM is possible by using HPM‑100-50 detectors and Ti:Sapphire laser excitation. Different than for multiphoton FLIM, the Ti:Sapphire laser is used as a one-photon excitation source .
Fig. 24: Pig skin samples stained with 3,3’-diethylthiatricarbocyanine. Excitation at 780nm, detection 800nm to 900nm
With the Zeiss LSM NLO OPO systems near-infrared FLIM can be performed by two-photon excitation. The fluorescence signals are detected by HPM-100-50 NIR detectors and non-descanned detection. With excitation wavelengths in the range of 1000 to 1330 nm, the typical NIR dyes are excited at high efficiency [66, 64, 69]. Fluorescence is detected up to 900 nm. An example is shown in Fig. 25.
Fig. 25: Pig skin stained with Indocyanin Green. LSM 7 OPO system, two-photon excitation at 1200 nm, non-descanned detection, 780 to 850 nm. Depth from top of tissue 10 µm (left) and 40 µm (right). 512x512 pixels, 256 time channels.
The bh multispectral FLIM system detects simultaneously in 16 wavelength intervals . By using bh’s multi-dimensional TCSPC process it avoids any time gating or wavelength scanning. The systems thus reach near-ideal recording efficiency. Dynamic effects in the sample or photobleaching do not cause distortions of the spectra or decay functions. An example for confocal detection and one-photon (diode-laser) excitation is shown in Fig. 26.
Fig. 26: Confocal multispectral FLIM. Part of a water flee, excitation by 405 nm ps diode laser, LSM 710 confocal port, bh MW FLIM detector
bh’s MW FLIM is the world’s first simultaneously detecting multiphoton multispectral NDD FLIM system. It uses an optical interface that connects to the NDD ports of the LSM 710/780/880 NLO and LSM 7 MP microscopes , see Fig. 6, page 4. A typical result is shown in Fig. 27.
Fig. 27: Multiphoton Multispectral NDD FLIM. LSM 710 NLO, bh MW FLIM detector
Multispectral FLIM got a new push by the introduction of the new PML‑16 GaAsP 16-channel detector. This detector has five time the efficiency of the older PML‑16 detectors with conventional cathodes. Another improvement came from bh’s Megapixel FLIM technology. Multi-spectral FLIM can now be obtained at an image size of 512 x 512 pixels in each wavelength channel while maintaining the usual 256-channel time resolution .
For many years, Z-stack FLIM had been prevented by photobleaching and photodamage caused by the high excitation dose. With the LSM 710/780/880 family microscopes and the new GaAsP hybrid detectors photobleaching is no longer a problem. The FLIM system automatically acquires FLIM in consecutive Z planes and saves the data into a sequence of files. Z‑stack FLIM is particularly interesting in combination with the deep-tissue imaging capability of multiphoton NDD FLIM. It can, however, be used also in combination with diode-laser FLIM and Intune FLIM.
Fig. 28: Z-stack FLIM. Pig skin, autofluorescence, 2p excitation, 5 µm to 60µm below the surface
Sometimes it is desirable to project the tree-dimensional structure of a tissue sample into a single image. A typical example is neuronal tissue where neurons are not necessarily located within a particular image plane. In these cases the SPCM software is able to record a Z stack of the 3-dimensional structure of the sample, and then project a selected number images of different Z planes into a single FLIM data set. An example is shown in Fig. 29.
Fig. 29: Infinite-focus depth FLIM. A Z stack of a 3-dimensional sample is recorded, and the image of several planes are projected into a singe FLIM image. Brain tissue, LSM‑780 NLO, Simple-Tau 150 FLIM system.
Time-series FLIM is available for all system versions, and all detectors. With SPC-152 dual-channel systems time series as fast as 2 images per second can be obtained. A time series taken at a moss leaf is shown in Fig. 30. The fluorescence lifetime of the chloroplasts changes due to the Kautski effect induced by the illumination.
Fig. 30: Time-series FLIM, 2 seconds per image. Chloroplasts in moss leaf, lifetime change by Kautski effect
Mosaic FLIM is based on bh’s ‘Megapixel FLIM’ technology introduced in 2014. Mosaic FLIM records a large number of consecutive images into a single FLIM data array. The individual images within this array can represent the elements of a tile scan (x-y mosaic), images in different depth in the sample (z-stack mosaic), or images for different times after a stimulation of the sample (temporal mosaic). Spatial mosaic FLIM combines favourably with the Tile Imaging capability of the Zeiss LSM 710/780/880 systems. An example of an x-y mosaic is shown in Fig. 31. The complete data array has 2048 x 2048 pixels, and 256 time channels per pixel. Compared to a similar image taken through a low-magnification lens the advantage of mosaic FLIM is that a lens of high numerical aperture can be used, resulting in high detection efficiency and high spatial resolution.
Fig. 31: Mosaic FLIM of a Convallaria sample. The mosaic has 4x4 elements, each element has 512x512 pixels with 256 time channels. The entire mosaic has 2048 x 2048 pixels, each pixel holding 256 time channels. Zeiss LSM 710 Intune system with bh Simple-Tau 150 FLIM system. Total sample size covered by the mosaic 2.5 x 2.5 mm.
The Mosaic FLIM function can be used to record Z Stacks of FLIM images. As the microscope scans consecutive images planes in the sample the FLIM system records the data into consecutive elements of a FLIM mosaic. The advantage over the traditional record-and-save procedure is that no time has to be reserved for save operations, and that the entire array can be analysed in a single analysis run.
Fig. 32: FLIM Z-stack, recorded by Mosaic FLIM. Pig skin stained with DTTC. 16 planes, 0 to 60 um from top of tissue. Each element of the FLIM mosaic has 512x512 pixels and 256 time channels per pixel. Plane 8 is shown magnified on the right. LSM 7 MP OPO system, HPM-100-50 GaAs hybrid detector.
Mosaic FLIM can be used to record fast FLIM time series. An example is shown in Fig. 33.
Fig. 33: Time series acquired by mosaic FLIM. Recorded at a speed of 1 mosaic element per second. 64 elements, each element 128 x 128 pixels, 256 time channels, double-exponential fit of decay data. Sequence starts at upper left. Moss leaf, lifetime changes by non-photochemical chlorophyll transient.
Also here, the advantage is that no time has to be reserved for save operations during the recording sequence. A Mosaic-FLIM time series can therefore be made very fast.
The most important advantage of temporal Mosaic FLIM is that the data can be accumulated. A lifetime change in the sample is stimulated periodically, and a mosaic recording sequence started for each stimulation. Because the entire photon distribution is kept in the memory the photons from the subsequent runs are automatically accumulated. The result is that the signal-to-noise ratio no longer depends on the speed of the series. The only speed limitation is the minimum frame time of the scanner. For the Zeiss LSMs frame times of less than 40 milliseconds can be achieved. This brings the transient-time resolution down to the range where Ca2+ transients in neurons occur. An example is shown in Fig. 34, for details please see page 181.
Fig. 34: Temporal mosaic FLIM of the Ca2+ transient in cultured neurons after stimulation with an electrical signal. The time per mosaic element is 38 milliseconds, the entire mosaic covers 600 milliseconds. Experiment time runs from upper left to lower right, stimulation occurred in the 4th frame. Photons were accumulated over 100 stimulation periods. Zeiss LSM 7 MP multiphoton microscope and bh SPC‑150 TCSPC module. Data courtesy of Inna Slutsky and Samuel Frere, Tel Aviv University, Sackler Faculty of Medicine.
The elements of a FLIM mosaic can be assigned to information delivered to the TCSC modules via their routing inputs. The traditional application is multi-wavelength FLIM. Examples for multi-wavelength FLIM by the Mosaic FLIM function are shown in  and . A potential application is the combination of FLIM with electro-physiology. In that case, one or more electrophysiology signals would be measured by micro-electrodes, digitized, and used to control the mosaic element into which the photons are recorded. The result would be a mosaic of FLIM images for different physiological states of the cells.
FLITS records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond [66, 62]. Typical applications are recording of chlorophyll transients and Ca2+ transients in neurons or neuronal tissue [65, 181]. Examples are shown in Fig. 35 and Fig. 36.
Fig. 35: FLITS of chloroplasts in a grass blade, change of fluorescence lifetime after start of illumination. Experiment time runs bottom up. Left: Non-photochemical transient, transient resolution 60 ms. Right: Photochemical transient. Triggered accumulation, transient-time resolution 1 ms.
Fig. 36: FLITS of Ca2+ concentration in cultured neurons. Ca2+ sensor Oregon Green, LSM 7 MP, electrical stimulation, 100 stimulation periods accumulated. Transient-time resolution 2 ms.
The bh FLIM systems are able to simultaneously record fluorescence and phosphorescence lifetime images. The technique is based on modulating a high-frequency pulsed excitation laser synchronously with the pixel clock of the scanner. Photon times are determined both with reference to the laser pulses and the laser modulation pulse [34, 40, 61]. Fluorescence is recorded during the ‘on’ time, phosphorescence during the ‘off’ time of the laser. A typical result is shown in Fig. 37.
Fig. 37: Yeast cells stained with a Ruthenium dye. Left: FLIM image and fluorescence decay curve in selected spot. Right: PLIM image and phosphorescence decay curve in selected spot.
The bh GaAsP hybrid detectors deliver highly efficient FCS. Because the detectors are free of afterpulsing diffusion times are obtained from a single detector, without the loss in correlation events that occurs when the signals from two detectors are cross-correlated. FCS is be obtained with the diode-laser systems, the Intune system, and even with the multiphoton NDD systems.
Fig. 38: FCS with GaAsP hybrid detectors. Left to right: Confocal FCS with ps diode laser, Confocal FCS with Intune laser, Two-photon NDD FCS
The bh FLIM systems for the Zeiss LSMs use bh SPCImage data analysis software, see page 225 of this handbook. SPCImage runs a de-convolution on the decay data in the pixels of FLIM data. It uses single, double, or triple-exponential decay analysis to produce pseudo-colour images of lifetimes, amplitudes, or intensities of decay components, or of ratios of these parameters. An ‘incomplete decay’ model is available to determine long fluorescence lifetimes within the short pulse period of the Ti:Sa laser of a multiphoton system. Moreover, by extracting the instrument response function (IRF) from the FLIM data themselves, SPCImage avoids troublesome IRF recording.
The main panel of the SPCImage data analysis is shown in Fig. 321. It shows a lifetime image (left) a parameter histogram over the pixels of a region of interest (upper right), and the fluorescence decay curve in a selected spot of the image (lower right). The basic model parameters (one, two or three exponential components) are selected in the upper right.
Fig. 39: Main panel of the SPCImage data analysis (Version 5.4 and later)
Moreover, SPCImage can extract phosphorescence intensities from normal FLIM data, or distinguish regions with single-exponential decay from regions where the decay is multi-exponential. SPCImage is also able to display time-gated images, e.g. to extract SHG images from FLIM data, or to reject un-depleted fluorescence in STED-FLIM images. A batch-processing function and a batch export function are available for analysing a large number of FLIM data sets automatically and to convert them into bmp of tif images. Please see chapter 'SPCImage Data Analysis'.
SPCImage has a histogram function for the decay parameter selected for coulour-coding the lifetime image. The histogram shows how often pixels of a given parameter value (lifetime, lifetime of a decay component, amplitude of a component, or combinations of these values) occurs in the lifetime image. Histograms can be displayed for several regions of interest. An example is shown in Fig. 386.
Fig. 40: Decay-parameter histogram, for two different regions of interest
SPCImage has a function that displays two-dimensional histograms of decay parameters. 2D histograms display a density plot of the pixels over two selectable decay parameters. The decay parameters can be lifetimes, t1, t2, t3, amplitudes, a1, a2, a3, of decay components, amplitude or intensity-weighted lifetimes, tm or ti, or arithmetic conjunctions of these parameters. A histogram of the amplitude ratio, a1/a2, over the amplitude-weighted lifetime, tm, is shown in Fig. 387.
Fig. 41: Main panel of SPCImage showing a FRET cell (left) and 2D Histogram of lifetime versus amplitude ration of components
Areas of parameter combinations of a multi-exponential decay function can be selected in the 2D histograms and back-annotated in the images, see Fig. 42.
Fig. 42: Images and 2D histograms. Selection of different cell compartments in the decay-parameter histogram.
Version 6.0 SPCImage FLIM analysis software combines time-domain multi-exponential decay analysis with the phasor plot. In the phasor plot, the decay data in the individual pixels are expressed as phase and amplitude values in a polar diagram [147, 148]. Independently of their location in the image, pixels with similar decay signature form clusters in the phasor plot. Different phasor clusters can be selected, and the corresponding pixels back-annotated in the time-domain FLIM images, see Fig. 390. Moreover, the decay functions of the pixels within the selected phasor range can be combined into a single decay curve of high photon number. This curve can be analysed at high accuracy, revealing decay components that are not visible by normal pixel-by-pixel analysis, see Fig. 391. Please see chapter 'SPCImage data analysis'.
Fig. 43: Left: Lifetime image and lifetime histogram. Right: Phasor plot. The clusters in the phasor plot represent pixels of different lifetime in the lifetime image. Recorded by bh Simple-Tau 152 FLIM system with Zeiss LSM 880.
Fig. 44: Left: Selecting a cluster of phasors in the phasor plot. Middle: Combination of the decay data of the corresponding pixels in a single decay curve. Right: Display of the pixels corresponding to the selected cluster in the phasor plot.
The bh ‘Burst Analyser’ software is used for data analysis of single-molecule fluorescence. It uses parameter-tag data files recorded in the FIFO mode of the SPC‑630, SPC‑130EM, SPC‑150, SPC‑150N, or SPC‑160 TCSPC modules. Photon bursts from single molecules travelling through a femtoliter detection volume are identified in the parameter-tag data. Within the bursts, intensities, intensity variations, fluorescence lifetimes, and ratios of these parameters between several detection channels or different time windows of a PIE recording are determined, and time-traces and histograms of the parameters are calculated. The results are used to obtain histograms and time traces of FRET efficiencies, and to calculate FCS and FCCS data. The Burst Analyser is described in a separate handbook, see .
Fig. 45: bh Burst Analyzer software for single-molecule data analysis
From the user point of view, FLIM in a laser scanning microscope is performed by the same general procedures as conventional imaging. First, the sample is brought in focus by looking through the eyepieces and manually turning the microscope focus buttons. Then a fast repetitive scan is run in which the FLIM system displays fast preview images. While observing these images, the exact location and size of the scan area are selected, and the focal plane is fine-adjusted. Then an appropriate FLIM mode is selected from the predefined setup panel, and the final FLIM acquisition is started. A step-by-steps recipe of a basic FLIM measurement is given below. For complex FLIM procedures please see chapter ‘Advanced FLIM Techniques and Procedures’, page 109.
1. Turn on the FLIM system: First turn on the extension box, then start the laptop computer.
2. Start the ZEN software on the Zeiss computer. Start the SPCM application on the FLIM computer.
3. One-photon systems: Turn on the ps diode laser or the Intune laser in the Zeiss ZEN software. Two-photon systems: Turn on the Ti:Sa laser
4. Put the sample in the microscope. Turn on the microscope lamp. Switch to the Eyepiece beam path. Use the eyepieces to bring the sample in focus and in the right position. Turn the microscope lamp off.
5. In the Zeiss ZEN software, use an image size of 512 x 512 pixels. Confocal systems: Send the light to the confocal output. NDD systems: Send the light to the NDD output with the FLIM detector.
6. Enable the detectors in the SPCM software.
7. Click into the ‘predefined setup’ panel, and load the setup for fast preview. Start the measurement in the SPCM software, start ‘Continuous Scan’ in the ZEN software.
8. By looking at the images displayed, fine-adjust focus and sample position. Use the zoom function of the LSM to select the desired image area. Adjust the laser power or the pinhole size to obtain a CFD count rate between 5×104 and 5×106.
Should one or both detectors shut down by overload (right panel) decrease the laser power or reduce the pinhole size. Then click on the ‘Reset’ button of the DCC panel to re-activate the detector.
9. Stop the Preview. Load an appropriate FLIM acquisition setup from the predefined setup panel.
Start the scan, start the measurement. Let the measurement run until you are satisfied by the signal-to noise ratio.
10. When the images look good, stop the measurement in the FLIM system. Stop the scan in the LSM. Save the FLIM data by using ‘Main’, ‘Save’. Send the data to the SPCImage lifetime data analysis.
11. Set the cursors as shown in the SPCImage panel. Select the decay model and the fit parameters. Click on ‘Calculate decay matrix’. Let the calculation run until a lifetime image appears.
The bh FLIM technique makes use of the special properties of high-repetition rate optical signals detected by a high-gain detector, see Fig. 46. Fluorescence of a sample is excited by a laser of 80 MHz pulse repetition rate (a). The expected fluorescence waveform is (b). However, the detector signal (measured by an oscilloscope) has no similarity with the expected fluorescence waveform. Instead, it consists of a few pulses randomly spread over the time axis (c). The pulses represent the detection of single photons of the fluorescence signal.
Fig. 46: Detector signal for fluorescence detection at a pulse repetition rate of 80 MHz. Detection rate 107 photons per second
The photon detection rate shown in trace (c) of Fig. 46 is about 107 s-1. This is on the order of the maximum possible detection rate of most photon counting detectors, and far above the count rates available from a live specimen in a scanning microscope. Thus, the fluorescence waveform (b) has to be considered a photon density as a function of the time after the excitation pulses. Fig. 46 shows that the detection of a photon in a particular signal period is a relatively unlikely event [55, 66]. The buildup of the photon distribution is then a straightforward task: The arrival times of the photons after the excitation pulses would be measured, and a histogram over the arrival time be built up, see Fig. 47.
Fig. 47: Principle of time-correlated single photon counting
The principle shown in Fig. 47 is the classic principle of time-correlated single photon counting [354, 518]. The limitation of classic TCSPC is that it is intrinsically one-dimensional. To use it in a laser scanning microscope the scanner had to be stopped in every pixel until a fluorescence decay curve has been acquired. Such ‘slow scan’ procedures have indeed been used [106, 107] but are neither compatible with live cell imaging nor with the fast scan rates used in modern scanning microscopes.
The limitation of classic TCSPC has been overcome by a multi-dimensional TCSPC technique introduced by bh in 1993. The photons are characterised not only by a single parameter (the arrival time after the laser pulse) but by several parameters, such as the location in a scan area [46, 51], the wavelength [48, 53, 57], the time from the start of the experiment , or the time from a stimulation of the sample . The recording process builds up a photon distribution over these parameters [55, 66, 67]. The result is a multi-dimensional photon distribution as shown in Fig. 48.
Fig. 48: Multi-dimensional TCSPC. Each photon is characterised by several parameters, and a photon distributions over these parameters is built up.
Combined with a confocal or two-photon laser scanning microscope, multi-dimensional TCSPC makes a fluorescence lifetime imaging (FLIM) technique with near-ideal counting efficiency, picosecond time resolution, multi-wavelength capability, and the capability to resolve multi-exponential decay profiles [51, 55, 66, 67].
The basic architecture of a TCSPC FLIM device is shown in Fig. 49. The laser scanning microscope scans the sample with a high-frequency pulsed laser beam. The fluorescence from the sample is collected back through the beam path of the microscope and detected by a fast photon counting detector. For every detected photon the detector sends an electrical pulse into the TCSPC module. Moreover, the TCSPC module receives scan clock pulses (pixel, line, and frame clock) from the scanning unit of the microscope.
Fig. 49: Multidimensional TCSPC architecture for FLIM
For each pulse from the detector, the TCSPC module determines the time within the laser pulse sequence (i.e. in the fluorescence decay) and the location within the scanning area, x and y. The photon times, t, and the spatial coordinates, x and y, are used to address a memory in which the detection events are accumulated. Thus, in the memory the distribution of the photon density over x, y, and t builds up.
The FLIM data can be built up directly in the device memory of the TCSPC module, or the data of the individual photons and the scan clocks are transferred into the computer and the photon distribution is built up there.
The first technique has the advantage that it works up to extremely high count rates and scan speeds. It is applicable for resonance scanners, and ultra-fast polygon scanners . The disadvantage is that the size of the FLIM data is limited by the on-board memory capacity of the TCSPC device.
The second technique has the advantage of having the large memory of the computer available for the buildup of the FLIM data. It thus delivers FLIM data with large numbers of pixels and time channels. Moreover, in addition to building up FLIM data it keeps the full information about the individual photons available. Such ‘parameter tagged’ photon data can be used in various ways, such as for multi-parameter single-molecule spectroscopy , FCS , and phosphorescence lifetime imaging (PLIM) . bh TCSPC FLIM modules have therefore both principles implemented, please see  for details.
The result of a FLIM measurement is a data array representing the pixel array of the scan, with every pixel containing a large number of time channels with photon numbers for consecutive times after the excitation pulse, see Fig. 49, right. In other words, the result is an image that contains a fluorescence decay curve in each pixel.
The results are displayed as pseudo-colour images, see Fig. 50. The brightness represents the number of photons per pixel. The colour can be assigned to any parameter of the decay profile: The lifetime of a single-exponential approximation of the decay, a lifetime of a decay component, the amplitude of a component, a ratio of lifetimes or amplitudes of a multi-exponential decay, or the average lifetime of a multi-exponential decay.
Fig. 50: Lifetime image of a BPAE cell, stained with Alexa 488. FLIM data 512 x 512 pixels, 256 time channels per pixel. Fluorescence decay shown for two selected pixels. Zeiss LSM 710 Intune system with Becker & Hickl Simple-Tau 150 FLIM system.
It should be explicitly noted that multi-dimensional TCSPC does not require that the scanner stays in one pixel until enough photons for a full fluorescence decay curve have been acquired. It is only necessary that the total pixel time, over a large number of subsequent frames, is large enough to record a reasonable number of photons per pixel. Thus, TCSPC FLIM works even at the highest scan rates available in laser scanning microscopes. At pixel rates used in practice, the recording process is more or less random: A photon is just stored in a memory location according to its time in the fluorescence decay, its detector channel number, and the location of the laser spot in the sample in the moment of detection.
Simultaneous recording in several channels can be obtained by a ‘routing’ technique that uses a single TCSPC channel for several detectors [55, 66]. Although an elegant solution for multi-wavelength FLIM (see Fig. 52), routing does not increase the counting capability and the throughput of a FLIM system. Systems with no more than four detector channels are therefore increasingly built with parallel TCSPC channels, see Fig. 51. Parallel systems deliver high throughput rates. Another advantage is that the channels are independent. If one channel overloads the other ones still deliver correct data. Dual-channel systems have become standard for bh FLIM systems . Four-channel systems are easily feasible [52, 66], and an eight-channel parallel system has been demonstrated .
Fig. 51: Parallel-channel TCSPC system. The light is split in two wavelength channels, the signals of which are recorded by parallel TCSPC FLIM modules.
The principle described in Fig. 49, page 30, can be extended to simultaneously detecting in a large number of wavelength channels [48, 57]. As shown in Fig. 46, page 29, the count rate of the detector is far lower than the laser pulse rate. Thus, the probability of detecting several photons per period is negligible. Now consider the case that the light signal delivered to the detector is split spectrally and spread over a one-dimensional array of detectors. The total intensity for the whole array is the same as for a single detector receiving the undispersed signal. Thus, it is also unlikely that the whole array will detect several photons per signal period. In particular, it is unlikely that several detectors of the array will detect a photon in the same signal period. This is the basic idea behind multi-wavelength TCSPC. Although several detectors are active simultaneously they are unlikely to detect a photon in the same signal period. The times of the photons detected in all detectors of the array can therefore be determined in a single TCSPC channel.
To obtain multi-wavelength FLIM data it is sufficient to spread a spectrum of the fluorescence light over an array of detector channels, and determine the detection times, the channel number in the detector array, and the position, x, and y, of the laser spot for the individual photons. These pieces of information are used to build up a photon distribution over the time of the photons in the fluorescence decay, the wavelength, and the coordinates of the image [55, 66, 57, 82, 90, 91]. The technique is also known as ‘routing’ because the ‘channel’ signal routes the photons into different data blocks. The architecture of multi-wavelength FLIM is shown in Fig. 52.
Fig. 52: Principle of Multi-Wavelength TCSPC FLIM
As for single-wavelength FLIM, the result of the recording process is an array of pixels. However, the pixels now contain several decay curves for different wavelength. Each decay curve contains a large number of time channels; the time channels contain photon numbers for consecutive times after the excitation pulse.
Mosaic FLIM is part of bh’s new Megapixel FLIM (64-bit) technology. It records a sequence of lifetime images into a single, large photon distribution. The principle is shown in Fig. 53.
Fig. 53: Mosaic FLIM. The TCSPC system records a series of lifetime images in to a single, large photon distribution.
Mosaic FLIM is used to record image of large sample areas via the Tile Imaging function of the Zeiss LSMs, to record Z stacks, or to record fast time-series of FLIM images. Time series can even be recorded by triggered accumulation to resolve transient lifetime changes at a resolution down to less than 40 ms, see Fig. 34 in the introduction. Please see section ‘Mosaic FLIM’, page 118, for details.
Simultaneous FLIM and PLIM is based on on-off modulation of the FLIM laser and assigning two times to the photons. One is the time in the pulse period of the laser, the second one the time in the modulation period. The photon distribution over the times in the pulse period is the fluorescence lifetime image, the photon distribution over the times in the modulation period is the phosphorescence lifetime image. Details are described under 'Simultaneous FLIM / PLIM', page 131.
Fig. 54: Simultaneous FLIM and PLIM
The bh FLIM systems are also able to record FCS. The principle is shown in Fig. 55. For every photon, the TCSPC module determines the time in the excitation pulse period, t, and the time from the start of the experiment, T. From these data, the instrument software calculates the photon distribution over t, and the correlation function of the photons over T. The first one is the fluorescence decay curve, the second one the FCS curve. Cross-FCS is obtained by correlating the photons in one TCSPC channel versus the photons in the other .
Fig. 55: FCS Recording
The bh FLIM systems feature a high degree of modularity . Consequently, a large number of system configurations are possible for LSM 710/780/880 FLIM systems. The sample can be excited by one-photon excitation or by two-photon excitation. The fluorescence light may be detected by one detector, two detectors [50, 66], by three or four detectors, or by the 16 channels of a bh multi-wavelength detector [25, 48, 55, 30]. The FLIM systems may use a single TCSPC channel, or several TCSPC channels in parallel to increase the acquisition speed [52, 55, 66]. Different detectors may be used, and the detectors may be connected to the microscope through different optical interfaces.
To keep the variety of FLIM configurations at a reasonable level LSM 710/780/880 FLIM systems come in a two standard configurations:
FLIM systems for the LSM 710/789/880 NLO multiphoton microscopes have two HPM‑100 hybrid detectors and two parallel SPC‑150 TCSPC channels. Excitation is performed by the Ti:Sapphire laser of the microscope. The detectors are attached to the outputs of a Zeiss NDD T adapter module.
Confocal FLIM systems have two detectors and two parallel SPC‑150 TCSPC channels. Two HPM‑100-40 detectors are attached to the BIG port adapter of the scan head via a beamsplitter/filter module delivered by bh. Excitation is performed by the internal ps diode lasers of the LSM 710/780/880 or by the Intune laser.
Due to the modularity of the system and the compatibility of detector adapters a large degree of flexibility is maintained. Both multiphoton and one-photon systems can be delivered with only one detector and one TCSPC channel. The detectors can be moved from the NDD T adapter to a bh beamsplitter module attached to the BIG port of the scan head. More detector channels can be added by cascading beam splitters at the respective outputs of the microscope, and adding more detectors and TCSPC modules. Different detectors, such as the Zeiss BIG 2 detectors, or the NIR-sensitive HPM‑100-50 can be used. Multi-spectral FLIM can be achieved by replacing one detector with the bh MW-FLIM GaAsP multi-wavelength detector assembly.
Standard LSM 710/780/880 FLIM systems come as compact ‘Simple-Tau’ TCSPC systems. Single-channel systems contain one SPC‑150, SPC-150N or SPC-160 TCSPC FLIM module , dual-channel system contain two. Both versions contain one DCC‑100 detector controller module. Systems for PLIM also contain a DDG‑210 pulse generator card. Except for the different number of TCSPC modules and a different configuration of the SPCM user interface there are no significant differences between the two Simple-Tau systems, see Fig. 56.
Fig. 56: Simple-Tau 150 system with one TCSPC channel (left) and Simple-Tau 152 system with two channels (right).
The small size of the Simple-Tau systems is an advantage especially when a FLIM system has to fit into a crammed laboratory environment. However, with the introduction of bh’s megapixel FLIM technology resolution and pixel numbers in FLIM have increased enormously. The Simple-Tau systems are therefore available with large screens and external keyboards. The ‘Simple-Tau 152 LS’ with a 27” screen and 1920 x 1080 pixels resolution is shown in Fig. 57.
Fig. 57: Simple-Tau 152 LS system
The entire FLIM system is controlled by SPCM data acquisition software. Since 2013, the SPCM software uses 64 bit technology. As a result, the bh FLIM systems are able to record FLIM data with unprecedented pixel numbers and time channel numbers. Moreover, entirely new multi-dimensional recording principles, such as spatial or temporal Mosaic FLIM, or multi-wavelength FLIM at full resolution of large fields of view have become available. The data acquisition software is described in section ‘SPCM Software’, page 53, the data analysis in section ‘SPCImage Data Analysis Software’, page 225.
The HPM‑100-40 is the standard detector of the bh FLIM systems. The HPM‑100-40 uses hybrid detector technology, and combines high time resolution, excellent efficiency, large area, easy alignment, and absence of afterpulsing and afterpulsing background [66, 60]. The GaAsP cathode has a sensitivity about 5 times higher than conventional multi-alkali photocathodes. The sensitivity advantage over conventional PMTs is further enhanced by a perfect transfer efficiency of the photoelectrons from the photocathode to the avalanche diode.
The HPM-100-40 module is based on a Hamamatsu R 10467-40 tube. The has been integrated in a common housing with the high-voltage generators and a low-noise preamplifier, see Fig. 58, left. The HPM‑100-40 module is operated directly from the DCC‑100 detector controller of the FLIM system. Due to the 8 kV acceleration voltage the time resolution is very good, with a clean, smooth IRF shape, see Fig. 58, middle and right.
Fig. 58: HPM‑100 hybrid PMT module. The housing contains the tube, the high-voltage power supplies, and a low-noise preamplifier. Middle and right: IRF of the HPM‑100‑40, linear and logarithmic scale.
Due to its 3 mm diameter active area, the HPM-100 detector can be used at the non-descanned ports of the Zeiss multiphoton microscopes and at the confocal outputs from the scan head. When used as a confocal detector, it does not require accurate alignment, and can be used efficiently even with large pinhole diameters.
The perhaps most significant advantage of the HPM‑100-40 is the absence of afterpulsing. Afterpulsing is the major source of background in FLIM experiments. In comparison to conventional PMTs a HPM therefore delivers a substantially better dynamic range of the decay curves, see Fig. 59, left. As a result, fluorescence decay parameters can by derived from a given number of photons at a higher accuracy [33, 60, 66, 267].
FCS measurements are free of the typical afterpulsing peak, see Fig. 59, right. Thus, FCS can be obtained from a single detector, without the need of cross correlation. The result is a substantial increase in the signal-to noise ratio of the FCS data.
Fig. 59: FCS of fluorescein molecules in water, obtained by a single GaAsP hybrid detector
The superior performance of the hybrid detector becomes most obvious when it is used as a non descanned detector in a multiphoton microscope. With an active are of 3 mm in diameter it fits perfectly to the NDD optics of the LSM 710/780/880 NLO family microscopes. It not only outperforms standard PMTs by a factor of 5 in detection efficiency, it also yields a better lifetime accuracy from a given number of detected photons. Fig. 60 shows data recorded in the two channels of an LSM 710 NLO multiphoton FLIM system. The sample was a mouse kidney section, Molecular Probes F24630, stained with DAPI, Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidin. The left image shows the fluorescence of the two Alexa dyes. The lifetime image not only separates the two dyes but also shows variation due to the inhomogeneous local environment. The DAPI image is shown on the right. The DAPI does not display noticeable lifetime variation. The lifetime histograms of both images are shown on the right. As expected, the Alexa image shows a broad lifetime distribution. The DAPI lifetime histogram is narrow and symmetrical, showing that the FLIM system obtained near-ideal timing accuracy.
Fig. 60: Lifetime images recorded by two HPM‑100-40 hybrid PMT modules, Simple-Tau 152 dual channel parallel FLIM system. Mouse kidney section stained with DAPI, Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidin, excitation 780 nm. Left: Alexa and DAPI image. Right Lifetime distributions. Note the narrow lifetime distribution of the DAPI.
The HPM-100-50 detectors have a spectral range up to about 900 nm. They are used for FLIM with NIR dyes in combination with OPO excitation and one-photon excitation with a Ti:Sapphire laser. Please see section ‘Near-Infrared FLIM’, page 109 for details.
bh FLIM systems can be equipped with the new ultra-fast HPM-100-06 or -07 hybrid detectors of bh . In combination with Ti:Sa laser systems and bh SPC-150N, SPC-150NX or SPC-160 FLIM modules the FLIM systems achieve instruments response widths of less than 20 ps fwhm (full width at half maximum). Even with ps diode lasers, especially those of 405 nm and 445 nm wavelength, the IRF width is less than 40 ps, see Fig. 61
Fig. 61: IRF of an HPM-100-06 with a femtosecond laser (left) and with a bh picosecond diode laser (right)
Potential applications are metabolic FLIM via the bound and unbound components of NAD(P)H, see ‘FLIM with Ultra-Fast Detectors’, page 12, and quantitative FRET experiments with resolution of the bound and unbound donor components.
Becker & Hickl deliver cooled versions of the HPM‑100-40 and -50 hybrid detectors. The cooling reduces the dark count rate by about a factor of 5. Although this sounds promising the effect on the practically achieved quality of FLIM data is normally minuscule. The reason is that the daylight pickup of the microscope optics is usually much larger than the dark count background of even the uncooled detectors. Cooling is therefore only recommended for the near-infrared (-50) versions of the HPMs, and, possibly, for a PLIM measurement with phosphorescent compounds of extremely low emission intensity. Also then, a noticeable effect is only obtained if special provisions against pickup of ambient light are taken.
The Zeiss BiG-2 detector contains two PMTs with GaAsP photocathodes. As described for the HPM‑100-40 hybrid detectors, the GaAsP photocathode provides an efficiency about 5 times higher than a conventional photocathode. It is sensitive from about 350 nm to 700 nm. The BiG-2 detector can be used both at the confocal ports and at the non-descanned ports of the LSM 710/780/880 family confocal or multiphoton microscopes. The light is split on the two detectors by commonly used Zeiss-type microscope beamsplitter cubes. The cubes can be equipped with user-specific dichroic beamsplitters and filters to match the spectral properties of the fluorophores to be observed. The BiG-2 detector has an electrical output for the detector signal. It can therefore be used as a detector for TCSPC FLIM. The output amplitude for a single photon is on the order of 300 mV, perfectly compatible with the bh TCSPC FLIM systems. The output signals of the BiG‑2 are connected to the ‘CFD’ inputs of the two SPC‑150 modules via A-PPI-D pulse inverters.
The IRF of the BiG-2 detector is shown in Fig. 62. The full-width at half maximum (FWHM) is about 250 ps. The logarithmic display (top right) shows that there is a slight shoulder before the main peak, and a secondary peak about 1.5 ns later. Such secondary IRF structures are common for conventional PMTs [55, 66]. Their presence in the BiG-2 detector is therefore not surprising. The amplitude of the secondary peak is moderate and has little influence on the accuracy of fluorescence lifetime analysis. It may, however, complicate the extraction of low-amplitude decay components from double- or triple-exponential decay profiles.
Fig. 62: Top: IRF of the two channels of the BiG-2 detector recorded by the SPC-150 modules of the Simple-Tau system. Left: Linear scale. Right: Logarithmic scale. Horizontal scale 0 to 6 ns. The IRF width is about 250 ps. Please compare with IRF of hybrid detector, shown in Fig. 58.
Due to its excellent efficiency the BiG detector works well also in FCS applications. Fig. 63 shows decay curves and FCS curves of Atto 425 excited with the 405 nm ps diode laser. Although FCS at short excitation wavelength is not easy decay curves and FCS curves were recorded at high efficiency and without noticeable artefacts. Afterpulsing remains at a tolerable level and does not impair the FCS data at correlation time larger than 1 µs. Please see  for detailed discussion of the BIG 2 FLIM and FCS performance.
Fig. 63: Fluorescence decay functions and FCS curves detected in the two channels of the BiG‑2 detector. Atto 425, excitation by 405 nm ps diode laser. bh Simple-Tau 152 dual-channel FLIM system. The red curve is a fit with two diffusion terms and a triplet term.
The MW FLIM detection system  detects the fluorescence simultaneously in 16 wavelength channels . The optical principle is shown in Fig. 437, left. The fluorescence light is focused into the slit plane of a polychromator. The polychromator projects a spectrum of the fluorescence light on a 16-channel PMT tube inside a bh PML‑16 multichannel detector . Until 2013, multi-wavelength systems used the PML‑16C, based on a 16-channel PMT with a conventional multi-alkali cathode. Since 2014, the multi-wavelength detector is available with a GaAsP-cathode PMT, providing about 5 time the sensitivity of the PML‑16C.
Multi-wavelength FLIM uses the multi-dimensional recording process described in section ‘Multi-Wavelength FLIM’, page 32. For every photon, the routing electronics of the PML‑16 delivers a timing pulse and a ‘channel’ data word. These signals are, together with the scan clocks, used by the TCSPC module to build up a multi-dimensional photon distribution over the image coordinates, the time in the fluorescence decay, and the wavelength [55, 66]. In other words, the TCSPC module ‘routes’ photons of different wavelength into separate lifetime images. Apart from unavoidable loss in the polychromator, the recording process itself works without loss of photons. For electronic principles please see  or . A photo of the MW FLIM assembly is shown in Fig. 64, right.
Fig. 64: Multi-wavelength FLIM detector assembly. Left: Optical principle. Right: Photo
As the other FLIM detectors, the MW FLIM detector has its own internal high-voltage generator. No external high-voltage power supply unit is required. The detector is controlled via the DCC‑100 detector controller module which provides for power supply, gain control, and overload shutdown. An example of a multi-spectral FLIM results is shown in Fig. 65.
Fig. 65: Mouse kidney sample stained with DAPI, Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidin. Zeiss LSM 710, bh Simple-Tau 150 FLIM system. Two-photon excitation at 800nm, detection from 400nm to 625nm
The potential of multi-wavelength FLIM has dramatically increased with the introduction of 64‑bit SPCM data acquisition software. Previously, the maximum image format for multi-wavelength FLIM was 128 x 128 pixels, and 256 time channels. With 64‑bit software multi-wavelength images with 16 wavelength channels, and 512 x 512 pixels and 256 time per wavelength channel can be recorded [66, 457].
The PMZ-100, and its LSM 510 equivalent, the PMC‑100 have been used in large quantities in the different bh FLIM systems. The PMZ‑100 detector is shown in Fig. 66. The IRF width is about 160 ps, the cathode is of the multialkali type. The preamplifier and the PMT power supply are integrated. The PMZ detector is operated directly from the DCC‑100 card of the FLIM system, and connected directly to the input of the SPC modules.
Fig. 66: PMZ‑100 detector. The interface to Zeiss NDD T adapter and the Zeiss BIG port is on the left
Since the HPM‑100‑40 GaAsP hybrid detectors were introduced the PMZ detectors have almost entirely been replaced with the more sensitive HPM‑100 detectors.
The NDD FLIM detectors are normally attached to the NDD port of the microscope via a Zeiss T Adapter. Fig. 67 shows the T adapter at an upright (left) and an inverted microscope (middle). Both microscopes are shown with one FLIM detector attached to the 0° output of the NDD T Adapter. A second detector is attached to 90° output of the T Adapter, see Fig. 67, right.
Fig. 67: NDD detection at the LSM 710/780/880 microscopes. Left: Upright microscope. Middle: Inverted microscope. Right: NDD beamsplitter cube with FLIM detector at the 0° output. The 90° output is shown with the detector removed.
Inside the T adapter, a beamsplitter/filter cube splits the light into two spectral components. The cube can be replaced and configured for different detection wavelengths. The T adapter contains also the laser blocking filter. The optical principle is shown in Fig. 68.
Fig. 68: Right: Optical principle of the T Adapter
There are a few possible modification of this setup. Of course, single-channel FLIM systems can attach the detector either to the 0° or to the 90° output, see Fig. 69, left. The unused output of the T adapter must be closed by a blind cover. Another possibility to attach a single FLIM detector is at the 0° output of the Zeiss NDD detector module.
Fig. 69: NDD FLIM systems with one detector. Left: The FLIM detector is attached to one output of the NDD T adapter. Right: The FLIM detector is attached to the 0° output of a Zeiss NDD detector module.
Of course, the right beamsplitter / filter cubes of the Zeiss detector module must be used to transmit light of the desired spectral range to the FLIM detector. One or both beamsplitter cubes may also be removed. Please note that the laser blocking filter must remain in the beam path.
It is sometimes requested to attach the T-Adapter to the 0° output of a Zeiss NDD detector module, see Fig. 70. The problem of this configuration is that the input lens of the T adapter is designed to collimate the light beam at the output of the microscope, not the beam at the output of the Zeiss NDD module. The configuration therefore does not deliver optimum sensitivity. The same problem occurs if the Zeiss NDD detector module is attached to an output of the Zeiss T adapter, see Fig. 71.
Fig. 70: T-Adapter attached to the 0° output of the Zeiss NDD detector module (not recommended)
Fig. 71: Zeiss NDD detector module attached to 0° output of Zeiss T adapter (not recommended)
The bh multi-wavelength FLIM detector is coupled to one of the optical outputs of the NDD T adapter or to the output of a Zeiss NDD detector module by a fibre bundle. Details of the optical interface are shown in Fig. 72. The NDD optics projects the fluorescence light leaving the back aperture of the microscope objective lens on the input of a fibre bundle (Fig. 72, left and second left). The input of the bundle is circular, the output has the shape of a slit. The fibre bundle thus transfers the light into the input slit of a polychromator. Fig. 72, second right and right, shows the input of the polychromator with the holder of the fibre bundle.
Fig. 72: Details of the LSM 710 NDD MW-FLIM system. Left: Polychromator with adapter for fibre bundle. Right: Fibre bundle, polychromator end and shutter end.
In contrast to two-photon excitation one-photon excitation generates fluorescence within the entire depth of the sample. Reasonable FLIM with one-photon excitation (not quite correctly called ‘VIS-FLIM’) therefore requires confocal detection to suppress of out-of focus light (see Fig. 247, page 155). In other words, for one-photon excitation the FLIM detector must be installed at a confocal optical output port of the scan head.
Recent versions of the LSM 710/780/880 microscopes have (or can be ordered with) a confocal output with a ‘BIG’ type adapter. The BIG adapter is identical with the NDD port adapters. The BIG adapter has two advantages over the older ‘DC’ port adapter (section below). The first one is that it provides a mechanically solid connection to the scan head. FLIM detectors connected to the BIG port therefore do not need any alignment. A second advantage is that the BIG adapter is mechanically and optically identical with the adapters at the NDD outputs of the multiphoton (NLO) microscopes. FLIM detectors can therefore be moved from the confocal port to an NDD port and back without changing adapters.
A single HPM-100, PMZ‑100 or MW-FLIM GaAsP detector can be attached directly to the BIG port of the microscope, as shown in Fig. 73. An additional filter can be screwed directly into the input of the detector.
Fig. 73: Left: BIG port adapter of the LSM 710/780 scan head with HPM-100 hybrid detector attached. Right: Front end of the HPM‑100 with BIG / NDD adapter.
The FLIM detectors of a dual-channel FLIM system are connected to the BIG port via a bh beamsplitter assembly. Two HPM‑100 detectors with this beamsplitter are shown in Fig. 74.
Fig. 74: Two HPM‑100-40 detectors with bh beamsplitter assembly and BIG port adapter
The beamsplitter assembly is also recommended if a single FLIM detector is used and the filter in front of it has to be replaced frequently. I that case, the beamsplitter assembly is just used as an easy way to insert filters between the scan head and the detector, see Fig. 75.
Fig. 75: Single HPM detector connected via bh beamsplitter module. The beamsplitter module provides a simple way to insert an additional filter in the beam path.
In the LSM 880 the confocal output from the scan head is used for the ‘Airy-Scan’ detector. It is therefore not directly accessible. For FLIM detectors Zeiss offers a beam switch that is inserted between the output of the scan head and the Airy Scan detector, see Fig. 76, left. The 90° position of the switch directs the beam to a vertical (BiG-type) port. This port can be used both for the BiG-2 detector and for the bh hybrid detectors, see Fig. 76, right.
Fig. 76: LSM 880 scan head with BiG-2 detector (left) and with two bh HPM‑100 hybrid detectors (right). The detectors are connected to a beam switch between the Airy Scan detector and the scan head.
Older LSM 710 microscopes may have a different version of the confocal port. The port is called ‘DC’ port. The detector adapter has an extension tube that opens a flap inside the scan head when the detector is attached. When the detector is removed the flap closes to maintain laser safety. The disadvantage of the DC port adapter is that it only loosely inserts in the scan head. The detector is therefore not automatically aligned with the beam path. Detectors with the DC port adapter are shown in Fig. 77. If needed, a filter can be inserted between the DC adapter and the detector, please see ‘System Setup’, page 277.
Fig. 77: Detectors for one-photon (confocal) FLIM, with adapter for LSM 710 DC port. Left to right: PMZ‑100 PMT module, HPM‑100 hybrid PMT module, MW FLIM multi-spectral FLIM detector.
DC-Port detectors are not directly compatible with NDD detectors. They can, however, be converted by replacing their front plates. Please see ‘System Setup’, page 277.
TCSPC FLIM requires excitation by a high-frequency pulsed laser. Although bh have a variety of picosecond diode lasers that are perfectly suitable for FLIM excitation these are not normally part of the bh FLIM system for the Zeiss microscopes. The reason is that the lasers must be integrated in the optical system and in the control routines of the microscope. For warranty and laser safety reasons this has to be done by the microscope manufacturer who also takes responsibility for the functionality and safety of the entire laser scanning system.
Femtosecond Ti:Sapphire lasers are used as excitation sources in the LSM 710/780/880 NLO multiphoton microscopes. The microscopes use a multiphoton (usually two-photon) absorption process to excite fluorescence in the sample [144, 206]. The laser wavelength is tuneable from about 680 nm to 1000 nm. The two-photon process excites fluorophores which are otherwise excitable at (one-photon) wavelengths in the range from 340 nm to 500 nm. Multiphoton excitation has a number of advantages over conventional one-photon excitation:
- Direct absorption and scattering at the near-infrared wavelength of the laser is low. Therefore fluorescence can be excited in deep layers of biological tissue.
- Fluorescence is excited preferentially in the focal plane of the laser. Excitation outside this plane is negligible. Therefore no confocal pinhole is required to suppress out-of-focus fluorescence. The fluorescence is diverted from the excitation beam directly behind the microscope lens and be transferred to a large-area detector. The principle is called ‘non-descanned detection (NDD), and is able to record even photons which are scattered on the way out of the sample.
- Because there is no out-of-focus excitation there is also no out-of focus photodamage or photobleaching. Two-photon excitation is therefore benign to live samples.
- A fourth advantage is that fluorophores with absorption in the UV can be excited without the need of UV optics. An important application is NADH imaging by two-photon excitation, and tryptophane imaging by three-photon excitation.
With its short pulse width and wide tuning range the Ti:Sapphire laser is an almost ideal excitation source for FLIM. The repetition rate is 78 MHz or 80 MHz (some versions had 90 MHz). The high repetition rate is sometimes considered a disadvantage for FLIM with fluorophores with lifetimes longer than a few ns. However, it is less a problem than commonly believed. Most of the fluorophores used for FLIM have fluorescence lifetimes shorter than 4 ns, and incomplete decay of the fluorescence within the laser pulse period can taken into account in the SPCImage data analysis.
The LSM 710/780/880 microscopes are available with one or two picosecond diode lasers. The lasers are modified versions of the bh BDL‑405 SMC (405 nm) and BDL‑445 SMC (445 nm) lasers [29, 66]. They are built in cooperation of Becker & Hickl, Berlin, and Lasos, Jena. The lasers are fully integrated in the LSM 710/780/880 hardware and the LSM 710/780/880 ZEN software by Zeiss. The ps diode lasers can be operated both in the ps pulsed mode and in the CW mode. They can not only be used for FLIM but also for the conventional (steady-state) imaging modes of the LSM. Thus, the full functionality of the LSM 710/780/880 systems is maintained with the ps diode lasers installed.
Please note that the diode lasers excite fluorescence via the conventional one-photon excitation process. That means the laser scanning microscope works reasonably only with confocal detection. That means the FLIM detectors have to be connected to a confocal optical output from the scan head.
The optics of BDL-SMC lasers correct both for beam profile and astigmatism. This results in highly efficient fibre coupling. The maximum optical power at the fibre output, i.e. at the input of the scan head, is on the order of 0.5 to 1 mW in the ps mode and 15 to 20 mW in the CW mode. In almost any conceivable FLIM experiment of less than 100 µW is needed. That means that the laser power is rather too high than too low. Therefore, the lasers have electrically operated optical attenuators which can be used in addition to electronic power control.
The optical pulse width of the BDL-SMC lasers is between 45 and 100 ps FWHM. The pulse width varies over the range of the electronic power control. The shortest pulse width is obtained at the high-power end of the control range, see Fig. 78.
The high coupling efficiency of the BDL-SMC lasers allows the laser diodes to be operated at a power where the pulse profile remains close to gaussian. The long tails typical of ps diode lasers at high power are thus avoided. Such tails substantially impair the accuracy of the fluorescence lifetime analysis, and, despite of the high power, result in a decrease in the signal-to-noise ratio of the lifetime data.
Fig. 78: Pulse shape for a BDL-405 SMC laser for 20%, 60%, and 100% power. Repetition rate 50 MHz.
The repetition rate can be switched between 20 MHz, 50 MHz, and 80 MHz . Higher repetition rate yields higher average optical power. However, the fluorescence may not completely decay between the pulses. For most of the fluorophores, including the GFPs, 50 MHz repetition rate is the best compromise.
With the two available excitation wavelengths, 405 nm and 445 nm, a large number of fluorophores can be excited. It is not required that the excitation wavelength matches exactly the absorption maximum of a fluorophore. There is usually enough absorption at the short-wavelength side of the absorption band to obtain sufficient excitation. Fig. 79 shows approximate excitation spectra for a number of commonly used fluorescent proteins. The curves were taken from  and . The most important ones are ECFP (Cerulean is almost similar), mTFP, and EGFP because these are good FRET donors. As can be seen from these curves the 445 nm laser excites ECFP and the new mTFP  near their excitation maxima. It also excites EGFP at reasonable efficiency. The 405 nm laser yields good efficiency for EBFP and ECFP, and even excites the EGFP at about 20% of its maximum absorption. The EYFP and the DsRed are normally used as FRET acceptors. FLIM FRET does not need to record the acceptor fluorescence. Excitation of EYFP and the DsRed is therefore not needed and not desirable.
Fig. 79: One-photon excitation spectra of commonly used fluorescent proteins. Curves after  and .
Examples of FLIM with the ps diode lasers are shown in Fig. 21, page 13, and Fig. 26, page 15.
bh BDL-SMN and BDS-SM ps diode lasers can be operated externally of the Zeiss LSM 710/780/880 microscopes and fibre-coupled to the scan head. In that case, the lasers are controlled from the bh SPCM software, see DCC-100 Detector and Laser Control, page 87. Lasers are available for the range from 375m to 515 nm, and from 635 nm to 785 nm. All lasers can be operated at 20 MHz, 50 MHZ, 80 MHz, and CW, and can be modulated for FLIM / PLIM operation. In addition to the control signals from the SPCM software, the lasers receive a beam blanking signal from the LSM. Thus, the lasers turn off in the beam flyback phases of the scanner, and when the scanning is stopped.
Fig. 80: Pulse shapes for 488 nm and 640 nm BDS-SM lasers. The indicated power is for 50 MHz repetition rate, and refers to free laser beam. Coupling efficiency into the fibre is about 50%.
The ‘Intune’ laser is a tuneable fibre laser. The tuning range is 488 to 640 nm. The laser delivers pulses with a duration of a few picoseconds at a repetition rate of 40 MHz. The Intune system comes with a scan head configuration that offers a much wider range of main dichroic beamsplitters than the standard 710/780 scan head. Thus, the system can be almost perfectly matched to the excitation spectra of the fluorophores used. Moreover, the beamsplitters have extremely steep transitions, and extremely low leakage. Contamination of the fluorescence signals by scattered laser light is almost entirely avoided, even if the system is operated without additional fluorescence filters. The high efficiency of the optics together with plenty of laser power available allows one to use the system with small pinhole sizes, and thus obtain diffraction-limited image quality. Examples are shown in Fig. 22 and Fig. 23, page 13.
Due to the high efficiency, the high laser power available, the high spectral purity of the laser, and the wide variety of excitation and detection wavelengths available the InTune system is also an excellent solution to FCS experiments. Please see ‘FCS Measurements’, page 138.
Unfortunately, the Intune laser systems have been discontinued by Zeiss in 2015. Existing ones can still be upgraded with FLIM.
TCSPC FLIM needs reference pulses from the lasers to determine the times of the photons in the laser pulse period, see Fig. 49, page 30. All pulsed lasers used in the Zeiss LSMs deliver synchronisation pulses. Ti:Sapphire lasers deliver a reference signal at a BNC connector at the back panel of the laser housing. Diode lasers and the Intune laser deliver reference at a connector inside the Zeiss laser rack. For the Ti:Sa lasers and for the diode lasers the polarity has to be reversed by a passive pulse inverter, see Fig. 432, page 302. The correct cable connections are installed during the system setup.
A problem can occur for some Ti:Sa lasers from Spectra Physics. The lasers deliver extremely low amplitudes of the reference signal. This is especially the case at wavelengths longer than 920 nm and shorter than 750 nm. Although the bh TCSPC modules synchronise to amplitudes as low as 20 mV such low amplitudes bear the risk of noise pickup from external RF sources. If a sufficient SYNC amplitude cannot be obtained, an external SYNC detector is available from bh, see Fig. 404, page 282. The detector is built into the AOM box of the LSM 710/780/880 NLO microscope. For cable diagrams of the complete FLIM systems, please see Fig. 408 to Fig. 412, page 285 to 288.
bh provide a USB-controlled delay switch box for the Sync signal to the SPC modules. The box allows the user to adjust the temporal relationship of the detector signals and the laser reference pulses . The delay box is also able to switch between two sources of the Sync signal. Such changes may be required if the FLIM system is used with different lasers, with different detectors, or in different optical configurations. Setups for the different configurations can then be defined, and be stored in the ‘Predefines Setup’ panel of the SPCM software. The correct Sync delay is set when the setups are loaded. The delay box is shown in Fig. 81. It is accessed via the ‘Sync’ settings in the main panel, or via the Sync section of the system parameter panel.
Fig. 81: Delay switch box. It is used to set the correct temporal relationship between the detector signals and the synchronisation signals from the lasers. The correct Sync delay for different system configurations is set by loading setup data via the SPCM software.
The ‘SPCM’ software is an integral part of the bh TCSPC systems. It not only controls the TCSPC hardware, it also reads the data from the TCSPC modules, builds up the results of the measurements, and controls the measurement procedure. There are operation modes for recording single decay curves, time-controlled sequences of decay curves, single FLIM images, multi-wavelength FLIM images, time-series and z stacks of FLIM images, mosaics of FLIM images, combined FLIM / PLIM images, FLITS images, fluorescence correlation (FCS) curves, time-series of FCS curves, photon counting histograms (PCH), and parameter-tagged single photon data for single-molecule spectroscopy .
Since 2014 the SPCM software uses Windows 7 or Windows 8 64-bit technology. 64‑bit SPCM software not only runs on 64‑bit computers, it is a real 64-bit application. It thus takes full advantage of the capabilities of the 64-bit Windows environment. The most significant one is that a large amount of memory can be addressed. As a result, FLIM data can be recorded with unprecedented numbers of pixels and time channels. More importantly, the large memory space allows advanced multi-dimensional FLIM techniques to be used without compromising spatial resolution. Multi-spectral FLIM can be recorded at unprecedented pixel numbers, the image area can be increased by spatial mosaic recording, Z stacks can be efficiently acquired without the need of intermediate data save actions, fast triggered time series of FLIM data can be accumulated by temporal mosaic FLIM, and PLIM can be recorded simultaneously with FLIM. As a result, the bh FLIM systems have capabilities far beyond the recording of simple FLIM images - they are capable of recording the complex behaviour of a biological system in a multi-parameter space.
The details of the measurement procedure are controlled by the SPCM software via a number of measurement control functions and measurement control parameters. The selection of the operation mode and measurement control mode acts both on the hardware and the software. To adapt to the various combinations of operating modes and measurement procedures the SPCM software has a flexible user interface. The entire set of hardware control parameters, software parameters, and user interface parameters is saved with the measurement data or into a separate setup file. The system setup can thus be reproduced by simply loading a data or a setup file.
The setup parameters for different hardware and software configurations and user interfaces are stored in a ‘Predefined Setup’ panel.
Switching between different instrument configurations is thus only a matter of a single mouse click.Please see ‘Switching Between Different Instrument Configurations’, page 79, and ‘Creating Predefined Setups’, page 93.
The SPCM software also controls the DCC-100 module that runs the detectors, the DB‑32 delay switch box, the DDG-210 pulse generator used for modulating the laser for PLIM, and a number of other experiment control modules . The parameters for these modules are stored together with the TCSPC hardware and software parameters. Therefore, also these functions are restored if a setup or data file is loaded either individually or from the Predefined Setup panel.
The Zeiss LSMs, the bh TCSPC devices and, consequently, the combination of both are complex multi-modal systems which deliver data in a complex multi-parameter space. The data produced by the systems can be single fluorescence decay curves, multiple decay curves for different wavelength or different times after the start of an experiment, combinations of decay curves and FCS curves, fluorescence decay data along a line in the sample, fluorescence and phosphorescence intensity images, multi-wavelength lifetime images, or even arrays of lifetime images for different sample position in x,y or z, or different experiment times after the start of an experiment or times after a periodic stimulation of the sample. To run all these different experiments and display the results in a user-compatible way the bh SPCM software has a user interface which is configured according to the particular operation mode of the TCSPC system and the requirements of the experiment the user is performing. There are up to eight display windows for TCSPC data. The data in the windows may be fluorescence decay curves, time-series of decay curves, decay and FCS curves, images from different TCSPC channels or within different time gates of the same TCSPC channel, images within different wavelength intervals of a multi-wavelength detector, or arrays of images from different spatial locations or different times within an experiment or two-dimensional projections of such data. Examples are shown in Fig. 82.
Fig. 82: Examples of user interface configurations of the bh SPCM software
The 3D Trace Parameters, Window Parameters and Display Parameters define the display of multidimensional TCSPC data in the SPCM software.
The 3D trace parameters define how many display windows are opened, which kind of data are displayed in the individual windows, from which TCSPC module the data are taken, and in which way multidimensional data are projected into the individual display windows. Examples are shown in Fig. 83 and Fig. 84.
Fig. 83 shows a 3D trace parameter setup for displaying images of different data type. The first two windows, W1 and W2, display intensity data of ps FLIM data from two TCSPC modules, M1 and M2. The next two windows, W3 and W4, display the same data as lifetime images. W5 and W 6 display MCS mode (PLIM) images from M1 and M2. Finally, W7 and W8 display intensity images from the direct counter channels of two SPC-160 modules. For all windows, the display mode is F(x,y), which means that the coordinates of the display windows are identical with the spatial coordinates of the data.
Fig. 83: 3D Trace parameters for the display of FLIM intensity images, FLIM lifetime images, MCS (PLIM) images, and intensity images from the fast counter channels of two SPC-160 modules
The problem that FLIM or PLIM data are actually three-dimensional x-y-t arrays is solved by projecting the data into the x-y plane.
For each pixel in the x-y plane, the data along the t axis are compressed into a single variable. Depending on the T Window selected this can be the total photon number (the result is an intensity image) or the photon number in a selectable time window (the result is a time-gated intensity image). The brightness and/or colour of the image represents one of these intensities. When ‘Lifetime Display’ is selected (W3 and W4) the colour of the image represents the lifetime, the brightness the total or gated intensity.
So far, things are still straightforward. However, TCSPC data can easily become four-dimensional. This happens when the routing function is used. Data in the individual routing channels then represent different detection wavelengths, different excitation wavelengths, or different levels of an external signal applied to the sample. The ‘Routing Window’ then defines a range of routing channels the data of which are projected into individual FLIM images and displayed as described above.
In some cases it is desirable to directly display the decay functions in spatially two-dimensional data. In that case, the x-y-t data must be projected into the t-x or the t-y plane. 3D Trace parameters for these projections are shown in Fig. 84. Display Mode is F(t,x) or F(t,y). The results are sequences of decay curves along the x or y axis. The data along the other spatial axis, y or x, are the sum of the decay functions over selectable Y windows for F(t,x) and over selectable X windows for F(t,y). Eight such windows can be defined, and can be selected individually for the eight display windows.
Please note that time windows cannot be defined for intensity image from the SPC-160 counters and for lifetime display. The counter images do not contain temporal data, and lifetime images are not f(t,x) or f(t,y) functions.
Again, different Routing windows can be defined for systems that use the routing function. The data displayed in the individual display windows are then for different detection wavelength, different excitation wavelength, or for whatever measurement parameter the routing signal represents.
Fig. 84: 3D Trace parameters for the display of decay functions over one spatial coordinate of spatially two-dimensional data
The window parameters define time windows for FLIM, routing windows, time windows for PLIM (MCS windows) and spatial windows in X and Y for displaying multi-dimensional data. Fig. 85 shows definitions for 8 windows along each coordinate.
Fig. 85: Window Parameters. Definitions shown for 8 FLIM time windows, 8 routing windows, 8 PLIM time windows, and 8 windows in x and y.
Basic FLIM and PLIM images are often displayed without the gating functions of the SPCM display routines. In that case, it can be enough to define just one windows along each coordinate, covering the entire time axis, all routing channels, and the entire image in x and y. Definitions for data of 256 FLIM time channels, 256 PLIM time channels, and 256 x 256 pixels without routing are shown in Fig. 86.
Fig. 86: Window parameter definitions for 256 FLIM and PLIM time channels 256 x 256 pixels for FLIM and PLIM display without gating functions.
The display parameters define how the data are displayed in the individual display windows of the SPCM software. Every display window has its own set of display parameters. The parameters define the display range, the colours, the display style (curves or colour-intensity plot), the display mode (f(x,y), f(t,x or f(t,y), and the t, x and y windows in which the data are displayed. Examples are shown in Fig. 87.
Fig. 87: Influence of display parameters on the data display. Left: Image displayed in a selectable time window. Middle: Decay functions over x coordinate in a selectable y window displayed in colour-intensity mode, Right: Decay functions over x coordinate in a selectable y window displayed in curve mode
For lifetime display (‘LIFET’ selected in the 3D trance parameters) the display parameter panel is expanded by definition for the lifetime display. The definitions include the lifetime range, a reference moment of the IRF, and contrast and brightness of the image displayed. An example is shown in Fig. 88.
Fig. 88: Display parameters for lifetime image display
2D trace parameters, window parameters, and display parameters for frequently used imaging modes are shown in the sections below. For description of the of the parameters of the SPCM display functions please see section above, or refer to the ‘Software’ section of the bh TCSPC Handbook .
Fig. 89 to Fig. 91 show the parameters for displaying an intensity image from the combined photon numbers in all time channels of a single TCSPC channel. In the 3D Trace parameters, Fig. 89, one single display window (W1) is enabled. The data type to be displayed is ps FLIM, the data come from TCSPC module M1, and the display mode is F(x,y). The Window parameters, Fig. 90, define a single Time Window, from time channel 1 to 256, and two spatial windows, both from pixel 1 to pixel 1024. In other words, the windows incorporate the entire FLIM data array.
The Display Parameters, Fig. 91, define a colour-intensity image with linear intensity scale. The intensity is coded by colour, the colour scale goes from black over red to white. The image is displayed for Time Window 1 - this is the only time windows defined (see Window Intervals). It contains the photons of all time channels (from 1 to 256). The SPC Main panel with an image defined by these parameters is shown in Fig. 92.
Fig. 89: 3D Trace parameters for display of a single image
Fig. 90: Window Parameters for display of a single image
Fig. 91: Display parameters
Fig. 92: SPCM Main panel with the image defined by the parameters shown above
Fig. 93 shows the parameters for the display of four gated images of one TCSPC channel. The 3D trace parameters define four display windows for the data of module M1 in four time windows, 1 to 4. The window parameters define the start and the end (time) channels for the individual time windows.
Fig. 93: 3D Trace parameters (top) and Window parameters (bottom) for the display of four time-gated images of SPC module M1.
The individual images have separate display parameters, see Fig. 94. The SPCM main panel with the four gated images is shown in Fig. 95.
Fig. 94: Display parameters for the four gated images
Fig. 95: Four gated images in the main panel of SPCM
The parameter setup for the display of two images from separate SPC modules is shown in Fig. 96. The Trace Parameters define two display windows. They contain data from two SPC modules, M1, and M2. The window parameters are the same as for a single image - there is only one time window, containing the photons of all TCSPC time channels from 1 to 256.
Fig. 96: 3D Trace parameters and Window parameters for the display of the images from two SPC module, M1 and M2
The two images are displayed with separate display parameters, see Fig. 97. The SPCM main panel with the two images is shown in Fig. 98.
Fig. 97: Display parameters for the two images defined by the parameters in Fig. 96
Fig. 98 SPCM Main panel. Two images for separate SPC modules.
The display of multi-wavelength FLIM data with a 16-channel detector is shown in Fig. 99 trough Fig. 101. The trace parameters define eight display windows. Data type is ps FLIM. The individual images in the display windows are derived from subsequent ‘Routing Windows’. Each routing window contains the data of two subsequent routing channels, see Window Parameters. This way, data of every two wavelength channels are combined in one image.
Fig. 99: Trance parameters and window parameters for multi-wavelength FLIM with the PML-16C or PML-16 GaAsP detector
As usual, the individual images have separate display parameters. The display parameters for the first three images are shown in Fig. 100. They contain the colour definition for the individual images, and the Routing Window. Since the intensities in different wavelength channels can vary over a wide range Autoscale is turned on for all images. The main panel of SPCM with the eight images is shown in Fig. 101.
Fig. 100: Display parameters in the first three display channels of a multi-wavelength measurement
Fig. 101: SPCM Main panel for multi-wavelength FLIM
Fig. 102 and Fig. 103 show the definition of the parameters for run-time lifetime display for a dual-channel SPC system. In the 2D trace parameters, two display windows are defined, one for each module. Data Type is ‘Lifetime’.
Fig. 102: 3D trace parameters for run-time display of lifetime images
The display parameters are shown in Fig. 103. As usual, there is a separate set of display parameters for each image. In the lower part of the panels, the lifetime range, the direction of the colour bar, the brightness and the contrast, and a reference moment for the IRF is defined. The reference moment can be determined by SPCImage (see SPCImage Data Analysis Software, page 225) or calculated from a reference FLIM file of a sample with known fluorescence lifetime. For principle of run-time lifetime calculation please see bh TCSPC Handbook . The main panel of the SPCM software with run-time lifetime images is shown in Fig. 104.
Fig. 103: Display parameters for run-time lifetime display
Fig. 104: SPCM Main panel with run-time lifetime calculation
Simultaneous FLIM/PLIM is based on on-off modulation of a pulsed excitation laser. FLIM is recorded in the laser-on phases by the normal FIFO Imaging procedure. PLIM is recorded in the laser-off phases by ‘MCS’ recording, for details please see ‘Simultaneous FLIM / PLIM’, page 131. To better separate the photons from the laser-on and laser-off phases a routing signal from the laser modulation is sent into the SPC module. Laser-on photons are stored into routing channel 1, laser-off photons into routing channel 2.
Fig. 105 shows the trace and window parameter setup. The 3D Trace parameters define three display windows. The first one is the FLIM window, the second and the third one are PLIM windows. Data type for the first window is ps FLIM, for the second and third windows it is MCS imaging. The window intervals define three routing windows. The first one contains the photons from the laser-on phases, the second one the photons from the laser-off phases. The third routing window contains the photons from both phases.
Fig. 105: 3D Trace parameters and window parameters for simultaneous FLIM/PLIM
The display parameters for the three windows are shown in Fig. 106. The parameters for W1 display an intensity image for the FLIM data. The data are from window 1, i.e. from the ‘laser on’ phases. Colour goes from black over green to white, autoscale is set, intensity scale is linear.
The parameters for W2 display an intensity image of the phosphorescence in the laser off phases. Routing window is 2 (laser off), the colour goes from black over red to white. Window W3 displays the entire signal in both phases of the laser modulation, therefore ‘Routing window’ is 3. Unlike W1 and W2, W3 does not display an image. Instead, it displays a series of phosphorescence decay curves over the horizontal axis of the image. Display mode is ‘3D Curves’ and F(t,x), the scale is logarithmic. The curve window helps the user set the correct time scale for PLIM recording. Phosphorescence decay times can vary over orders of magnitude, and the best recording time scale is not a priori known.
Fig. 106: Display parameters for fluorescence image, phosphorescence image, and phosphorescence decay curves
The SPCM main panel with these setup parameters is shown in Fig. 107. The fluorescence image is displayed on the left, the phosphorescence image in the middle, and the phosphorescence decay curves on the right.
Several modifications are possible to this setup. Often the fluorescence and the phosphorescence are recorded at different wavelength, and by different SPC modules. This can be accounted for by changing the Module from M1 to M2 in the trace parameters. It is also possible to activate the run-time lifetime display for the fluorescence and for the phosphorescence. Just change to ‘Lifetime’ in the trace parameters, and set an appropriate lifetime range and IRF moment in the display parameters.
Fig. 107: SPCM main panel for simultaneous FLIM/PLIM
When working in the ‘Single’, ‘Oscilloscope’, or ‘FIFO’ mode the SPC modules record decay curve or other optical waveforms and displays them in a 2-dimensional curve window. The SPCM software can display up to 16 decay curves or other optical waveforms simultaneously. The curves on the screen are referred to as 'Traces'. The ‘Trace Parameters’ define which information the traces contain and in which style and colour they are displayed. An example is shown in Fig. 108.
Fig. 108: Left: Trace parameters for a single SPC module. Right: Trace parameters
The 2D trace parameter panel contains the definitions for up to 16 traces. For each trace the display can be switched on and off by the ‘Active’ button, and different colours can be defined.
‘Module’ is the TCSPC module from which the displayed trace comes from, ‘Curve’ is the routing channel, and ‘Page’ is one of several memory pages in the internal memory of the TCSPC module. ‘Page’ can also be the step of a page stepping sequence, see ‘Measurement Control Parameters’ in the ‘System Parameters’ section.
The ‘System Parameters’ contain the complete set of hardware and measurement control parameters of the TCSPC module. If your system has been set up by a bh engineer you need not change any of these parameters. For users who like to setup a FLIM system on their own bh deliver a number of setup files for different FLIM configurations. If you start from these you need only adapt the TAC parameters and signal delays to the special requirements of your microscope. The following paragraph should therefore be considered supplementary information for advanced users.
The system parameters are accessible by clicking into ‘Parameters’, ‘System Parameters’. The system parameter panel is shown in Fig. 109. The parameters shown are for the FIFO imaging mode, 512x512 pixels, 256 time channels. A detailed description of the system parameters is given in . For parameters of typical FLIM experiments please see page 95 of this handbook.
Fig. 109: SPCM System parameter panel
The following paragraphs give an overview about the available operation modes and the parameters controlling the FLIM acquisition.
The operation mode selection panel of the bh TCSPC modules is shown in the figure below. The mode used for FLIM recording is ‘FIFO Imaging’. Other modes may be used for special application of a FLIM system.
The ‘Single’ mode records one decay curve for each of the detectors connected to the SPC module. It can be used for fluorescence decay measurement with the laser beam being parked in a pixel of interest. If used in combination with scanning it delivers an average decay curve over the complete scan area.
The ‘Oscilloscope’ mode performs a repetitive measurement and displays the results like an oscilloscope. The mode is an excellent tool for setup, maintenance and alignment purpose.
The F(t,T) mode runs a time-controlled sequence of ‘Single’ measurements. It is useful for single-point photobleaching experiments, experiments of photodynamic therapy, and for recording chlorophyll transients.
The F(t,EXT) mode is implemented for recording sequences of curves in connection with external experiment control. The Fi(T) and Fi(EXT) modes record time-gated intensity curves.
The ‘Scan Sync In’ mode is one of the two FLIM modes used in combination with the Zeiss LSMs. The SPC module records a photon distribution over the time in the laser period and over the coordinates of the scan area. The photon distribution is built up in the memory of the SPC module. The computer is not involved in the data acquisition process. Thus, the acquisition speed is not limited by the bus transfer rate or any software response times. This was important at early times of FLIM when the computers were not fast enough to read single-photon data from the TCSPC module and simultaneously build up the photon distribution. The downside is that the image format (the product of pixel number and time channels) is limited by the size of the on-board memory of the SPC module. The Scan Sync In mode is still used in a number of older LSM 512 FLIM system with SPC‑730 modules. In the LSM 710/780/880 FLIM systems it is used for the ‘Fast Preview’ mode, taking advantage of the fact that there is no saturation of the data transfer between the SPC module and the computer.
In the SPC‑150 the Scan Sync In mode can be combined with ‘Continuous Flow’ operation. In this mode the memory is split in two banks. Wile the module is recording in one bank the other bank is read. Then the banks are swapped and the measurement continues. Thus, fast sequences of images can be recorded without spending time on the readout on the data.
‘Scan Sync Out’ is an imaging mode that actively controls a scanner. It was implemented mainly for scanning with piezo-driven scan stages. The mode is not used for FLIM acquisition with the Zeiss LSMs. It can, however, be used to record and accumulate fast triggered sequences of decay curves. With a large number of accumulation cycles, sequences as fast as a few microseconds per curve can be recorded. The mode can be used to record photochemical quenching transients in chlorophyll , possibly also effects of electro-physiological stimulation in membranes and neurones. Please see  for details.
The ‘FIFO’ mode differs from the other modes in that the SPC module does not build up a photon distribution in its memory. Instead, the FIFO mode delivers information about each individual photon and transfers it into the computer. FIFO-mode data are also called ‘time-tagged’ or ‘parameter-tagged’ data. Parameter tagged data contain the time of the photon in the laser period (micro time), the time since the start of the experiment (macro time), and, if several detectors are used, the number of the detector that detected the photon. The memory of the SPC module works as a first-in-first-out (‘FIFO’) buffer. The photon data are buffered and continuously transferred into the computer. The computer either processes the data online or saves them into single-photon (.spc) files. The FIFO mode is the key to single-molecule techniques. It can be used to record FCS curves  in combination with fluorescence decay curves, photon counting histograms, or BIFL (burst-integrated fluorescence lifetime) data [55, 56, 165, 382]. The FIFO mode is normally used in combination with the ‘Point Scan’ function of the LSM 710/780/880 scanner.
‘FIFO Imaging’ is the standard FLIM mode for the LSM 710/780/880 FLIM systems. Compared to the ‘FIFO’ mode it not only records parameter-tagged single photon data but also the pixel, line, and frame synchronisation pulses from the scanner. The data are buffered in the FIFO and read out by the computer. The SPCM software analyses the incoming data and builds up FLIM images. Because the FLIM images are built up in the main memory of the computer large pixel numbers and large numbers of time channels can be used . With the introduction of 64‑bit SPCM software the available memory space has increased again [36, 457]. As a result, not only images of megapixel size but also complex photon distributions like multi-wavelength FLIM or spatial and temporal mosaic FLIM can be recorded.
The FIFO Imaging mode records photon times not only with reference to the laser pulses but also to the scan clock pulses or other external events. This allows photon distributions to be built up over two different time coordinates simultaneously. This ‘dual-time base’ operation is used to simultaneously record fluorescence and phosphorescence lifetime images. Please see ‘Simultaneous FLIM / PLIM’, page 131.
Different than in the Scan Sync In mode, the maximum count rate in the FIFO imaging mode can be limited by the bus transfer rate of the computer and by the speed of the software. In practice this is not a problem. The FIFO imaging mode easily records average count rates of several MHz per TCSPC module. In typical images the average count rate is rarely higher than this. Substantially higher count rates may, of course, occur in isolated spots of the image. However, these are buffered by the FIFO buffers of the TCSPC modules so that the photons are not lost.
The memory of the SPC modules provides memory space for a large number of decay curves. The memory may even hold data of several measurement blocks. Each block can contain a large number of decay curves. There may even be enough space to store the data of several images of moderate numbers of pixels and time channels. The individual memory blocks are termed ‘pages’. By defining a number of ‘steps’ greater than one a sequence of recordings can be defined that automatically steps through subsequent pages. A measurement sequence may also be repeated for several measurement ‘cycles’. The results of the cycles can be accumulated, written into subsequent memory pages, or saved into subsequent files.
The result of a measurement or the results of the individual cycles can be automatically saved into subsequent data files (‘autosave’). The results of the cycles can also be accumulated (‘accumulate’ button). In the Scan Sync In mode the cycle and accumulate function is used to read and display FLIM data during the during measurement, see Fig. 122, page 79.
By activating the ‘repeat’ button the complete measurement cycle is repeated until it is stopped by user interaction.
The repeat function is used to for the ‘Fast Preview’ function of FLIM. The image is defined with a moderate number of ‘scan pixels X’ and ‘scan pixels Y’, and an ADC resolution of ‘one’. With only one ADC channel the recorded image is a pure intensity image of moderate data size. This keeps the time for the data readout at a negligible level. With a fast scan rate and a collection time on the order of one second a sufficiently fast update rate for adjusting the focus or selecting an image area of the sample is obtained.
The start of a measurement, the steps of a page stepping sequence, or the cycles of a measurement sequence can be triggered  by an external ‘experiment trigger’. In the bh FLIM systems for the Zeiss LSMs the trigger function is used to record Z stacks or microscope-controlled time-series. Please see pages 113 and 114 of this handbook.
Collection time is the acquisition time for the measurement. For measurements running only for one cycle or step it is the time for the complete measurement. If cycles or steps are defined it is the time for the cycles or steps. Please note the ‘Collection Time’ stops the measurement only if the ‘Stop T’ button is activated.
The measurement can be stopped at any time by an operator stop command. You may therefore run the measurement simply until you are satisfied by the signal-to-noise ratio of the images displayed.
Important: After a stop by the collection timer or an operator stop command the internal scanning machine completes the current frame. Artefacts by recording incomplete frames are thus avoided.
These parameters control the constant fraction discriminators at the inputs of the detector and laser synchronisation signal, and the time conversion circuitry in the TAC. CFD Limit Low and CFD Threshold and SYNC Threshold and Sync Zero Cross control the discriminators for the detector signal and for the synchronisation signal from the laser. TAC gain and TAC offset determine the time-conversion range of the measurement. We discourage to change these parameters unless you are familiar with the internal function of the TCSPC electronics. If you need changes of the parameters, please refer to the bh TCSPC handbook  or contact bh.
In the Scan Sync In mode the SPC module records the FLIM photon distribution directly in its internal memory. Thus, the acquisition speed is not limited by the bus transfer rate or any software response times. The downside is that the image format (the product of pixel number and time channels) is limited by the size of the on-board memory of the SPC module. The Scan Sync In mode is still used in a number of older LSM 512 FLIM system with SPC‑730 modules. In the LSM 710/780/880 FLIM systems it is used for the ‘Fast Preview’ mode, taking advantage of the fact that images are recorded displayed up to the highest count rates the detectors can deliver.
The number of time channels per pixel and the number of pixels in X- and Y-direction is specified by the parameters ‘ADC Resolution’, ‘Scan Pixels X’ and ‘Scan Pixels Y’ in the Data Format and Page Control’ sections of the system parameter panel. A few combinations are shown in Fig. 110. The setups shown left and middle are typical of older (until 2003) LSM 510 FLIM system that used SPC‑730 TCSPC modules. The setup on the right delivers a pure intensity image, and is used for the Fast Preview mode of the LSM 710/780/880 FLIM systems.
Fig. 110: Three combinations of time channel and pixel numbers for the Scan Sync In mode. Applies to SPC-150 and SPC-730, for SPC‑830 the pixel numbers in x and y can be doubled. The setup on the right delivers a pure intensity image. It is used for the ‘Fast Preview’ mode of the LSM 710/780/880 FLIM systems.
The synchronisation of the recording process in the SPC module with the operation of the scanner requires a number of hardware parameters to be defined. To define these parameters, click on ‘More Parameters’. This opens the panel shown in Fig. 111. The parameters shown in Fig. 111, left, result in a FLIM images of the same pixel number as the LSM scan. The setup shown Fig. 111, right, bins the FLIM image down to 1/4 of the LSM scan pixels.
Fig. 111: Scanning parameters of the Scan Sync In mode. Left: Without pixel binning, the FLIM image is recorded with the same number of pixels as the LSM scan. Right: Pixel binning 2x2. The image is recorded with 1/4 of the pixels in the LSM scan. The setup on the right is used for the ‘Fast Preview’ mode of the LSM710/780/880 systems.
The meaning of the scanning parameters is as listed below:
X Sync Polarity: Polarity of X Sync Pulses, (Positive for Zeiss LSMs)
YSync Polarity: Polarity of Y Sync Pulses, (Positive for Zeiss LSMs)
Pixel Clock Polarity: Polarity of Pixel Clock Pulses, (Positive for Zeiss LSMs)
Line Predivider: Pixel binning factor in Y
Pixel Clock Divider: Pixel binning factor in X
Upper Border: Number of lines which are not recorded at the start of each frame. Used to reject invalid lines at the beginning of the frames
Left Border: Number of pixels which are not recorded at the start of each line. Used to reject invalid pixels at the beginning of the lines
Pixel Clock: Source of the pixel clock. Must be ‘External’ for Zeiss LSMs.
Please refer to  for a detailed description of the Scan Sync In mode parameters.
All SPC‑150/152/154 modules, all SPC-160/162/164 modules, and SPC‑830 modules manufactured later than May 2007 have a ‘FIFO Imaging’ mode built in. The mode records fluorescence lifetime images with pixel numbers or time-channel numbers beyond the limit set by the memory of the SPC module. Moreover, it is able to record MCS lifetime images on the microsecond or millisecond time scale. In terms of photon recording the FIFO Imaging mode is similar to the standard FIFO mode, i.e. it records parameter-tagged data of the individual photons and transfers them into the computer for further processing. However, the FIFO imaging mode also records synchronisation pulses from a scanner via the event marker inputs of the SPC module and builds up FLIM images, PLIM images, combined fluorescence and phosphorescence lifetime (FLIM/PLIM) images, and intensity images from the parallel counter channel of the SPC‑160.
The types of images to be built up are defined in a panel ‘Select & Configure Histograms’, see Fig. 112 right. It opens by a click on the ‘Configure’ button in the measurement control part of the system parameter panel, see Fig. 112, left.
Fig. 112: Configuring the types of images to be built up in the FIFO Imaging mode.
The ‘Picosecond FLIM’ button selects normal FLIM. ‘Intensity Image’ selects the build-up of an intensity image from the parallel counter channel of the SPC‑160, see . The MCS FLIM button activates a multi-channel scaler (MCS) imaging mode. Instead of the TAC times it uses macro-time differences of the photons to a reference pulse at one of the marker inputs. In laser scanning microscopes the MCS FLIM mode is used to record PLIM by the procedure described in section ‘Simultaneous FLIM / PLIM’, page 131. All three functions can be activated simultaneously, as shown in Fig. 112.
Images of different data types are recorded simultaneously and can be displayed simultaneously. Display windows for the images are activated in the ‘3D Trace Parameters’, please see ‘Examples for Display’, page 59.
The general measurement control parameters of the FIFO Imaging mode are the same as for the basic FIFO mode.
The basic setup for FIFO imaging is shown in Fig. 113, left. The operation mode is ‘FIFO Imaging’, the cycle, repeat, and autosave functions are turned off. The .spc (parameter-tagged photon data) are not saved. Instead, the SPCM software analyses the FIFO data online. What the software does with the data is defined in the ‘Configure’ menu, as described above. Stop T is not set, the measurement is stopped by the operator when an appropriate image quality has been reached. The collection time, display time, and repeat time parameters have the same effects as in the other operation modes. The Display Time defines how often intermediate images are displayed.
From the point of view of the user the measurement procedure is virtually the same as in the Scan Sync In mode, except for the fact that larger images, more time channels, other types of images, or more detector channels are available. The display parameters, window intervals, and 3D Trace parameters are the same as for the Scan sync In mode (see Examples for Display, page 59).
Fig. 113: Measurement control parameters for FIFO Imaging. Left without, right with saving of time-tag data.
For special data analysis procedures it may be desirable to save not only the final FLIM images but also the parameter-tagged photon data. A measurement control parameter setup with saving of the time-tag data is shown in Fig. 113, right. Because time-tag data can be extremely voluminous the file size is limited by ‘Limit .SPC File Size’. When this file size is reached the recording stops. The name of the FIFO data file is specified in the ‘Spec Data File’ field. To change the file name, click on the disc symbol right of the name field. A list of previously used data file names is available by clicking on the symbol. Important: Before starting a new measurement define a new file name for the FIFO data. If you don’t do so you are in danger of overwriting the FIFO data of an earlier measurement.
Before the data are written to the hard disk they are buffered in the main memory of the computer. The buffer size is defined by ‘Max Buffer Size’. The SPCM software checks whether enough memory is available; buffers sizes too large are rejected.
Every time the buffer is filled the data are stored on the hard disc. If possible, the buffer size should therefore be large enough to buffer the data of the entire measurement. If not enough buffer is available the computer has to periodically transfer the buffered data on the hard disc. The hard-disk actions may slow down the readout from the FIFO memory of the TCSPC module. It may then happen that the FIFO memory of the TCSPC module overflows. If this happens it may be better to reduce ‘Max Buffer Size’ to a few Megabyte. The data are then written in smaller portions, and within time intervals that are buffered by the FIFO. The better solution is, however, to upgrade your computer with more memory.
Both the start and the stop of a FIFO imaging measurement are synchronised with the frame clock. This avoids intensity steps in the images by recording incomplete frames. An unconditional stop of the measurement can be forced by a click on the stop-measurement button followed by a second click.
The number of time channels, number of routing channels, and the number of pixels are defined in the Data Format and Page Control section of the system parameters. Examples for the 32-bit version of SPCM are shown in Fig. 114.
1024 time channels 256 time channels 64 time channels 256 time channels 64 time channels
1 detector 1 detector 1 detector 16 detector channels 16 detector channels
The 64-bit version of the SPCM software achieves much larger image formats than the 32-bit version. The available image size depends on the amount of memory installed in the computer. Combinations of pixels numbers, time-channel numbers, and numbers of routing channels are shown in Fig. 115. Images with up to 2048 x 2048 pixels can be recorded while maintaining the FLIM-typical ADC resolution of 256 time channels.
Fig. 115: Time-channel, pixel, and detector channel number of typical FLIM data formats. 64 bit SPCM software.
When SPCM starts it does not automatically allocate memory for the maximum possible image size. The amount of memory Windows reserves for SPCM can be changed manually by clicking on Max Image Size in the Data Format definition. This opens the panel shown in Fig. 116.
Fig. 116: Definition of maximum amount of memory reserved for image recording
SPCM 64 bit has a ‘Mosaic Imaging’ function to record arrays of lifetime images. The array can represent a spatial array of images scanned with the ‘Tile Imaging’ function of a laser scanning microscope, images in different depth of a sample, or a time-series of images. Please see page 118 and following for details. The mosaic can also represent images of different wavelength recorded by multi-wavelength FLIM. The definition of the mosaic FLIM parameters is shown in Fig. 117. Every element of the mosaic has the format defined in the Data Format section, see Fig. 115. With the parameters shown in Fig. 117 an array of 64 images of 256x256 pixels would be recorded, see Fig. 117, right. Mosaic data can be accumulated to record fast time series for repeatable processes. The restart of the mosaic recording can be triggered by a signal at the trigger input of the TCSPC module or by a pulse edge at the Marker 3 input.
Fig. 117: Definition of a FLIM mosaic
The hardware parameters controlling the synchronisation with the scanner are defined under ‘More Parameters’. The setup panel is shown in Fig. 118.
Fig. 118: Scan synchronisation parameters of the FIFO Imaging mode. Left and second left: External pixel clock, Synchronisation by frame, line and pixel clock, pixel binning 1:1 and 2:1. Right: Internal pixel clock, Synchronisation by frame and line clock only. Pixel binning 1:1 and 2:1
In most FLIM applications recording in the SPC module is synchronised with the scanner via Frame Clock (Y Sync), Line Clock (X Sync) and Pixel Clock pulses. The corresponding scan-parameter setup is shown in Fig. 118, left and second left. The panel allows you to define the polarity (active edge) of the synchronisation pulses pixel and line binning factors, (line and pixel clock predividers), and a shift of the left and upper border of the recorded image area against the line and frame clock.
You can define operation with unidirectional (Scan type unidirectional) and bidirectional scanning (Scan type bidirectional). Scanners running in the bidirectional mode normally deliver a line clock at the beginning of the forward scan and at the beginning of the backward scan. 'Second Line Clock' must be set to 'Use'. Scanners running in the unidirectional mode may deliver a line clock at the beginning of each line or at the beginning and at the end of each line. In the second case 'Second Line Clock' must be set to 'Skip' to avoid the recording of ghost images during the beam flyback.
For scanners which do not deliver a pixel clock an internal pixel clock can be used, see Fig. 118, right. The SPCM software then calculates the position of the current photon within a line from the macro time difference between the photon and the previous line clock. The disadvantage of using an internal pixel clock is that the Pixel Time has to be individually selected for different scanner speed.
The san clock pulses are detected via the ‘Event Marker’ inputs of the SPC‑150 and SPC‑830 modules. Normally, Marker 0, 1, and 2 are used for pixel, line, and frame clock, respectively. They are enabled automatically. The fourth marker input (Marker 3) is not normally used for FLIM and therefore not enabled, see Fig. 118. It can, however, be used for FLITS (see page 124). FLITS requires that the source of the frame clock be switched from the scanner to the stimulation source. This can be conveniently achieved by connecting the scanner frame clock and the FLITS trigger to Marker 2 and Marker 3, respectively, and selecting between both via the ‘Y Sync (Frame)’ source parameter in the scan synchronisation panel. Switching from FLIM to FLITS is then only a matter of loading the appropriate predefined setup.
A FIFO measurement can be repeated for a defined number of cycles, and the results saved into individual data files. For using these functions, please set Stop T to stop the individual cycles after the defined collection time.
Routing works the same way as in the photon-distribution modes. The number of routing channels and the routing delay are defined in the system parameter panel under ‘page Control’, see Fig. 119.
Fig. 119: Definition of routing in the FIFO Imaging mode
Starting form version 9.0 the SPCM software has a ‘Triggered Accumulation MCS’ option in the FIFO mode. It is available for all SPC‑150 modules, all SPC‑160 modules, and SPC‑830 modules manufactured later than May 2007. The MCS mode uses the macro times of the photons to record decay data at the microsecond or millisecond time scale. The timing reference for the photons is a pulse at one of the ‘Marker’ inputs of the SPC card. The triggered MCS procedure can also be used to record microsecond or millisecond lifetime images in the FIFO Imaging mode. ‘MCS FLIM’ is defined in the lower part of the runtime definition panel, see Fig. 120.
‘Trigger’ defines which of the marker inputs is used as a timing reference. It is usually Marker 3 because Markers 0, 1, and 2 are used for the scan clocks. However, often the excitation pulses are synchronous with the pixels. ‘Trigger’ can then be identical with the pixel clock, i.e. Marker 0. Please make sure that the marker input used for trigger is enabled in the ‘More Parameters’ panel of the System Parameters, see, Fig. 120 right.
The time channel width (Time per point) can be any multiple of the macro time clock period. It is defined by a number of Macro Time units. The number of points of the curves is defined by ‘Points No.’. The time range of the curves recorded is then given by the product of Time per Point and Points No. It is displayed under ‘Time range’. The recording can be shifted by applying and ‘Offset’ to the photon times. Both positive and negative offsets are possible.
Fig. 120: Left: Definition of MCS FLIM parameters. Right: Definition of the macro time clock source and the marker clock edge
Normally MCS FLIM recording is done with the internal macro time clock. However, for special applications the SYNC frequency can be used. Using the SYNC frequency does, of course, require that the SYNC signal has a constant period, and is continuously present.
MCS FLIM works in combination with normal FLIM recording, see option ‘ps FLIM’ in the configuration panel. The combination of both requires that a high-frequency pulsed laser is turned on and off at a period in µs or ms range. Please see ‘Simultaneous FLIM / PLIM’, page 131. MCS FLIM works also in combination with routing.
Images can be displayed at the end of a FLIM measurement and in regular intervals within the measurement. During FLIM acquisition in the FIFO Imaging mode, images are displayed in intervals of ‘Display Time’. The definitions shown in see Fig. 121, left, run a measurement for 100 s, and display intermediate results in intervals of two seconds. The display routine does not synchronise the update of the screen with the scan. Thus you may see the scan rolling over the image.
Fig. 121, right, show the parameters for a fast online sequence of images. A short collection time and repeat time are defined in the Time section of the system parameters. In the measurement control section the repeat function is activated. The measurement then repeats with the shortest possible repeat time. Please note that the start and the stop of the measurement is synchronised with the beginning and the end of the frames of the scanner. Therefore, the measurement cannot repeat faster than the frame rate of the scanner.
Fig. 121: Control parameters for online-display in the FIFO Imaging mode. Left: Display of intermediate results during the measurement. Right: Display of single images of a repetitive measurement.
The way the data are displayed is controlled by the Trace Parameters, Window Parameters, and display parameters. Please see page 55.
Display of intermediate data during a Scan Sync In measurement is achieved by defining a collection time of the desired display period, a large number of measurement ‘cycles’ and activating ‘accumulate’ and ‘display each cycle’ in the system parameters of the SPCM software, see Fig. 122, left. The setting shown runs 100 cycles of the specified ‘collection time’, accumulates the data, and displays the accumulated data every four seconds. The display of the data in the display windows of the main panel is controlled by the Trace Parameters, Window Parameters, and Display Parameters. Please see page 55.
Display of a fast repeated Scan Sync In measurement is achieved by setting a short collection time and turning on ‘Repeat’ in the measurement control parameters. Also here, the acquisition is synchronised with the beginning and the end of the frames of the scanner. The display rate therefore cannot be faster than the frame rate.
Fig. 122: System control parameters for on-line display in the Scan Sync In mode. Left: Display of intermediate results during the measurement. Right: Display of single images of a repetitive measurement.
To facilitate on-line adjustments the essential hardware and measurement control parameters are also accessible directly from the main panel, see Fig. 123.
Fig. 123: Access to system and control parameters from main panel
The complete system parameter set is accessible under ‘System Parameters’, see page 67. The system parameters contain the operation mode, the measurement control parameters, and all hardware parameters of the particular SPC module.
The entire system parameter set, including the user interface configuration, is restored when the corresponding measurement or setup data are loaded. To simplify switching between different configurations the SPCM software has a ‘predefined setup’ panel, see Fig. 124. Setups of frequently used system configurations are stored in this panel, and then recalled by a single mouse click, see Fig. 125. We recommend to keep the predefined setup panel constantly open, see Application Options, page 80. For definition of predefined setups, please see page 93.
Fig. 124: Predefined-Setup panel. You can change between different instrument configurations by a single mouse click, see figure below.
Fig. 125: Switching the instrument configuration via the ‘Predefined Setup’ panel
Additional software configuration data can be defined in the ‘Application Options’ panel, see Fig. 126. These parameters are not stored in the SPCM data or setup files but in the registry of Windows. They are thus specific of your FLIM system, not of the data recorded.
In the left part you can define which control panels should open automatically when the SPCM application is started. For the Zeiss FLIM systems you should turn on ‘DCC‑100 detector control’ and ‘Predefined Setup Panel’.
In the middle part the main window fields for the TAC parameters, the CFD parameters, and the SYNC parameters can be enabled or disabled.
The ‘Scanner Control’ defines features of the scanner control in systems using piezo scanners or the bh DCS-120 scan head. These parameters have no influence on Zeiss FLIM systems.
The load options (bottom left) allow you to maintain the current scan parameters of the bh DCS scanner when files are loaded. The parameters have no influence on the Zeiss FLIM. ‘Do not change display colours’ and ‘Do not change display size and position’ may be useful when data files are loaded on other computers with different screen resolution. On the FLIM computer these options should be turned off. The correct display sizes are loaded from the predefined setups.
Software versions starting from version 9.4 have an option to compress FIFO imaging data files. Compressing results in a substantial reduction in file size. However, file compression takes time. You may therefore like switch off the compression when recording fast FIFO imaging time series. Please use ‘Do not compress FIFO imaging data sets’ in this case.
Fig. 126: Application Options
‘Send data to SPCImage’ contains options for sending FLIM data to the SPCImage data analysis. You can select whether the data of all display windows are sent to the SPCImage data analysis, or only the data from the active display window. You also can configure the SPCM software to automatically send the data to SPCImage at the end of a FLIM measurement. SPCImage itself has an option to start the data analysis automatically when it receives data, see page 233. Thus, an automatic process from the start of the measurement to the processed FLIM image can be created. Whether you want this is a different question: Not every FLIM measurement delivers the desired result. Running a FLIM analysis automatically after any FLIM measurement may therefore not always be appropriate.
To make the windows of the main window resizable click into ‘Display’ and select ‘Scale Contents on Resize’. To resize a window, seize the edge of the window with the mouse cursor and drag the panel to the desired size. To shift a window, seize the top bar of the window and shift it into the desired position, see Fig. 127.
Fig. 127: Resizing and shifting windows of the main panel
Clicking into a display window area by the right mouse key opens the select panel shown in Fig. 128. ‘Proportional Graph’ sets the display proportions according to the ‘Scan Pixels X’ and ‘Scan Pixels Y’ of a Scan measurement. ‘Full Size Graph’ spreads the display window over the maximum available area. The panel also allows you to enable or disable the cursors, and to access the Display Parameters and Trace Parameters.
Fig. 128: Select panel for display size, cursor display, and display and trace parameters
It can happen that a display window has disappeared behind the edge of the screen or otherwise got out of control. (This can happen if a file from a dual-screen system is loaded in a computer with only one screen.) In that case, click into ‘Display’, and ‘Default Size and Position’. When the window is back in the screen area, set it to ‘Scale Contents on Resize’, see Fig. 129.
Fig. 129: Left: Setting window sizes and positions to default. Right: Window sizes user-definable
Cursors in the display windows of the main panel are enabled by clicking into the window with the right mouse key. This opens a small panel in which the cursors functions can be enabled or disabled, see Fig. 130, left. A display window with cursors is shown in Fig. 130, middle. Two cursors and a ‘data point’ are available.
When the cursors are enabled a window with the cursor settings opens, see Fig. 130, right. The cursor settings window can be placed anywhere in the screen area. It can be closed by clicking on the ‘close’ symbol in the upper right corner, and re-opened by a right mouse click into the display window and selecting ‘Cursor Settings’.
Fig. 130: Cursors in the display windows of the main panel
The cursors and the data point can be shifted by the mouse cursor, or by changing the cursor positions in the cursor settings window. The style and the colour of the cursors can be changed and a zoom function is available.
You can use the ‘data point’ to display the photon number in a selected pixel. The photon number at the data point position is displayed in the lower right of the cursor-settings panel, see Fig. 131.
Fig. 131: Displaying the photon number in the data-point location
A second way to display the number of photons is to use the autoscaling of the display function. When ‘autoscale’ is active the number of photons in the brightest pixel is displayed as ‘Max Count’, see Fig. 132. Please note that there are separate display parameters for the individual images. Click into the top bar of the image you are interested in to see the correct photon count.
Fig. 132: Displaying the photon number in the brightest pixel. Open the display parameter panel and select the display window you are interested in.
The SPCM data acquisition software has a direct link to the SPCImage data analysis. Data are sent to the data analysis by clicking on ‘Main’, ‘Send Data to SPCImage’. For a number of instrument configurations several data sets may have been recorded. In the ‘Application Options’ (see page 80) you can select whether you want to send the data of all display windows to SPCImage or only the data of a selected one.
For sending selected data click on the display window that shows the data to be analysed, and then click on ‘Send Data to SPCImage’, see Fig. 133. Please note that you can also send the data automatically after the end of the measurement, see ‘Application Options’, page 80.
Fig. 133: Sending data to the data analysis. Click on data set to be sent (left), click on ‘send data to SPCImage’ (middle), SPCImage opens with the data selected (right)
Clicking on the 'Trace Statistics' button opens a window which displays information about the data shown in the curve windows. For decay curves or other waveforms the FWHM values, the peak counts, the total counts and the first moment of the photon distribution are displayed, see Fig. 134, left. For an FCS window the trace statistics window displays the results of the fit, see Fig. 134, right.
Fig. 134: Trace statistics panel. Left for a waveform window, right for FCS window
The window can be placed anywhere in the screen area. Please note that the trace statistics window works also in the oscilloscope mode. The window is thus an efficient tool to adjust the system for best time resolution, correct signal transit time, counting efficiency, or IRF stability.
A measurement is started and stopped by the start and stop buttons in the command bar on top of the SPCM main panel.
The SPCM software can stop the measurement either by an operator command, or after a defined acquisition time. For the reasons explained above we recommend to stop the acquisition by an operator command. Of course, the image brightness (or photon number) in images of arbitrary acquisition time are not comparable. This is rarely problem because the brightness information is not normally used in FLIM experiments. Exceptions are, for instance, that you want to compare the expression level in different cells, or compare a cell expressing a fluorescent protein with a cell not expressing it. In that case, define the desired ‘Collection Time’ and set the ‘Stop T’ button, see lower right part of the SPCM main panel.
The start and the stop of a FLIM acquisition is automatically synchronised with the scanning. No matter whether you start the FLIM system first or the scanning: The measurement will not start until the TCSPC system receives the first frame clock pulse from the scanner. When you stop the measurement the FLIM acquisition continues until the scanner has finished the current frame. Consequently, the FLIM data always contain a number of complete frames. Horizontal steps in brightness caused by recording incomplete frames result are thus avoided.
In may, however, happen that the scan stops within the acquisition time of the FLIM system. The reason can be that the scanning has been stopped unintentionally, or a limited number of frames have been defined in the ‘Acquisition’ window of the ZEN software. You can stop the FLIM acquisition in this situation. However, the FLIM system will wait for the current frame to be completed, and display a corresponding message in the status window. A second click on the ‘Stop’ button resolves the conflict by forcing an unconditional stop of the measurement. The FLIM data obtained in this situation are correct. However, if the scan stopped within a frame the image may contain a horizontal step in brightness. Please note that this has only aesthetic consequences: The decay data in the image are correct.
The general answer is: As long as the sample allows you to do so. The accuracy of the fluorescence lifetime (or, more accurately, of the fluorescence decay parameters) increases with the square root of the number of photons recorded. For a single-exponential fit and background-free recording, the standard deviation of the lifetime is very close to N1/2 [197, 267], see ‘Signal-to-Noise Ratio of FLIM’, page 160. Thus, the acquisition time required to record useful data depends on your requirements to the lifetime accuracy and on the count rate at which the measurement is run. It also depends on which part of the sample is interesting to you: Is it a bright part of the image, or are you more interested in dim features of the sample.
Of course, the acquisition time also depends on the number of pixels used. Filling more pixels with a given number of photons per pixel takes more time. A bit surprisingly, the required acquisition time indirectly depends on the effective optical resolution. If you record at high zoom factors and use large pixel numbers the Airy disc (point spread function) of the microscope lens (the ‘Airy disc) becomes over-sampled. Pixels within the diameter of the airy disc contain highly correlated lifetime data. The data analysis takes advantage of this correlation: It allows you to bin the decay functions of several pixels within a selectable area, see SPCImage Data Analysis Software, Binning, page 241. An oversampling factor of, for example, five, allows you to bin the decay data of 5x5 pixels without impairing the spatial resolution. The result is an increase in the effective number of photons by a factor of 25, or a corresponding decrease in the acquisition time. Please see ‘Acquisition Time of FLIM’, page 164.
The conclusion is that you have to develop a feeling of how good the raw images should look like for your special application. The software cannot decide this, it can only support you in deciding whether to stop or to continue, see section below.
When a measurement has been completed the SPCM software can save the data automatically into a file. The file name is defined in the Measurement Control section if the SPCM System Parameters. Define Autosave ‘End of Measurement’ and specify a file name. The file name will be extended by a number as more files are saved.
Fig. 135: Auto-saving FLIM data at the end of the measurement
Auto saving may look convenient but has also a few disadvantages. The first one is that not every FLIM measurement yields good data. The cell may have moved during the acquisition time, the focal plane may have changed, or the image got corrupted otherwise. The autosave function will save all these ‘junk data’, and you have to seek out the good images from the junk later. (Please note that the SPMC software has a ‘Multi-File View’ function for that.) Moreover, the autosave function does not let you type in sample information in the file info field. Written information about the individual measurements is thus not included in the files.
The FLIM data can be transferred to the SPCImage data analysis at the end of the measurement. Please see ‘Application Options’, page 80. However, please note that the raw data are not automatically saved in this case. We therefore suggest to use the automatic data transfer in combination with the Autosave function. Moreover, the automatic transfer does not decide whether the data you recorded are good images or not. It will also transfer the ‘junk’ images to the data analysis, and possibly drive you crazy with stubborn attempts to analyse them.
The Multi SPC Software is able to control up to four TCSPC modules. The information shown in the status window of the SPCM software belongs to the only one of the modules that is selected in the ‘Select SPC’ panel, see figure right. The ‘Select SPC’ panel can be placed anywhere in the screen area.
Fig. 136: Module-select panel. Parameters, count rates, and status information are displayed for the selected module
The count rates are displayed in the status window of main panel of the SPCM data acquisition software, see Fig. 137.
Fig. 137: Count rates (left) status information (upper middle)
The ‘SYNC’ rate is the repetition rate of the laser. The Ti:Sapphire laser of a multiphoton microscope has a repetition rate between 78 and 92 MHz. Fluctuations of the SYNC rate fluctuates or deviations from the nominal repetition rate are indications that the laser mode-locking is not running properly. The FLIM system may still record images under these conditions but the efficiency and the time resolution may be severely impaired.
The repetition rate of a picosecond diode laser is 20, 50, or 80 MHz. When a diode laser is used in combination with an LSM 510 (see Appendix: LSM 510 FLIM Systems) the SYNC rate display shows the corresponding values. In the LSM 710/780/880 the laser is integrated in the beam blanking system of the microscope. That means, the laser emission is turned off during the beam flyback, and when the microscope is not scanning. The indicated SYNC rate is therefore lower than the laser repetition rate.
The CFD, TAC and ADC rates indicate the rate of the detected, converted, and stored photons, respectively . The rates are thus direct indicators of the progress of a FLIM measurement. The count rates may fluctuate due to inhomogeneous intensity in the scan area, due to the beam blanking during the beam flyback, and due to suppression of photons outside the useful scan area.
When a FLIM measurement is running, from time to time take a look at the CFD and TAC count rate. A gradual decrease in the count rates indicates photobleaching. Photobleaching can change the recorded lifetimes. It is not only that different fluorophores or fluorophores in different binding states bleach at different rate, the photobleaching products may also fluoresce themselves. As long as the total decrease in count rate does not exceed 20 % the effect on the lifetimes may still be negligible. If the drop is larger you either have to reduce the laser power or to increase the detection volume by increasing the pinhole size, see below, ‘Photobleaching’.
Both the TCSPC module and the detectors work well up to a detected (CFD) count rate of about 8 MHz. Although there is about 50% loss of photons at a count rate this high systematic errors in the recorded lifetimes are still small [55, 66]. You should, however, take into regard that the displayed count rates are averages over the whole scan area, and the whole scanning cycle, including the beam flyback. The count rate within small bright spots may be substantially higher than the average count rate. With the sample reasonably filling the image area you can use CFD count rates up to a few million photons per second (see ‘Counting Loss and Pile-Up Effects’, page 165).
The 'Device state' field of the SPCM main panel (see Fig. 137) displays information about the status of the SPC module. It shows the presence of the SYNC signal from the laser and the scan clocks, and shows whether or not a measurement is running. The information displayed includes progress of the measurement, the cycle number in a time-series, or the number of photons recorded in the FIFO or FIFO Imaging mode.
The ‘Scan Clocks’ lamp turns on when all scan control pulses are present. To display the state of the individual clocks, move the mouse cursor on the ‘Scan Clocks’ lamp. This opens a window with indicators for the individual clocks.
The software checks the Scan Clock status every one second. For frame rates slower than one frame per second it may therefore happen that the frame clock status changes between red and green, This does not indicate a malfunction, it just means that the software does not see a frame clock every one second.
The DCC-100 detector controller [26, 66] delivers operating voltages and gain control voltages to the detectors and provides for overload shutdown in case of excessive light intensity. It also controls external ps diode lasers of the LSM 170/780/880 system. In the LSM 510 NLO (multiphoton) FLIM systems the DCC-100 controls also the shutters of the NDD detectors.
The DCC software panel can be configured according to the function the DCC-100 is performing in the system. Fig. 138, left, shows the panel for controlling two detectors of an LSM 710/780/880 FLIM system.
Fig. 138: DCC-100 panel, control of two detectors (LSM 710/780/880). Left: Standard HPM-100 detectors. Right: Detectors with coolers
After the start of the DCC‑100 software the outputs of the DCC‑100 are disabled. This is a safety function. It avoids unintentionally switching on a laser, the output voltage of a high-voltage power supply, or the power supply of highly sensitive detectors. Please note: The DCC software can be configured to turn on the outputs automatically (Option ‘Enable Outputs on Startup’). This option is not intended for operation of PMTs and lasers, and should not be used in conjunction with the bh FLIM systems.
The power supply of the detectors can be turned on and off. Turning off the power supply may be required when a detector is replaced, or when a filter on front of a detector has to be replaced.
Thermoelectric coolers of HPM-100-C or of PMC-100 detectors are driven by the DCC-100 as well. The DCC-100 panel with the cooling driver turned on is shown in Fig. 138, right. The cooler current and the maximum cooler voltage can be set independently. The values act on the coolers of both detectors. For detectors without coolers ‘Cooling’ the settings have no effect.
The gain of the detectors is set by the sliders under ‘Connector 1’ and Connector 2’. When changing the detector gain, please remember that the detectors work in the photon counting mode. In other words, the detector delivers a pulse for every photon detected. The data acquisition system counts these single-photon pulses. The light intensity is proportional to the number of pulses per time interval. A change in detector gain changes the amplitude of the single-photon pulses, not their frequency. The detector gain is adjusted in order to obtain a single-photon pulse amplitude well above the threshold of the input discriminator of the TCSPC card (the CFD threshold). With the right combination of CFD threshold and detector gain the count rate (and thus the intensity of the image recorded) becomes almost independent of the detector gain. Please see ‘Adjusting the CFD Parameters and the Detector Gain’, page 294. Additional information can be found in , , and .
Please note: The detector gain cannot be used to control the intensity of the recording, or to avoid overload shutdown at high intensity. Reducing the detector gain results in loss of photons, i.e. a decrease of the signal-to-noise ratio of the lifetime images. It is not equivalent to a reduction in the detector gain of a conventional laser scanning microscope.
If the light intensity at one or both detectors is too high the DCC-100 shuts down the gain and the +12 V supply voltage. If shutters are in the detector beam path (as there are in the LSM-510 NLO systems) it also closes the shutters. The DCC‑100 panel after an overload shutdown is shown in Fig. 139.
Fig. 139: DCC‑100 panel after an overload shutdown
If an overload shutdown has occurred, first remove the source of the overload. Then click on the ‘Reset’ button. The detector then resumes normal operation. Please do not attempt to avoid overload by decreasing the detector gain. This may result in poor counting efficiency, distortion of the decay curves, and, in a multi-wavelength system, poor channel uniformity .
The DCC-100 is also able to control shutters. Shutters are used in the LSM 510 NLO systems with NDD detectors. Please see Appendix: LSM 510 FLIM Systems, Fig. 449 to Fig. 458.
Once the correct detector gain has been determined, we recommend to lock the detector setup, see Fig. 140. This avoids unintentional changes of the detector gain.
Fig. 140: Locking the detector setup
The DCC panel for controlling two external bh ps diode lasers of an LSM 710/780/880 FLIM system is shown in Fig. 141, left. For each laser, the power can be controlled electronically and optically. Electronic control is performed via the ‘Laser Power’ sliders, optical power control via the > Power and < Power buttons. Optical power control changes the laser power via a variable optical attenuator. Electrical power control changes the laser power by changing the amplitude of the electrical pulses injected in the laser diode. Electrical power control is proportional to the value selected in the DCC panel. However, it also changes the shape and the width of the laser pulses. We therefore recommend to run the lasers at an electrical power level that yields best pulse shape, and adjust the power mainly with the optical attenuators. For systems having only one detector and one laser the control of both can be performed via the same DCC-100 module. The corresponding DCC panel is shown in Fig. 141, right.
Fig. 141: Control of bh ps diode lasers via the DCC-100. Left: Control of two lasers. Right: Control of one laser and one detector from one DCC-100
The ‘Save’ panel is shown in Fig. 142. It contains fields to select different file types, to select or specify a file, to display information about existing file, and to select between different save options.
Fig. 142: Save panel
You can chose between ‘SPC Data’ and ‘SPC Setup’. The selection refers to different file types. With ‘SPC Data’ files are created which contain both measurement data and system parameters. When this file is loaded not only the measurement data are restored but also the complete system setup. With ‘SPC Setup’ files are created that contain the system parameters only. When such files are loaded the system setup is restored, but no data are loaded. Files created by ‘SPC Data’ have the extension ‘.sdt’, files created by ‘SPC Setup’ have the extension ‘.set’.
A file name can be written into the ‘File Name’ field. ‘Select File’ opens a dialog box that allows you to change or create directories. Moreover, it shows the names of existing files. These are ‘.sdt’ files or ‘.set’ files, depending on the selected file format. If you want to overwrite an existing file you can select it in the ‘File Name’ field. A history of previously saved files is available by clicking on the button.
After selecting the file text can be written into the ‘Author’, ‘Company’ and ‘Contents’ fields. Both for ‘SPC data’ and ‘SPC setup’ the file information is saved in the file. The file information helps considerably to later identify a particular measurement among a large number of data files. We therefore strongly recommend to spend a few seconds on typing in a reasonable file information.
If you have selected an existing file the file information contained in it is displayed in the ‘File info window’. If you want to overwrite this file you can edit the existing file information.
Under ‘What to Save’ the options ‘All used data sets’, ‘Only measured data sets’ or ‘Selected data blocks’ are available.
The default setting is ‘All used data sets’, which saves all valid data available in the memory of the SPC modules. These can be measured data, calculated data or data loaded from another file. Except for special cases (see) we recommend to use the ‘All used data sets’ option.
The ‘Load’ menu is shown in Fig. 143. It contains fields to select different file types, to specify a file, to display information about the file selected, and to select different load options.
Fig. 143: Load panel
You can chose between ‘SPC Data’ and ‘SPC Setup’. The selection refers to different file types. With ‘SPC Data’, .sdt files are loaded. These files contain both measurement data and system parameters. Thus the load operation restores the complete system state as it was in the moment when the file was saved.
If you chose ‘SPC Setup’, .set files are loaded. These files contain the system parameters only. The load operation sets the system parameters, but the actual measurement data are not influenced.
Note: Measurements in the ‘FIFO’ (time tag) mode deliver an .spc file that contains the micro time, the macro time, and the detector channel for each individual photon. These files are loaded by using the ‘Convert’ routines, see .
The file to be loaded can is selected in ‘File Name’ field. ‘Select File’ opens a dialog box that displays the available files. These are ‘.sdt’ files or ‘.set’ files depending on the selected file format. A history of previously loaded files is available by clicking on the button.
The file info window displays information about the file selected. The first three lines of the file info are inserted automatically when a file is saved. The last three items can be typed in by the operator, see ‘Saving and Loading Setup and Measurement Data’.
Activating a data block in the ‘Block Number in File’ field enables a ‘Block Info Button’. Clicking on this button opens a list that contains the device number of the SPC modules by which the data were recorded, the time and data of the recording, and all system parameters, see Fig. 144. At the end of the block information the minimum and maximum count rates of the corresponding measurement are shown (see Fig. 144, right). The block info often helps to recover the exact recording conditions of an older measurement.
Under ‘What to Load’ the options ‘All data blocks & setup’, ‘Selected data blocks without setup’ or ‘Setup only’ are available. The default setting is ‘All data blocks & setup’, which loads the complete information from a previously saved data file. Except for special cases (see below) we recommend to use the ‘All data blocks & setup’ option.
Older software versions may contain less system parameters than newer ones. Therefore, loading older files into a newer software (or vice versa) can cause warnings of missing or unknown parameters. To load the file, click on the ‘Continue’ button until the file is loaded. Unknown parameters are ignored, and missing parameters are replaced with default values. To avoid further problems with such a file, we recommend to save it in the current software version (Use option ‘All used data blocks’, see ‘Saving and Loading Setup and Measurement Data’).
Setups of frequently used system configurations can be added to a list of ‘predefined setups’. Changing between these setups then requires only a mouse click. To use the predefined setup option, click on ‘Main’, ‘Load Predefined Setups’. This opens the panel shown in Fig. 145, left. A setup is loaded by clicking on the button left of the name of the setup.
To add or delete setups to or from the list, or to change the names of the setups, click into one of the name fields with the right mouse key. This opens the panel shown in Fig. 145, middle.
To add a setup, click on the disc symbol right of the ‘File Name’ field and select a ‘.set’ file or a ‘.sdt’ file. Select the files you want to put into the list of predefined setups, and click on the ‘Add’ button. In principle, you can select any .sdt or .set file in any directory of the computer. We do, however, discourage to use files in such locations for the simple reason that they can be overwritten. To avoid unintentional overwriting of files used for the setup panel the SPCM software has a directory ‘Default Setups’, see Fig. 145, right. Files that are to be used as predefined setups should be saved or copied into this directory. If a file in the Default Setups directors has to be replaced, either copy it from another directory by using the Windows Explorer or delete the old file before you save the new one by the SPCM software.
Every setup has a user-defined ‘nickname’. The default nickname is the file name of the file. To change the nickname, click into the nickname filed and edit the name. Then click on ‘Replace’.
Fig. 145: Editing the list of predefined setups
You can add both ‘.set’‘ files and .sdt’ files to the setup list. A .set file contains only setup parameters, a .sdt file contains both setup parameters and measurement data. You can define whether a .sdt file is loaded with or without the data by the ‘load with data’ button.
The Acquisition Mode parameters of the LSM 710/780/880 ZEN software for FLIM acquisition are shown in Fig. 146. Scan Mode is ‘Frame’, Frame Size is 512 x 512 pixels. You can change the frame size, but this requires changes in the SPCM FLIM acquisition parameters as well. We therefore recommend to leave the frame size of the LSM scan on 512 x 512 unless you have good reasons to change it. Such reasons may be that you need a particularly fast scan, or want to record FLIM data with 1024 x 1024 or 2048 x 2048 pixels.
You can use any scan speed for FLIM. Nevertheless, we recommend to work at or close to the maximum scan speed available. This reduces triplet population and possible excitation of higher triplet states. Moreover, fast scan speeds make working with FLIM more comfortable. The fast preview function of FLIM can only be as fast as the frame time. Fast scanning also minimises the start and stop delay of the FLIM acquisition: When FLIM is started, the hardware waits for the start of the next frame to start the acquisition, and, after stop, continues until the end of the current frame. These times are reduced by fast scanning.
‘Averaging’ must be turned off by setting the number of averaged lines to ‘1’. ‘Direction’ must be ‘unidirectional’, as shown in Fig. 146.
Fig. 146: Acquisition mode parameters of the LSM 710/780/880. The settings are defined for FLIM.
The ‘Scan Area’ selection (see bottom of Fig. 146) works the same way for FLIM as it does for standard imaging. The FLIM image adjusts automatically to the scan area selected. To select a scan area we recommend to run a fast preview in the FLIM system and change the scan area until the desired image is obtained. The final FLIM acquisition is then performed with this scan area.
The laser and beam path configuration for one-photon FLIM is shown in Fig. 147. The laser is either the InTune laser or a diode laser in the ps mode. The beam path configuration is shown right. The DC port is marked with ‘Fibre Out’ is the confocal (DC or BIG) port, and a ‘Plate’ must be used to send the light to it.
Fig. 147: Laser selection and beam path configuration for one-photon FLIM
The setup for multiphoton NDD FLIM is shown in Fig. 148. The laser is ‘Chameleon’, and the ‘Non Descanned’ port is active.
Fig. 148: Laser selection and beam path configuration for multiphoton NDD FLIM
The typical main panel for single-detector FLIM in the FIFO Imaging mode is shown in Fig. 149. An image is displayed on the left, the display parameters are kept open on the right. For switching between different user interface configurations, operation modes or scan formats, the predefined setup panel is kept open in the lower right.
Please make sure that the ‘Autoscale’ function of the display parameters is active. TCSPC has an enormous dynamic range; with autoscaling you can see anything from single photons to extremely bright images. ‘Reverse Y’ must be turned on to obtain the FLIM images in the same orientation as the intensity images displayed in the ZEN software.
Fig. 149: Main panel for a single-detector system. 512 x 512 pixels.
The ‘System Parameters’ panel is shown in Fig. 150, left. Operation Mode is ‘FIFO Imaging’. STOP T is not set, the acquisition runs until it is stopped by the operator. The size of the recorded image is 512 x 512 pixels, 256 time channels. The scan synchronisation parameters are shown in Fig. 150, right. The setup is for an LSM scan of 512x512 pixels. The FLIM image format is the same as the LSM scan, therefore both the line predivider and the pixel predivider are ‘1’. The ‘left border’ parameter defines the x location of the recorded part of the scan. The parameter is different for different LSM and ZEN software versions. For some versions it must be 48, for other versions it must be 0. If you get images badly shifted in x please change the ‘Left boarder’ parameter.
Fig. 150: System parameters, FLIM image 512 x512 pixels
The ‘Window parameters’ are used to display different subsets of multidimensional data. Images can be displayed in different time windows, or decay curves can be displayed over one of the image coordinates and in selected windows of the other coordinate. The panel is shown in Fig. 151. For simplicity, only one time window and one X and one Y window have been defined. For displaying time-gated images and curves within x and y windows please see ‘3D Trace, Window and Display Parameters’, page 55 and ‘Parameter Setup for Imaging Modes’, page 70.
Fig. 151: Window Intervals: Only one time window has been defined.
The SPCM software is able to display several images simultaneously. The images may come from different TCSPC channels, different channels of a multi-wavelength detector, or be created within selected time-windows of the data. These definitions are made in the ‘3D Trace parameters’, see Fig. 152. Also here, only one display window has been activated for simplicity.
Fig. 152: 3D trace parameters: Only one active display window has been defined.
Setups for different pixel numbers are normally available from the Predefined Setups’ panel, see ‘Switching Between Different Instrument Configurations’, page 79. For definition of other pixel formats, the pixel numbers and the pixel binning parameters can be defined under ‘More Parameters’ in the system parameter panel. Example for FLIM formats of 256 x 256 pixels, 256 time channels, and 1024 x 1024 pixels, 256 time channels are shown in Fig. 153. The definitions for the 256 x 256 pixel image are for the default (512 x 512 pixel) scan in the LSM. The definitions for the 1024 x 1024 pixel image require a 1024 x 1024 pixel scan in the LSM.
Fig. 153: Left: Definition of a 256 x 256 x 256 FLIM format, LSM scan 512 x512 pixels. Right: Definitions for a 1024x1024x256 FLIM format, LSM scan 1024x1024 pixels.
Most FLIM experiment can be perfectly performed with a time-axis resolution of 256 time channels (see Fig. 153). The time channel width (for a 10 ns observation interval) is then 40 ps. An exception may be FLIM experiments with the new ultra-fast HPM-100-06 and -07 hybrid detectors. These detectors have an instrument-response width of 20 ps. To fully exploit the time resolution a time-channel width of about 10 ps is necessary, which means that for the same 10 ns observation-time interval 1024 time channels should be used. The definition in the system parameters is shown in Fig. 154, left. In combination with scan formats of 1024 x 1024 pixels it can be necessary to explicitly allocate more memory to the SPCM application. To do so, please click into ‘Max Image Size’ and select the maximum memory space available in your computer, see Fig. 154, right.
Fig. 154: Left: Data format definition for an image of 1024 x 1024 pixels and 1024 time channels. Right: Allocating memory space for large FLIM data formats.
The online lifetime display of SPCImage is activated by selecting ‘Lifetime’ in the 3D trace parameters, see Fig. 155.
Fig. 155: 3D trace parameters for online lifetime display
The SPCM main panel with lifetime display is shown in Fig. 156. The lifetime image is shown on the left, the display parameters on the right. For lifetime display, the display parameters have an additional part that defines the colour coding of the lifetime scale and a reference value for the lifetime calculation. The lifetime calculation is based on the first moment of the decay curves in the individual pixels. It therefore requires a reference moment for the IRF position. The reference moment can be obtained by analysing data (with similar timing parameters) by SPCImage, or from a reference data file of a sample with known lifetime. Please see  or  for details.
Please note that online lifetime display does not require changes in the FLIM data format. The data can still be recorded with a large number of time channels, and a large number of pixels. That means that precision multi-exponential data analysis can be applied to the data as usual.
Fig. 156: SPCM Main panel of a single-channel system, online-lifetime display function activated
The Main panel of the SPCM software for a dual-channel FLIM system is shown in Fig. 157. Images for both detector channels are displayed. The predefined setups and the detector control panel have been placed at the lower right. There is also a ‘Select SPC’ panel. It allows you to switch the count rate display between the two TCSPC channels.
Fig. 157: Main panel for a dual-channel TCSPC system
The system parameters for the two SPC modules of the system are the same as for a single-channel system, see above, Fig. 150. The only difference is that you can select whether you want common system parameters for the two SPC modules or separate ones. The selection button is in the lower right of the system parameters. Please use ‘Common’. Separate parameters are required only for special systems, e.g. systems with different detectors.
Fig. 158: Standard dual-channel FLIM systems use ‘Common’ system parameters for both channels.
The 3D Trace parameters are defined to display the images recorded in both TCSPC modules, see Fig. 159.
Fig. 159: 3D Trace parameters for a dual-channel FLIM system
There are separate sets of display parameters for the two display windows of the main panel. Thus, different colour, different intensity scaling, or different T windows can be assigned to the individual images. The display parameters for the two display windows of Fig. 157 areas shown in Fig. 160.
Fig. 160: Display parameters for the two display windows of the main panel shown in Fig. 157
Online Lifetime display is activated as shown for the single-channel system. Open the 3D Trace parameters and select ‘Lifetime’ for both modules, see Fig. 161. Lifetime display in one module can be combined with intensity display in the other. It is also possible to activate more display windows, and to display both intensity and lifetime images for both channels. Please see also ‘Examples for Display of Imaging Data’, page 59.
Fig. 161: Trace parameters for lifetime display in both channels of a dual-channel system
An example of the SPCM main panel for dual-channel lifetime display is shown in Fig. 162. The two images have independent display parameters, as shown in Fig. 163. This makes it possible to assign different colour coding of the lifetime, different reference moments, and different contrast and brightness to the individual images.
Fig. 162: SPCM main panel for online-lifetime display, dual channel system
Fig. 163: Display parameters for the two lifetime images shown in Fig. 162. Different colour coding, contrast, and brightness can be assigned to the individual images.
The SPCM main panel for multi-wavelength FLIM is shown in Fig. 164. Every two of the 16 wavelength channels of the multi-wavelength detector are combined into one of eight display windows. Each display window has its own set of display parameters. In Fig. 164 the colours of the images were selected according to the real wavelengths of the channels.
Fig. 164: SPCM main panel for multi-wavelength imaging
The TCSPC system parameters of a FLIM measurement with the MW-FLIM detector are shown in Fig. 165. The measurement is run in the FIFO imaging mode. The image size is 256 x 256 pixels, 64 time channels. 16 images in 16 wavelength intervals are recorded simultaneously. ‘Routing Channels X’ is 16, corresponding to the number of channels of the PML‑16 detector module of the MW-FLIM assembly.
Fig. 165: System parameters for multi-wavelength imaging with the bh MW FLIM detector
The number of images to be displayed is defined in the 3D trace parameters, see Fig. 166. In the present case, all eight possible display windows are activated. For each of the images a ‘Routing X’ window, i.e. a range of wavelength channels is defined. Which wavelength channels the windows contain is defined in the Window Parameters, see Fig. 167. With the definitions shown each window consists of two subsequent wavelength channels. Other windows definitions, such as single wavelength channels or wider, even overlapping, wavelength intervals can be used.
Fig. 166: 3D trace parameters for multi-wavelength FLIM
The window parameters are shown in Fig. 167. With the definitions shown each window contains the combined data of two subsequent wavelength channels. Other window definitions, such as single wavelength channels or wavelength intervals containing more than two channels can be defined if needed.
Fig. 167: Window intervals for multi-wavelength FLIM. Each window contains two subsequent wavelength channels
The fast preview continuously records images of short acquisition time and displays them in short intervals. The SPCM system parameters for the fast preview are shown in Fig. 168, left. The operation mode is Scan Sync In. (The FIFO imaging mode works as well, but may saturate at excessively high count rates). ‘Repeat’ must be set. Important for a fast update rate is that the collection time is 1 second or shorter, and that the ADC resolution is set to ‘1’.
The scan parameters are shown in the middle. Line and pixel divider are set to 2 to obtain an image of 256x256 pixels with a 512x 512 LSM scan.
‘Autoscale’ in the display parameters (Fig. 168, right) must be switched on to make images visible independently of the number of photons they contain.
Fig. 168: System parameters (left), scan parameters (middle) and display parameters (right) for preview mode
The main panel should is the same way as for the FLIM acquisition modes, see Fig. 149 and Fig. 157.
Fast lifetime preview is possible by using the FIFIO Imaging mode with lifetime display, in combination with short acquisition time and the repeat function of the SPCM software. A typical main panel is shown in Fig. 169. It shows images in two channels of a dual-channel FLIM system. Both images are calculated by the online-FLIM function of the SPCM software. The images size is 512 x 512 pixels.
Fig. 169: SPCM main panel, fast lifetime preview. Lifetime range for both images 1800 ps to 2200 ps.
The system parameters are essentially the same as for the normal FIFO imaging setup, see ‘Dual-Detector System’, page 100. The only difference is that ‘Stop T’, and ‘Repeat’ are active, and that a short ‘Collection’ and a short ‘Repeat’ time are defined. The setup shown in Fig. 170 records and displays images of 512 x512 pixels. Limited by the speed of the scanner, the fastest image rate that can be reasonably obtained with 512 x512 pixels is about 1 image per second. Faster image rates can be reached if the number of pixels both in the LSM scan and in the SPCM software is reduced. The scanner then runs at higher frame rates, and less photons are required to fill the pixels with photons. With 256 x 256 pixels about two images per second can be obtained, with 128 x 128 pixels about four images per second.
Fig. 170: System parameters for fast lifetime preview
The Trace and Window Parameters can be defined as shown in Fig. 166 and Fig. 167, page 104. For the Display Parameters the settings shown in Fig. 168 can be used. In practice, an adaptation of the colour range to the lifetime range of the particular sample may be required. The display parameters for the images shown in Fig. 169 are shown in Fig. 171. Identical display parameters were used for both channels.
Fig. 171: Display parameters used for the images shown in Fig. 169. Identical parameters were used for both channels.
The Oscilloscope Mode records fluorescence decay curves and displays them in short periods of time. The Oscilloscope Mode is not directly used in FLIM experiments. It is, however, an extremely useful tool for system alignment, setup and troubleshooting.
The main panel of a dual-channel (SPC‑152) system in the oscilloscope mode is shown in Fig. 172. The fluorescence decay curves detected in both channels are displayed in the upper left. The display parameters, the detector control panel, and the predefined setup panel are open on the right. The Application Options have been set to display the TAC, CFD, and SYNC parameters at the bottom of the panel.
Fig. 172: Main panel for the Oscilloscope Mode
The system parameters are shown in Fig. 173. The setup is the standard one for a system with two SPC modules without routing. For the function of the Oscilloscope mode Stop T and a conveniently short Collection Time have been set.
Fig. 173: System parameters for the Oscilloscope Mode
The trace parameters are shown in Fig. 174. One trace for Module 1 (M1), and a second one for Module 2 (M2) has been enabled.
Fig. 174: Trace parameters for the oscilloscope mode. One trace for Module 1, and another for Module 2 are active.
There is an increasing interest in fluorescence imaging in the wavelength range above 700 nm. Activities are driven by the fact that biological tissue is almost free of autofluorescence in this range, and by the fact that near-infrared dyes are used in diffuse imaging of small animals [260, 300] and clinical applications . For these applications it is important to have information about binding of the dyes to proteins, DNA, collagen, and other cell constituents available. It is also important to know whether the dyes change their fluorescence lifetimes on binding, whether these lifetime changes depend on the binding targets , and whether the lifetime reports biologically relevant parameters.
Near-infrared FLIM can be performed with the Zeiss LSM 710/780/880 NLO multiphoton systems. The titanium-sapphire laser is used for conventional one-photon excitation of the fluorophores. The standard HPM‑100-40 detector of the FLIM system is replaced with an HPM‑100-50. Fluorescence is then detected up to wavelength of 900 nm. Because one-photon excitation is used the detector must be attached to the confocal output of the scan head, see Fig. 73, page 45. The internal main beamsplitter wheel of the scan head does not contain dichroics for the near-infrared range. However, there is an 80/20 beamsplitter that can be used to reflect the fluorescence signal. The 80% loss in the excitation path is no problem because enough excitation power is available. A laser blocking (long-pass) filter of the appropriate cutoff wavelength must be inserted in front of the detector. Please see [66, 64] and  for further technical details.
The FLIM system is used in the normal FLIM configuration, with system parameters as described under ‘System Parameters for Basic FLIM Experiments’, page 95. Advanced procedures like time-series recording, Z-stack imaging and Mosaic FLIM can be used as described later in this section. Two FLIM images obtained from a pig skin sample stained with DTTCC (3,3’-diethylthiatricarbocyanine) are shown in Fig. 175.
Fig. 175: Pig skin samples stained with DTTCC. Zeiss LSM 710 NLO, Ti:Sa laser used for one-photon excitation. Excitation wavelength 780 nm, detection wavelength 800 nm to 900 nm. HPM‑100‑50 hybrid detector, Simple-Tau 152 FLIM system. Lateral size of the images 212x212 µm, depth about 30 µm from surface. Note the high contrast of the images.
The excitation wavelength range of multiphoton FLIM can be extended to wavelength longer than 1000 nm by using an optical parametric oscillator (OPO) as an excitation source. A suitable system based on the LSM 710/780/880 NLO microscope family is available from Zeiss .
An HPM-100-50 detector is attached to the NDD output of the microscope. To be able to detect fluorescence up to 900 nm the standard two-photon beamsplitter in the beam path at the back of the objective lens is replaced with a 980 nm (short-pass) dichroic mirror. The beamsplitter is not critical, an 80/20 wideband beamsplitter can be used as well. The standard 700 nm short pass (laser blocking) filter in the Zeiss T adapter is replaced with a 980 nm short pass filter.
A FLIM image obtained from a pig skin sample stained with Methylene Blue is shown in Fig. 176, left. The sample was excited at 1200 nm, the fluorescence was detected from 680 nm to 780 nm. Excitation was surprisingly efficient. No more than 4 percent of the available OPO power were needed to obtain a count rate on the order of 1 MHz. Decay curves from characteristic spots are shown in Fig. 176, right.
Fig. 176: Pig skin stained with methylene Blue. Left: Lifetime image, double-exponential decay model, amplitude-weighted lifetime. Two-photon excitation at 1200 nm, 512 x 512 pixels, 256 time channels. Right: Decay curves in characteristic spots of the image.
FLIM Images of a similar sample stained with ICG are shown in Fig. 177. The data were analysed by a double-exponential decay model. The lifetime shown is the amplitude-weighted average of the decay components. The fluorescence was excited at 1200 nm, fluorescence was detected from 780 nm to 850 nm. As for the Methylene Blue, about 3 % of the available excitation power was sufficient to obtain a count rate on the order of 1 MHz. Please see [37, 66, 64] and  for further details.
Fig. 177: Pig skin stained with Indocyanin Green. Two-photon excitation at 1200 nm, detection from 780 to 850 nm. Amplitude-weighted lifetime of double-exponential decay. Depth from top of tissue 10 µm (left) and 40 µm (right). 512x512 pixels, 256 time channels.
A time-series of FLIM recordings can by programmed by defining a number of measurement ‘cycles’ and activating the ‘autosave’ function of the SPCM software. The control section of the SPCM system parameters is shown in Fig. 178.
Fig. 178: Recording a FLIM time series
The parameters shown in Fig. 178, left, record a time-series in the FIFO imaging mode, the parameters shown right in the Scan Sync In mode.
With the settings shown in Fig. 178 the FLIM data are acquired with the collection time of 10 seconds per image. The measurement is repeated for 40 ‘cycles’. The result of each recording cycle is displayed, and the data are automatically saved into subsequent files. The file names are defined under ‘Spec data file’. The software automatically adds a number to the file name, i.e. ‘time-series_c01.sdt’, ‘time-series_c02.sdt’, until ‘time-series_c40.sdt’.
For extremely fast time series it can happen that the standard 512 x 512 pixel scan of the LSM is not fast enough. The number of pixels in the LSM scan must then be reduced. This requires other line and pixel binning factors to be used in the FLIM recording. The parameters for a 128 x 128 pixel FLIM time series recorded from a 128 x 128 pixel LSM scan are shown in Fig. 179.
Fig. 179: Page control and binning parameters for a series of 128x128 pixel images recorded with a 128 x 128 LSM scan
If the sample delivers sufficient count rate the acquisition time per image can be made as short as 1 second. It may be useful to turn off the display of each cycle in that case. This gives the computer more time to save the data. A typical result of a fast time series is shown in Fig. 180.
Fig. 180: Time series with an acquisition time of 2 seconds per image. Chloroplasts in a moss leaf, the lifetime decreases with the time of exposure.
If you want to record single frames of the scan set the collection time to a very small value, e.g. 50 ms. Since every measurement cycle waits for the beginning of the next frame and runs the measurement until the end of this frame the sequence records one frame per step.
Extremely fast time-series can be obtained by ‘Continuous Flow’ option of the Scan Sync In Mode. The system parameters are shown in Fig. 181. With this setup the memory of the SPC‑150 is split into two halves or ‘banks’. While the recording runs in one bank of the memory the data of the other bank are read. After each cycle the memory banks are swapped and the process continues. Thus, images are obtained without any time gaps between the recordings [44, 66, 256]. Of course, a high count rate is essential to obtain reasonable data within a time shorter than a second. Continuous Flow Imaging is therefore especially interesting in combination with dual-SPC‑150 systems. The system parameters are shown in Fig. 181.
Fig. 181: System parameters for Continuous Flow Imaging. 50 Images are acquired. Because the collection time is shorter than the frame time each image contains one frame.
Because the SPC‑150 memory is split into two banks the maximum image size or the maximum number of time channels is only one half compared with a normal Scan Sync In measurement. For the setup shown above a fast LSM scan of 256x256 pixels was used. The FLIM data size is 256 x 128 x 64 time channels, i.e. two lines of the 256 x 256 pixel scan were binned into one line of the image. The binning is obtained by setting ‘Line predivider’ = 2 in the ‘More parameters’ panel.
The collection time in the SPCM software is 80 ms. This is shorter than the frame time of the LSM scan. Consequently, the SPC-150 module records each individual LSM scan into one separate FLIM data file. Results of the Continuous Flow imaging mode can be found in  and .
There are situations in which a time-series has to be recorded over a long period of time, and with long rest periods between the individual recordings. In these cases the recording can be controlled from the microscope. The corresponding settings in the Zeiss ZEN software are shown in Fig. 182. Select ‘Time-series’ in the window under the ‘Live’ and ‘Continuous buttons (Fig. 182, left). In the ‘Acquisition Mode’ window, select a Scan Speed and an ‘Averaging Number’ that yields the desired acquisition time per step of the sequence (Fig. 182, middle). In the ‘Multi-Dimensional Acquisition’ window, select the number of cycles and the time interval between the steps of