Confocal and Multiphoton FLIM
7th edition, December 2017
Becker & Hickl GmbH
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Principle of Data Acquisition
DCS‑120 FLIM Functions in Brief
Principle of the bh FLIM Technique
Simultaneous FLIM and PLIM
Description of System Components
DCS‑120 Scan Head
Data Acquisition System
Excitation Light Sources
Picosecond Diode Lasers
DCS Switch box
SYNC Delay Box
Control Elements of the Scan Head
Data Acquisition Software
Synchronisation and Count Rates
Running a FLIM Measurement
Electronic Pinhole Alignment
Display of FLIM Data
3D Trace, Window and Display Parameters
Examples for Display of Imaging Data
Display of Fluorescence Decay Curves
Overview on Operation Modes
Measurement Control Parameters
CFD, SYNC and TAC Parameters
Parameter Setup for Imaging Modes
Scan Sync In Mode
FIFO Imaging Mode
Control of the Online Display Functions
System Parameter Setting from the Main Panel
Switching Between Different Instrument Configurations
Resizing and Positioning the Display Windows
Cursors in the Display Windows of the Main Panel
Link to SPCImage Data Analysis
Starting and Stopping a Measurement
Automatic Actions at the End of the Measurement
Module Select (Multi-SPC Systems)
Synchronisation and Count Rates
General Scan Parameters
Park Beam Function
Assigning Names to the Laser Buttons
Ti:Sa Laser and AOM Control
Saving and Loading Setup and Measurement Data
Creating Predefined Setups
System Parameters for Basic FLIM Experiments
Basic Dual-Channel FLIM System
Fast Preview Mode
Fast Lifetime Preview
Advanced Techniques and Procedures
Main Panel Configuration
DCS Box Settings
Record and Save Procedures
Time-Series FLIM by Continuous-Flow Procedure
Z Stack FLIM by Record-and-Save Procedure
X-Y Mosaic FLIM
Z Stack Recording by Mosaic FLIM
Time-Series Recording by Mosaic FLIM
FLITS: Fluorescence Lifetime-Transient Scanning
Example: Chlorophyll transients
Excitation Wavelength Multiplexing
Scanner and Laser Control Parameters
Configuration of DCS Switch Box
Display of the Images
Simultaneous FLIM / PLIM
SPCM System Parameters
Scan and Laser Control Parameters
DCS Box Configuration
Synchronous Start of Several TCSPC Modules
Selecting a Position for FCS from an Image
Variants of the DCS‑120 System
DCS-120 WB Wideband Version
Swapping Diode Lasers
Tuneable Excitation FLIM
DCS‑120 MP Multiphoton Excitation System
General System Architecture
Coupling the Ti:Sa Laser into the Scanner
Main Dichroic Beamsplitter
Beam Path to Non-Descanned Detectors
Control of Detectors and Shutter
Control of Ti:Sa Laser and AOM
Multiphoton FLIM Procedure
Failure to Record Images?
Examples of Two-Photon FLIM
NADH FLIM with Ultra-Fast Detectors
Laser Safety Recommendations
DCS-120 MACRO System
Scanning Through Endoscopes
A Few Things FLIM Users Should Know
What is Important for FLIM of Biological Objects?
The Scanning Advantage
One-Photon FLIM versus Multi-Photon FLIM
Effect of Fluorescence Depolarisation on Measured Decay Data
Signal-to-Noise Ratio of FLIM
Why does FLIM need more photons than ‘normal’ imaging?
Acquisition Time of FLIM
Counting Loss and Pile-Up Effects
Why Use FLIM
Measurement of Molecular Environment Parameters
Recording Ca++ Transients in Live Neurons
Fluorescence Resonance Energy Transfer (FRET)
Single-Exponential FLIM FRET
Double-exponential FLIM FRET
Using Information from the Acceptor Decay Function
FRET between Endogenous Fluorophores
FRET in Diffraction-Limited Spots
Practical Hints for FRET Measurements
NAD(P)H and FAD Lifetime Imaging
Review of NAD(P)H / FAD FLIM Literature
Unmixing NAD(P)H / FAD Fluorescence Components
Two-Photon FLIM of Mammalian Skin
FLIM of Other Endogenous Fluorophores
Diffusion of Nanoparticles in Skin
Autofluorescence of the Ocular Fundus
Oxygen Sensing by Phosphorescence Lifetime Measurement
Oxygen Sensing by PLIM
Simultaneous Recording of Oxygen and NAD(P)H Images
Delayed Fluorescence of PpIX
SPCImage Data Analysis Software
Analysing FLIM Data with SPCImage
SPCImage Software Panel
Loading of FLIM Data
Calculating the Lifetime Image
Saving the Data
Export of Data
Analysing Single-Curve Data
Initial ‘Hot Spot’ Selection
Region of Interest (ROI) Selection
Lines of Interest
Fit Definition Parameters
General Model Parameters
Model Options and Fit-Control Parameters
How Many Exponential Components Are Needed?
Calculation of Multi-Wavelength Lifetime Images
Mosaic FLIM Data
Analysis of Phosphorescence Lifetime Images
Display of Lifetime Images
Selection of Decay Parameter
Instrument Response Function
Synthetic IRF: Calculating an IRF from fluorescence decay data
IRF from scattering data
IRF from SHG data
Two-Dimensional Histograms of Decay Parameters
Setup and Training Service
DCS Switch Box
SYNC Delay Box
Connecting the Lasers to the Scan Head
Aligning the Fibre Couplers of the Scan Head
Aligning the Fibre Couplers of the Laser
Replacing the Main Beamsplitter
Attaching or Replacing Detectors
System Parameter Setup and Optimisation
General System Checks
Adjusting the CFD Parameters and the Detector Gain
Main Dichroic Beamsplitters
The DCS-120 systems are complete confocal laser scanning microscopes for fluorescence lifetime imaging. The systems use bh’s multi-dimensional TCSPC FLIM technology [58, 68, 69] in combination with fast laser scanning and confocal detection or multi-photon excitation . DCS-120 systems are available with various inverted and upright microscopes, see Fig. 1. The DCS-120 scan head with the associated control and data acquisition electronics can also be used to upgrade a conventional microscope with FLIM recording. A ‘DCS-120 MACRO’ system is available for FLIM of centimetre-size objects, see Fig. 2.
Fig. 1: The DCS‑120 system with a Zeiss Axio Observer microscope (left) and Zeiss Axio Examiner (right) .
Fig. 2: DCS‑120 MACRO system and DCS-120 MP multiphoton system (right)
In the basic configuration, the DCS-120 uses excitation by two ps diode lasers and records in two fully parallel detector and TCSPC channels. The 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 initiated a breakthrough in FLIM recording . Another step was made by the introduction of 64-bit data acquisition software [42, 446]. 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, temporal Mosaic FLIM, wavelength-multiplexed FLIM, combined fluorescence and phosphorescence lifetime imaging (FLIM/PLIM), and fluorescence lifetime-transient scanning (FLITS). Due to its high sensitivity, the system can also be used for FCS recording and single-molecule spectroscopy. 16-channel multi-wavelength FLIM is available as an option. It uses a new multi-wavelength detector with a GaAsP cathode. Due to the high efficiency of the detector and the large memory space available in the 64 bit environment multi-wavelength FLIM can be recorded with unprecedented pixel numbers . Advanced versions of the DCS-120 system are available for multiphoton excitation and tuneable excitation sources [34, 36].
This handbook describes the system architecture of the DCS‑120 system, the system components, and the optical and electronic principles of FLIM data acquisition. 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 370 references related to the bh FLIM systems. As supplementary literature we recommend the bh TCSPC Handbook , which presents additional 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 continuously 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 of its detection. The recording process builds up a photon distribution over these parameters, see Fig. 3. 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. 3: Principle of TCSPC FLIM
The recording process delivers a near-ideal photon efficiency, excellent time resolution, and is independent of the speed of the scanner. 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 mosaics of FLIM images, multi-wavelength FLIM images, images of physiological effects occurring in the sample, and to record simultaneously fluorescence and phosphorescence lifetime images.
The principle of the scanner is shown in Fig. 4. Two laser beams are coupled into the scanner. They are combined by a beam combiner, pass the main beamsplitter, and are deflected by the scan mirrors. The scan lens sends the beam down the microscope beam path in a way that the scan mirror axis is projected into the back aperture of the microscope lens. The motion of the scan mirrors causes a variable tilt of the beam in the plane of the microscope lens. The laser is thus scanning an image area in the focal plane of the microscope lens. The scanning can be very fast - the line time can be as short as a millisecond, an entire frame can be scanned in less than a second.
The fluorescence light is collected back through the microscope lens, passes the scan lens, and is again reflected at the scan mirrors. The reflected beam is stationary, independently of the motion of the scan mirrors. It is separated into two spectral or polarisation components, and projected into confocal pinholes. The light signals passing the pinholes are filtered spectrally, and sent to the detectors. Only light from the excited spot in the focal plane of the microscope lens reaches the detectors. The result is a clear image from a defined depth inside the sample, without out-of-focus blur and lateral scattering.
Fig. 4: Principle of confocal scanning. Two lasers and two detection channels.
The DCS-120 MP version uses two-photon excitation by a titanium-sapphire laser. Due to the nonlinear nature of the two-photon process, excitation occurs only in a confined layer around the focal plane of the microscope lens. Two photon excitation has several advantages over one-photon excitation: First, the laser wavelength is in the NIR, where absorption and scattering coefficients are low. Consequently, deep layers of the sample can be reached. Second, fluorophores with excitation wavelengths in the UV can be reached without the need of UV optics. Third, since excitation occurs only in the focal plane, photochemical effects in the sample are reduced. A fourth advantage is that light scattered on the way out of the sample can efficiently be recorded without impairing the image quality.
Two-photon excitation occurs only in a thin layer around the focal plane of the microscope. Therefore, no pinhole is needed to suppress the detection of out-of focus fluorescence. Consequently, there is no need to send the fluorescence light all the way back through the scanner. Instead, the fluorescence is split from the excitation directly behind the microscope lens, and directly send to the detectors. The result is that even photons scattered on the way out of the sample have a chance to reach the detectors. The fact that scattered photons are detected does not impair the image quality - the data acquisition system automatically assigns them to the x-y position of the laser beam, not to the position where they left the sample. The result is high image quality and high detection efficiency from deep sample layers. The principle is shown in Fig. 5, for details please see section ‘DCS‑120 MP Multiphoton Excitation System’, page 191.
Fig. 5: Scanning with 2-photon excitation. Non-descanned detectors shown on the right.
On-photon excitation, two-photon excitation and descanned and non-descanned detectors can be combined in one system.
The DCS‑120 FLIM systems use the bh 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 [42, 68, 446].
The main panel of the SPCM data acquisition software is configurable by the user . Configurations for different modes of the DCS system are shown in Fig. 6. 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 . Frequently used operation modes and user interface configurations are selected from a panel of predefined setups.
Fig. 6: SPCM software panel. Top left to bottom right: FLIM with two detector channels, multi-spectral FLIM, combined fluorescence / phosphorescence lifetime imaging (FLIM/PLIM), fluorescence correlation (FCS), Z-Stack FLIM, excitation-wavelength multiplexed FLIM
Frequently used instrument configurations are stored in a ‘Predefined Setup’ panel. Changing between the different configurations and user interfaces is just a matter of a single mouse click, see Fig. 7.
Fig. 7: Changing between different instrument configurations: The DCS-120 system switches from a FLIM configuration into an FCS configuration by a simple mouse click
The scanner control is fully integrated in the SPCM data acquisition software. The zoom factor and the position of the scan area can be adjusted via the scanner control panel or via the cursors of the display window. Changes in the scan parameters are executed online, without stopping the scan. 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.
Fig. 8: Interactive scanner control
Depending on the frame format and the zoom factor, the DCS-120 scanner control automatically selects the maximum speed of the scanner. The scanner thus always runs at high pixel rate, resulting in fast acquisition, minimum triplet excitation, and minimum photobleaching.
Fig. 9: Automatic selection of scan speed
Even the slightest misalignment of the pinhole of a confocal system has a large impact on the sensitivity and image resolution. Therefore the DCS scan head has piezo-driven pinhole alignment in x, y, and z. Alignment can be done at any time, by simply maximizing the image intensity in the preview mode of the DCS system. Thus, the best possible images can be obtained for every laser wavelength, objective lens, and sample configuration.
Fig. 10: Effect of pinhole alignment on the image. Left to right: Differences between a slightly misaligned system, a system with near-perfect alignment, and a system with perfect alignment.
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. 11.
Fig. 11: SPCM software in fast preview mode, display rate one image per second.
The DCS-120 uses fast beam scanning by galvanometer mirrors. A complete frame is scanned within a time from 100 ms to a few seconds, with pixel dwell times down to one microsecond.
Compared with sample scanning, beam scanning is not only much faster, it avoids also induction of cell motion by exerting dynamic forces on the sample. Moreover, live cell imaging requires a fast preview function for fluorescence images for sample positioning and focusing. This can only be provided if the beam is scanned at a high frame rate. With its fast scanner and its multi-dimensional TCSPC process the DCS system achieves surprisingly short acquisition times, see Fig. 12.
Fig. 12: FLIM images recorded within 5 seconds acquisition time. 256 x 256 pixels (left) and 512 x 512 pixels (right), both with 256 time channels.
Fast scanning is also the basis of recording fast FLIM time series. With the DCS‑120 time-series can be recorded as fast as two images per second . An example is shown in Fig. 13.
Fig. 13: Bacteria in motion. Autofluorescence, acquisition speed 2 images per second, scan speed 6 frames per second
Starting from Version 9.72 SPCM software the DCS-120 system is able to display lifetime images online, both during the accumulation of FLIM data and for the individual steps of a fast image sequence . Lifetime images can be displayed at images rates as fast as 10 images per second. 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. Importantly, it does not require to reduce the time resolution (time channels per pixel) to obtain high calculation speed. 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.
Fig. 14: 256x256-pixel images obtained by the online FLLIM display function. Acquisition time 0.2s, 0.5s, and 2s.
The bh HPM‑100‑40 GaAsP hybrid detectors of the DCS‑120 combine SPAD-like sensitivity with the large active area of a PMT . The large area avoids any alignment problems, and allows light to be efficiently collected through large pinholes, see Fig. 15, and from the non-descanned beam path of the DCS-120 MP system . 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.
Fig. 15: Fluorescence lifetime images recorded with an HPM‑100-40 hybrid detector (left) and with an id‑100‑50 SPAD (right). Images and decay functions at selected cursor position.
The DCS-120 system can be equipped with the new ultra-high speed HPM‑100‑06 and -07 hybrid detectors. The time resolution (IRF width) of these detectors is less than 20 ps, full width at half maximum . Despite their slightly lower quantum efficiency these detectors deliver unprecedented accuracy for amplitudes and lifetimes of fast decay components of multi-exponential decay functions. The main application is metabolic imaging, where lifetimes and amplitude ratios of the decay components of NAD(P)H must be determined . To take full advantage of the detector speed the DCS-MP (two-photon excitation) system should be used for such applications. However, also with bh ps diode lasers an overall system response of 38 ps can be obtained.
Fig. 16: Left to right: IRF of HPM-100-06, 2-photon NADH image recorded with HPM-100-06, decay curve in selected pixel. The detector is so fast that the rise of the fluorescence signal is almost instantaneous, resulting in an extremely stable fit of fast decay components.
The control of the Ti:Sa laser and the AOM (acousto-optical modulator) of DCS-120 MP Multiphoton systems is integrated in the SPCM software. Both the laser wavelength and the laser power are controlled from the ‘Ti:Sa Laser and AOM Control’ panel of the software. The AOM is automatically tuned to the same wavelength as the laser. Please see page 110 for details.
Fig. 17: Ti:Sa Laser and AOM control panel
DCS-MP systems are available with NDD (non-descanned) optics and NDD detectors. The NDD optics efficiently transfers fluorescence light to the detectors, even if the photons are scattered on the way out of the sample. NDD thus allows the user to fully exploit the deep-tissue imaging capability of multiphoton excitation. The detectors for the NDD ports are the same as for the confocal ports. DCS systems with two TCSPC channels can be equipped with two non-descanned and two confocal detectors, either pair being active at a time.
With its two detection channels the DCS‑120 system records in two wavelength intervals simultaneously. The signals are detected by separate detectors and processed by separate TCSPC modules. Compared to a FLIM system with one TCSPC channel and a router, more than twice the photon rate can be processed. There is no intensity or lifetime crosstalk due to counting loss or pile up [58, 68]. Even if one channel overloads the other one is still able to produce correct data.
Fig. 18: Dual-wavelength detection. BPAE cells stained with Alexa 488 phalloidin and Mito Tracker Red. Left: 484 nm to 560 nm. Right: 590 nm to 650 nm.
The two ps-diode lasers of the DCS‑120 system can be multiplexed on a pixel-by-pixel, line-by-line, or frame-by-frame basis. With the two detection channels of the DCS system, images for three or four combinations of excitation and emission wavelength are obtained. An example is shown in Fig. 19.
Fig. 19: Excitation wavelength multiplexing, 405 nm and 473 nm. Detection wavelength 432 nm to 510 nm and 510 nm to 550 nm. Mouse kidney section, stained with Alexa 488 WGA, Alexa 568 phalloidin, and DAPI.
In combination with the ultra-fast HPM-100-06 and -07 detectors, the DCS-120 MP multiphoton system achieves an instrument response function (IRF) of less than 20 ps FWHM . 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 . An NADH FLIM image recorded with the DCS-120 MP using an HPM-100-06 is shown in Fig. 20. Please see ‘NADH FLIM with Ultra-Fast Detectors’, page 202.
Fig. 20: Left: NADH Lifetime image, amplitude-weighted lifetime of double-exponential fit. Right: Decay curve in selected spot, 9x9 pixel area. DCS-120 MP with HPM-100-06 detector and SPC-160 TCSPC/FLIM module, FLIM data format 512x512 pixels, 1024 time channels, time-channel width 12ps.
With 64 bit SPCM software pixel numbers can be increased to 2048 x 2048 pixels, with a temporal resolution of 256 time channels. Two such images are recorded simultaneously in different wavelength channels. Fig. 21 and Fig. 22 (facing page) show an example.
Fig. 21: BPAE sample (Invitrogen) scanned with 2048 x 2048 pixels. Green channel, 485 to 560 nm
Large pixel numbers are not only important for tissue imaging. They are also useful in cases when a large number of cells have to be investigated and the FLIM results to be compared. Megapixel FLIM records images of many cells simultaneously, and under identical environment conditions. Moreover, the data are analysed in a single analysis run, with identical IRFs and fit parameters. The results are therefore exactly comparable for all cells in the image area.
Fig. 22: BPAE sample (Invitrogen), scanned with 2048 x 2048 pixels. Red channel, 560 to 650 nm
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). An example of an x-y mosaic is shown in Fig. 23. 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. 23: 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. DCS‑120 MP multiphoton system with motorised sample stage.
With the bh multispectral FLIM detectors the DCS‑120 records FLIM simultaneously in 16 wavelength channels [52, 60, 68]. The images are recorded by a multi-dimensional TCSPC process which uses the wavelength of the photons as a coordinate of the photon distribution. There is no time gating, no wavelength scanning and, consequently, no loss of photons in this process. The system thus reaches near-ideal recording efficiency. Moreover, dynamic effects in the sample or photobleaching do not cause distortions in the spectra or decay functions. Multi-wavelength FLIM got an additional push from the new 64-bit SPCM software, and from the introduction of a highly efficient GaAsP multi-wavelength detector. 64-bit software works with enormously large photon distributions, and the GaAsP detector delivers the efficiency to fill them with photons. As a result, images in 16 wavelength channels can be recorded at a resolution of 512x512 pixels and 256 time channels. An example is shown in Fig. 24.
Fig. 24: Multi-wavelength FLIM with the bh MW-FLIM GaAsP 16-channel detector. 16 images with 512 x 512 pixels and 256 time channels were recorded simultaneously. Wavelength from upper left to lower right, 490 nm to 690 nm, 12.5 nm per image. DCS‑120 confocal scanner, Zeiss Axio Observer microscope, x20 NA=0.5 air lens.
Fig. 25 demonstrates the true spatial resolution of the data. Images from two wavelength channels, 502 nm and 565 nm, were selected form the data shown Fig. 24, and displayed at larger scale and with individually adjusted lifetime ranges. With 512x512 pixels and 256 time channels, the spatial and temporal resolution of the individual images is comparable with what previously could be reached for FLIM at a single wavelength. Decay curves for selected pixels of the images are shown in Fig. 26.
Fig. 25: Two images from the array shown in Fig. 24, displayed in larger scale and with individually adjusted lifetime range. Wavelength channels 502 nm (left) and 565 nm (right). The images have 512 x 512 pixels and 256 time channels.
Fig. 26: Decay curves at selected pixel position in the images shown above. Blue dots: Photon numbers in the time channels. Red curve: Fit with a double-exponential model.
In combination with the Zeiss Axio Observer Z1 microscope the DCS‑120 system is able to record z-stacks of FLIM images. The sample is continuously scanned. For each plane, a FLIM image is acquired for a specified ‘collection time’. Then the data are saved in a file, the microscope is commanded to step to the next plane, and the next image is acquired. The procedure continues for a specified number of Z planes. A Z stack of autofluorescence images taken at a water flee is shown in Fig. 27.
Fig. 27: Z stack recording, part of a water flee, autofluorescence. 15 steps in Z, step width 4 um.
Z Stacks of FLIM images can be recorded by the Mosaic FLIM function of the 64 bit SPCM software. 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. 28: FLIM Z-stack, recorded by Mosaic FLIM. Pig skin, autofluorescence. 16 planes, 0 to 30 um from top of the tissue. Each element of the FLIM mosaic has 512x512 pixels and 256 time channels per pixel.
The DCS-120 WB wideband version can be used with tuneable excitation. Images obtained with a Toptica Ichrome laser  are shown in Fig. 29.
Fig. 29: Tuneable excitation with DCS-120 WB and Toptica Ichrome laser. Left to right: Excitation 488 nm emission 525±15 nm, excitation 488 nm emission 620±30 nm, and excitation 580 nm emission 620±30 nm.
The DCS‑120 WB version is able to record lifetime images with near-infrared fluorophores. An image of a pig skin sample incubated with 3,3’-diethylthiatricarbocyanine is shown in Fig. 30.
Fig. 30: Near-Infrared FLIM. Pig skin sample stained with 3,3’-diethylthiatricarbocyanine, detection wavelength, detection wavelength from 780 nm to 900 nm.
A two-photon FLIM image of a convallaria sample recorded by the DCS-120MP system is shown in Fig. 31.
Fig. 31: Two-photon FLIM image of a convallaria sample. 2048 x 2048 pixels, 256 time channels
The DCS-120MP uses non-descanned detection . Combined with the large penetration depth of the NIR excitation clear images from deep tissue layers are obtained, see Fig. 32.
Fig. 32: Pig skin, autofluorescence, image in different depth in the sample. Amplitude-weighted lifetime of triple-exponential decay model. Excitation 805 nm, 512x512 pixels, 256 time channels. Zeiss Axio Observer Z1, Water C apochromate NA=1.2, non-descanned detection, HPM‑100-40 hybrid detector.
Time-series FLIM by the traditional record-and-save procedure is available for all DCS‑120 system versions. With the 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. 33. The fluorescence lifetime of the chloroplasts changes due to the Kautski effect induced by the illumination.
Fig. 33: Time-series FLIM, 2 images per second. Chloroplasts in a leaf, the fluorescence lifetime of the chlorophyll decreases with the time of exposure.
SPCM 64-bit software versions later than 2014 have a ‘Mosaic Imaging’ function implemented. For time-series recording, subsequent frames of the scan are recorded into subsequent elements of the mosaic. The sequence can be repeated and accumulated [68, 70]. The time per mosaic element can be as short as a single frame, which can be less than 100 ms. Another advantage is that the entire array can b analysed in a single SPCImage data analysis run. Fig. 34 shows the change of the lifetime of chlorophyll in plant tissue with the illumination.
Fig. 34: Time series of chloroplasts in a leaf recorded by Mosaic Imaging. 64 mosaic elements, each 128x128 pixels, 256 time channels. Scan time per element 1s. Experiment time from lower left to upper right. Amplitude-weighted lifetime of double-exponential decay.
Phosphorescence and fluorescence lifetime images are recorded simultaneously by bh’s proprietary FLIM/PLIM technique. The technique is based on modulating a ps diode laser synchronously with the pixel clock of the scanner [64, 68]. FLIM is recorded during the ‘On’ time, PLIM during the ‘Off’ time of the laser. The SPCM software delivers separate images for the fluorescence and the phosphorescence which are then analysed with SPCImage FLIM/PLIM analysis software. Please see page 283 for details.
Currently, there is increasing interest in PLIM for background-free recording and for oxygen sensing [18, 19, 147, 149, 237, 354, 469]. In these applications, the bh technique delivers a far better sensitivity than PLIM techniques based on single-pulse excitation. The real advantage of the FLIM/PLIM technique used in the DCS‑120 is, however, that FLIM and PLIM are obtained simultaneously. It is thus possible to record metabolic information via FLIM of the NADH and FAD fluorescence, and simultaneously map the oxygen concentration via PLIM. An example is shown in Fig. 268.
Fig. 35: Yeast cells stained with (2,2’-bipyridyl) dichlororuthenium (II) hexahydrate. FLIM and PLIM image, decay curves in selected spots.
PLIM can also be interesting for the investigation the luminescence properties of inorganic compounds. The emission can come almost entirely from energy levels of extremely long lifetime. An example is shown in Fig. 36.
Fig. 36: Phosphorescence lifetime imaging of a phosphor of a cathode-ray tube. Left: Lifetime image. Right: Phosphorescence decay curve at selected position within the image
FLITS records transient effects in the fluorescence lifetime of a sample along a one-dimensional scan. The technique is based on building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses, and the experiment time after a stimulation of the sample. 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 [65, 68].
Fig. 37: FLITS of chloroplasts in a grass blade, change of fluorescence lifetime after start of illumination. Left: Non-photochemical transient, transient resolution 60 ms. Right: Photochemical transient. Triggered accumulation, transient resolution 1 ms.
The technique has recently been introduced to record Ca++ transients in live neurons, see [67, 177].
The DCS MACRO version of the DCS system scans objects directly in the focal plane of the scanner. Objects up to a size of 12 mm can be imaged at high resolution.
Fig. 38: Lifetime Images of a Coccinella beetle, recorded by the DCS-120 Macro system.
The image area of the DCS MACRO can be extended by a motorised sample stage. Mosaic FLIM is used to record images of objects with dimensions in the 10-cm range, see Fig. 39.
Fig. 39: Mosaic FLIM image of a $20 bill. Combination of beam scanning with sample stepping by motor stage.
The DCS-120 Macro can be combined with endoscopes. Optical details are described in  and at page 207 of this handbook. Images of (benign) human skin lesions are shown in Fig. 326.
Fig. 40: Basal cell papilloma (left) and a keratomic lesion (right), scanned in vivo through a rigid endoscope. Excitation wavelength 405 nm, detection wavelength 480 to 560 nm. Excitation power 50 µW, acquisition time 10 seconds.
The bh GaAsP hybrid detectors deliver highly efficient FCS. Because the detectors are free of afterpulsing there is no afterpulsing peak in autocorrelation data . Thus, accurate diffusion times and molecule parameters are obtained from a single detector. Compared to cross-correlation of split signals, correlation of single-detector signals yields a four-fold increase in correlation events. The result is a substantial improvement in the SNR of FCS recordings.
Fig. 41: FCS curve recorded by a single HPM-100 detector. There is no afterpulsing peak, and the efficiency is four times higher than for commonly used cross-correlation of split light signals. Right: Dual-colour FCS, autocorrelation blue and red, cross-correlation green. Online fit with FCS procedures of SPCM data acquisition software.
The bh DCS-120 FLIM systems use bh SPCImage data analysis software , see page 283 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. 403. 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. 42: 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. 468.
Fig. 43: 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. 469.
Fig. 44: 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. 45.
Fig. 45: 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 [142, 143]. 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. 46. 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. 47. Please see chapter 'SPCImage data analysis'.
Fig. 46: 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. 47: 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. 48: bh Burst Analyzer software for single-molecule data analysis
The bh FLIM technique makes use of the special properties of high-repetition rate optical signals detected by a high-gain detector, see Fig. 49. Fluorescence of a sample is excited by a laser of high 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. 49: 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. 49 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 the photon density as a function of the time after the excitation pulses. Fig. 49 shows that the detection of a photon in a particular signal period is a relatively unlikely event [58, 68]. 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. 50.
Fig. 50: Principle of time-correlated single photon counting
The principle shown in Fig. 50 is the classic principle of time-correlated single photon counting [346, 507]. 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 [102, 103] 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 [50, 55], the wavelength [52, 54, 60], 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 [58, 68]. The result is a multi-dimensional photon distribution as shown in Fig. 51.
Fig. 51: 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 [55, 58, 68, 60].
The basic architecture of a TCSPC FLIM device is shown in Fig. 52. 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. 52: 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 can be 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. 52, 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. 53. 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. 53: 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.
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 [58, 68]. Although an elegant solution for multi-wavelength FLIM (see Fig. 55), routing does not increase the counting capability and the throughput of a FLIM system. bh FLIM systems with two detector channels are therefore not built with routing but with fully parallel TCSPC channels, see Fig. 54. Parallel systems deliver high throughput rates and short acquisition times. Another advantage is that the channels are independent. If one channel overload the other one still delivers correct data. Dual-channel systems have become standard for the bh FLIM systems [29, 68]. Four-channel parallel systems are easily feasible [56, 68], and an eight-channel parallel system has been demonstrated .
Fig. 54: 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. 52, page 30, can be extended to simultaneously detect in a large number of wavelength channels [52, 60, 68]. As shown in Fig. 49, 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 [58, 68, 60, 81, 86, 87]. 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. 55.
Fig. 55: 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.
In the DCS‑120 system FLIM can be combined with excitation wavelength multiplexing. The principle is shown in Fig. 56. Excitation at different wavelength is achieved by multiplexing (on/off switching) of the two ps diode lasers of the DCS system, or by switching the wavelength of the acousto-optical filter (AOTF) of a super-continuum laser. A multiplexing signal that indicates which laser (or laser wavelength) is active is fed into the routing input of the TCSPC module. The signal represents the excitation wavelength. The TCSPC module is running the normal FLIM acquisition process: It builds up a photon distribution over the coordinates of the scan area, the photon times, and the excitation wavelength. The result is a data set that contains separate images for the individual excitation wavelengths. It can also be interpreted as a single image that has several decay curves for different excitation wavelengths in its pixels. The multiplexing of the lasers is controlled by the GVD‑120 scan controller of the DCS system. The wavelength switching is synchronised with the pixels, lines, or frames of the scan and can thus be made very fast.
Fig. 56: FLIM with laser wavelength multiplexing
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. 57.
Fig. 57: Mosaic FLIM. The TCSPC system records a series of lifetime images in to a single, large photon distribution.
Mosaic FLIM is has originally be developed to record image of large sample areas by the bh FLIM systems in combination with the ‘Tile Imaging’ function of the Zeiss LSMs . It turned out, however, that Mosaic FLIM is also an excellent way to obtain FLIM Z stacks and fast FLIM time series. Mosaic 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. 236 in section ‘Time-Series Recording by Mosaic FLIM’.
Simultaneous FLIM and PLIM is based on on-off modulation of the excitation 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 167.
Fig. 58: Simultaneous FLIM and PLIM
The bh FLIM systems are also able to record FCS. The principle is shown in Fig. 59. 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. 59: FCS Recording
In a conventional fluorescence microscope fluorescence is excited throughout the full depth of the sample. The sharp image seen in the focal plane is therefore surrounded by out-of-focus haze from above and below this plane. A confocal microscope solves this problem by scanning the sample with a focused laser beam and detecting the signal only from a small volume element around the illuminated spot [129, 140, 331, 359].
However, scanning does more than that - it also avoids that light scattered in the sample causes crosstalk between the pixels. Surprisingly, this is rarely mentioned in the microscopy literature. Consider an imaging system that illuminates the entire sample at once and detects an image from the focal plane by a camera. In all pixels of this image the camera will detect scattered light not only from this pixel but from all other pixels in the illuminated area. A scanning system - scanning the sample by a focused laser beam and detecting light only from the excited spot - records only light from the current pixel. In other words, already scanning alone - without the help of confocal detection - improves the image quality, see ‘The Scanning Advantage’ page 212. The difference between wide-field images and scan images can be dramatic, see Fig. 330, page 213.
The principle of confocal detection is shown in Fig. 60. The basis is a pinhole in the upper focal plane of the microscope, see Fig. 60, left. Consider a point source located in the focal plane in the sample and on the optical axis. The light from this point (shown red) will be focused into a diffraction-limited spot in the upper focal plane of the microscope. A pinhole placed in this plane and on the optical axis will transmit the light from this particular point of the sample [129, 140, 331, 359, 497]. Light coming from points above or below the focal plane (shown light red) is focused into a plane below or above the pinhole. As a result, only a small fraction of light from outside the focal plane light can pass. The pinhole thus suppresses light from sample planes that are not exactly in the focus of the microscope lens.
Fig. 60: Left: Suppression of out-of-focus light by a confocal pinhole. Middle: Scanning system. Right: Separation of excitation and fluorescence light and projection into pinhole.
A system as the one shown in Fig. 60, left, does, of course, not deliver an image of the sample. To obtain an image the point in which the light is excited and detected must be scanned over the sample. Scanning can be achieved by moving the sample or by moving the light beam. Moving the sample has the merit that the point from which the system detects is always on the optical axis. The drawback is, however, that piezo scanning is slow, and exerts forces to the sample. Sample scanning is therefore not used in laser scanning microscopy for biological objects. Instead, confocal microscopes use optical beam scanning by galvanometer-driven mirrors (often called ‘galvos’). The principle is shown in Fig. 60, middle. The collimated laser beam is deflected by the galvanometer mirrors. It then passes the ‘scan lens’. This lens is the equivalent of the eyepiece of a conventional microscope. It focuses the laser into the upper focal plane, which is conjugate with the focal plane of the microscope objective lens in the sample. Thus, the angular motion of the galvos scans a focused spot of laser light over the focal plane in the sample.
Fluorescence light emitted in the focal plane is returned via the same beam path. After being reflected at the galvanometer mirrors the fluorescence signal from the focal plane forms a stationary, collimated beam of light. It is separated from the laser beam by a dichroic mirror, and focused into a stationary pinhole, see Fig. 60, right. Only light that passes the pinhole is transferred to the detector.
In the DCS system, the light passing the pinhole can further be split into two components of different wavelength or different polarisation. Each component passes additional filters to exactly define the detection wavelength intervals and to suppress residual laser light. As a result, clean images in a defined wavelength interval an from a defined plane in the sample are recorded. Examples are shown in Fig. 21 and Fig. 22 , page 12 and 13.
The internal design of the DCS‑120 scan head is shown in Fig. 61, a schematic drawing in Fig. 62.
Fig. 61: Internal Optics of the DCS‑120 scan head
Fig. 62: Schematics of the DCS‑120 scan head with filter characteristics of standard filters and dichroics
Diode lasers and other lasers for one-photon excitation are connected to the scan head via single-mode optical fibres. The DCS‑120 uses special fibres that have collimators at their outputs, see Fig. 63, left. The collimators are inserted into fibre manipulators  which allow the lateral position and the angular alignment to be changed in the sub-µm range see Fig. 63, middle. The adjustments are used to centre the laser beam on the back aperture of the microscope lens and to align the image of the excited spot of the sample with the pinholes. The benefit of this design is that slight realignments are possible without opening the scan head (see also Fig. 484, page 339). For multiphoton excitation, the beam of a titanium-sapphire laser is coupled into the scanner by sending it through the centre of the fibre manipulators, see Fig. 63, right.
Fig. 63: Coupling of the lasers into the DCS‑120 scan head. Lasers for one-photon excitation are coupled via single-mode fibres that deliver a collimated beam (left). The fibres are attached to the scanner via fibre manipulators (middle). For multiphoton excitation the beam of the titanium-sapphire laser is sent into the scanner directly (right)
Both laser input channels have neutral-density filter wheels. By these wheels the laser intensity can be varied within a range of more than 1:100. Both laser beams are combined via a dichroic mirror. A conventional mirror sends the combined laser beams down to the main dichroic beamsplitter assembly.
The main dichroic assembly consists of a solid block that holds a conventional mirror and the dichroic. A number of different main dichroics are available for the DCS‑120. For details please see ‘Filter Characteristics’, page 357. Customer specific versions can be made on request. There is also a ‘wideband version’ that allows the scanner to be used with a large number of different laser wavelengths without the need of replacing the main beamsplitter. Please see ‘DCS-120 WB Wideband Version’, page 183. The main dichroic assembly is shown in Fig. 64.
Fig. 64: Main dichroic beamsplitter assembly
After being reflected at the main dichroic the laser beam is deflected by the galvanometer mirrors. The galvanometer mirrors are shown in Fig. 65. The mirrors have small size and, consequently, small moments of inertia. Moreover, optimised electronic control and optimised flyback trajectories minimise mechanical resonance. The scanner thus achieves line times down to 0.6 ms and pixel times down to 0.6 µs, see page 106.
Fig. 65: Galvanometer mirrors of the DCS-120 scan head
The excitation light leaves the scanner through the scan lens. The lens focuses the laser into the upper focal plane of the microscope and simultaneously projects the galvo rotation axis on the back aperture of the microscope lens (see Fig. 60, right). To bring the image plane of the scan lens into coincidence with the upper focal plane of the microscope the complete scanner can be moved in and out with respect to the microscope.
The fluorescence light returned from the sample enters the scanner back through the scan lens. The scan lens collimates the light into a narrow beam. The motion of the beam is ‘descanned’ by reflection at the galvanometer mirrors. The descanned beam passes the main dichroic. The result is a stationary beam of fluorescence light that is largely free of the laser light.
The beam of fluorescence light passes a telescope that adds additional magnification to the optical path from the sample to the pinholes, see Fig. 66.
Fig. 66: Function of the telescope in the fluorescence light path. Together with the scan lens the telescope adds additional magnification to the system so that the physical pinhole sizes for diffraction-limited operation are in range from 0.25 to a few mm. Not to scale, aperture of light bundles exaggerated.
The advantage of this additional magnification is that the physical size of the pinholes becomes much larger than it would be in the upper image plane of the microscope. The alignment of the optical elements behind the telescope is therefore not critical.
Before the beam enters the pinholes it is split into two components by the secondary beamsplitter. This is a wheel containing two (in recent scanners three) dichroics, a polarising beamsplitter, a mirror, and a glass plate. Thus, the light can be split into two different wavelength components, components of 0° and 90° polarisation, or the full signal can be sent into either detection channel.
The pinholes of both channels are located in separate pinhole wheels. Each wheel contains 11 pinholes from 0.25 to 5 mm diameter. With the optical elements used in the DCS‑120 scan head the magnification, M, from the sample plane into the pinhole is:
(Mlens = Magnification of objective lens)
The diameter of the central peak of the Airy disc in the pinhole is
with λ = Detection (vacuum) wavelength, NA = Numerical aperture of objective lens, Mlens = Magnification of objective lens.
Thus, for the commonly used objective lenses the physical pinhole diameters from 0.25 to 5 mm correspond to approximately 0.5 to 10 Airy disc diameters in the focal plane of the microscope lens.
Standard DCS-120 data acquisition systems come as compact ‘Simple-Tau’ TCSPC devices . In the standard configuration, the systems contain two SPC‑150 TCSPC FLIM modules, a GVD‑120 scan controller card, and a DCC‑100 detector controller. A photo is shown in Fig. 67, left. The Simple-Tau systems are available with large screens and external keyboards. The ‘Simple-Tau 152 LS’ with a 27” screen is shown in Fig. 67, right.
Fig. 67: Simple-Tau 152 system with two TCSPC FLIM channels.
The same data acquisition system is available on a PC basis, see Fig. 68. These ‘Power Tau’ systems provide space for additional data storage media, and for additional TCSPC channels.
Fig. 68: Power-Tau 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 numbers of pixel and time channel. 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 67, the data analysis in section ‘SPCImage Data Analysis Software’, page 283.
The DCS‑120 systems use SPC‑150, SPC‑150N, or SPC‑160 TCSPC FLIM modules . The SPC‑150 and the SPC-150N are shown in Fig. 69.
Fig. 69: SPC‑150 (left) and SPC-150N module (right)
These modules fill only one computer slot, so that two of them can be placed in the Simple-Tau extension box, and four of them in the Power Tau.
The detectors of the bh FLIM systems are controlled via a bh DCC‑100 detector controller card [25, 68]. The card supplies the power to the detectors, drives the coolers of actively cooled detector modules, provides software control of the detector gain and shuts down the detectors in case of overload. The DCC‑100 card is shown in Fig. 70.
Fig. 70: DCC‑100 detector controller card. Control panel shown on the right.
The galvanometer mirrors of the DCS‑120 scan head are controlled via a bh GVD-120 scan controller card. The GVD‑120 generates the ramp signals for the x and y deflection. The signals are generated by the hardware of the GVD module, the software is only used to load different waveforms or scan parameters in the scan controller. The waveforms are therefore independent of the computer speed and of software reaction times. The DCS‑120 uses a cycloid waveform for the flyback portion of the signals (see Fig. 71, right). This minimises mechanical resonances and thus allows the DCS-120 to be operated at extremely high scan rates (see Table 1 and Table 2, page 107).
The GVD‑120 also generates the scan synchronisation pulses for bh TCSPC modules, and the beam blanking, multiplexing, and intensity control signals for the excitation lasers. The GVD‑120 card is shown in Fig. 71, left, a typical scan waveform in Fig. 71, right.
Fig. 71: GVD‑120 scan controller card (left) an scan waveforms (right)
Driving the galvo mirrors at high scan rates requires more electrical power electrical power than a PC card can deliver. The signals from the GVD‑120 are therefore amplified by a GDA‑120 dual channel galvo amplifier. The amplifier box is shown in Fig. 72.
Fig. 72: GDA‑120 dual-channel driver amplifier for galvanometer scanner
The DCS‑120 system can be used with a number of different detectors. The detectors are attached to the back of the scan head. The adapters of the detectors insert directly into the detector ports and are locked with a set screw. All FLIM detectors are controlled via the DCC-100 detector controller card, see Fig. 70.
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 [63, 68]. The GaAsP cathode detects from 350 to 700 nm, and 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 tube has been integrated in a common housing with the high-voltage generators and a low-noise preamplifier, see Fig. 73, 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. 73, middle and right.
Fig. 73: 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.
When used as a confocal detector, the HPM detector delivers a better sensitivity than even a fast single-photon avalanche photodiode (SPAD). It does not require accurate alignment, and can be used efficiently even with large pinhole diameters. A comparison is shown in Fig. 15.
Fig. 74: Fluorescence lifetime images recorded with an HPM‑100‑40 (left) and with an id‑100‑50 SPAD (right), pinhole 3 AU. Images with decay functions at selected cursor position. The HPM collected twice the number of photons
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. 75, left. Fluorescence decay parameters can by derived from a given number of photons at a higher accuracy [35, 63, 68, 259].
FCS measurements are free of the typical afterpulsing peak, see Fig. 75, 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. 75: 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 [29, 68]. Please see section ‘DCS‑120 MP Multiphoton Excitation System’, page 191.
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 187 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. 76
Fig. 76: 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 section ‘Autofluorescence FLIM’, 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.
Before the introduction of the HPM‑100‑40 hybrid detectors, the PMC-100 detector was used in large quantities in the bh FLIM systems . The PMC‑100 is shown in Fig. 77.
Fig. 77: PMC‑100 detector
It contains a small PMT, a cooler, the PMT power supply, and the preamplifier in a compact housing. The PMC‑100 delivers an IFR of 150 ps FWHM. The standard version is the PMC‑100-1, with a useful spectral range from 330 to 780 nm. For detection in the NIR the PMC‑100-20 can be used. The -20 version is sensitive up to 860 nm. In the last years, the PMC detectors have almost entirely been replaced by hybrid detectors.
Until recently, the Hamamatsu R3809U MCP PMT  has been the ultimate in time resolution. Its instrument response function (IRF) has a width of less than 30 ps (FWHM) [58, 68]. Together with the pulse width of the ps diode laser an IRF width of 50 to 70 ps is obtained. With the introduction of ultra-fast hybrid detectors the R3809U has become second in time resolution. Nevertheless, the R3809U can be used in the DCS-120 system. With SPC-150N modules it achieves a time resolution (IRF width) of about 22 ps - better than reported in most application papers of the past years.
The R3809U needs a high-voltage power supply and a preamplifier. Fig. 78 shows the R3809U with a bh HVM power supply module attached and the bh HFAC‑26 preamplifier.
Fig. 78: Left: R3809U MCP PMT with HVM-100 power supply module. Right: HFAC-26 preamplifier
The H7422P-40 detector  is a conventional PMT with a GaAsP cathode. For may years it was the only detector that could be used both for FLIM and FCS measurements . The IRF width of the H7422P-40 is 250 to 350 ps, with a long tail and a secondary peak at about 1 ns [58, 68]. The H7422‑40 has been entirely replaced by the new HPM‑100‑40 hybrid detectors.
The MW FLIM detection system  detects the fluorescence simultaneously in 16 wavelength channels . The optical principle is shown in Fig. 79, 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 [58, 68]. 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. 79, right.
Fig. 79: 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.
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 [42, 68, 446]. Please see Fig. 24 and Fig. 25, page 15.
Standard DCS‑120 confocal systems use bh BDL-SMN or BDS-SM picosecond diode lasers . The lasers are available with wavelengths of 375 nm, 405 nm, 440 nm, 473 nm, 488 nm, 515 nm, 640 nm, 685 nm, or 785 nm. The complete driver electronics is integrated; the power supply comes from the DCS‑120 switch box. Both lasers are shown in Fig. 80.
Fig. 80: BDL‑SMN (left) and BDS-SM picosecond diode lasers
The lasers can be switched between picosecond pulse operation and CW operation. In the picosecond mode, pulse repetition rates of 20 MHz, 50 MHz, and 80 MHz can be selected. The pulse width is in the range of 30 ps to 100 ps, depending on the wavelength version. The power in the pulsed mode is between 0.3 and about 5 mW. In the CW mode an output power of up to 40 mW is available. This is much more power than usually required for FLIM. Typical pulse shapes for a few laser wavelengths are shown in Fig. 81.
Fig. 81: BDL-SMN laser, pulse shapes for four typical laser wavelengths. 80 MHz, power measured in free beam.
The optical power is stabilised by an internal regulation loop both in the pulsed mode and in the CW mode. The result is low optical noise, excellent power stability both in the CW and in the pulsed mode, and a strictly linear power control characteristic.
The BDL and BDS lasers have fast on/off (multiplexing) capability. Fast on/off switching is used to turn off the excitation during the flyback phases of the scanner, when the scanner is not running, and during the phosphorescence-detection phases of simultaneous FLIM/PLIM. Fast switching is also used to multiplex two lasers for quasi-simultaneous recording at two excitation and emission wavelengths.
The lasers are controlled via the GVD-120 scan controller card and the switch box of the DCS‑120 system. The GVD‑120 provides a power control signal, a beam blanking signal during the flyback of the scanner, and an on/off signal for laser multiplexing and phosphorescence measurement.
Super-Continuum lasers with acousto-optical filters can be tuned over the entire range from 420 nm to 800 nm. For FLIM/PLIM the lasers can be on/off modulated via the filter. We have tested different devices in combination with the DCS‑120 scan head and generally obtained good results [37, 38, 68]. However, there can be some pitfalls. The pulses are not always as short as expected, especially at shorter wavelength. We have found pulse widths of up to 300 ps in the range below 480 nm. This is too broad to determine accurate lifetimes for fast effects, such as the bound component of FAD, or the interacting donor component in FRET experiments. Also, we occasionally encountered broadband leakage of the acousto-optical filter. This is not a problem in scanners with a single-wavelength dichroic beamsplitter. But in systems with wideband beamsplitters (which are most desirable for a tuneable excitation source) the leakage can make FLIM impossible. The only solution is then to put a cleaning filter in the excitation beam path. This has to be replaced when a different wavelength is used which is, at least, inconvenient in a tuneable-excitation system.
Titanium-Sapphire lasers are used in the DCS‑120MP multiphoton system. Titanium-Sapphire lasers deliver femtosecond pulses in the NIR range. Due to the short pulse width and high peak power high efficiency of multiphoton excitation is obtained. The lasers are tuneable over a wide range. The NIR radiation penetrates deep into biological tissue, and the short pulse width is ideal for FLIM. With the ultra-fast HPM‑100-06 and -07 detectors a TCSPC instrument-response function of less than 20 ps FWHM is obtained. The combination of these features is the reason that more than 50% of all bh FLIM systems are two-photon systems. Ti:Sa lasers cannot be modulated or power-controlled electronically. Therefore the DCS-120 MP system uses an AOM (acousto-optical modulator) to control the intensity and to modulate the laser for FLIM/PLIM. The control of the laser and the AOM is fully integrated in the SPCM data acquisition software. Please see 'Ti:Sa Laser and AOM Control', page 110.
The DCS switch box distributes scan clock signals, routing signals, and detector and laser control signals in the system, and switches between different sources and destinations of these signals. It also provides trigger and marker inputs to the TCSPC modules for special FLIM modes and FLIM procedures. Moreover, it provides the power supply to the ps diode lasers, and allows the user to switch between different laser pulse repetition rates and CW operation. A photo of the DCS switch box is shown in Fig. 82.
Fig. 82: DCS switch box
bh provide a USB-controlled delay switch box for the Sync signal to the SPC modules. The box allows the user to electronically 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. 483. It is accessed via the ‘Sync’ settings in the main panel, or via the Sync section of the system parameter panel.
Fig. 83: 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.
This section gives condensed instructions for the use of the DCS system. It provides the minimum of information needed to record data in the basic FLIM modes. Please note that the DCS system can do much more than this, see chapters ‘SPCM Software’, ‘Advanced Techniques and Procedures’, and ‘Variants of the DCS‑120 System’. Moreover, there are many ways a user can influence the quality of the recorded data. We therefore recommend to take a look also into chapter ‘A Few Things FLIM Users Should Know’.
The DCS-120 scan head has mechanical control elements for the laser attenuators, the secondary beamsplitter, the pinholes, and the filters of the two detection channels. The control elements are shown in Fig. 84.
Fig. 84: Control elements of the scan head
The laser power can be changed optically by rotating the ND filter wheels at the inputs of the scanner (Fig. 85, left), or electronically by moving the laser power sliders in the scan control panel (Fig. 85, right).
Fig. 85: Laser power control
Optical power control (see Fig. 85, left) has the advantage that the pulse shape remains unchanged. Electronic power control changes not only the power but also the shape of the laser pulses. An example for a 473 nm BDL‑SMC laser is shown in Fig. 86. Due to the good fibre-coupling efficiency of the bh ps diode lasers there is normally enough power reserve to operate the laser at a power that yields best pulse shape. This is usually the case at about 50% of the available power, see Fig. 85, right. Under no circumstances the electronic power control should be changed between a FLIM measurement and a corresponding IRF recording.
Fig. 86: Pulse shape for different average power for a BDL‑473 SMC laser
The laser power to be applied to a specific sample depends on various parameters. On the one hand, it should be high enough to obtain a sufficiently high count rate to accumulate enough photons within a reasonable acquisition time. On the other hand, it should be low enough to avoid photobleaching or other changes in the sample. As a rule of thumb, the laser power should be adjusted to obtain a detector count rate of 105 to 106 photons per second.
The secondary beamsplitter splits the fluorescence signal into the two detection channels. The beamsplitter wheel has 7 positions, A to G. Different generations of scanners may have different configurations of the secondary beamsplitter wheel. In the standard configuration (shown in Fig. 84) the wheel contains two dichroic beamsplitters, A and B, a polarizing beamsplitter, C, a glass plate, D, and a mirror, E. Positions F and G are for user-specific beamsplitters. Please note that the filters in the detection beam path (see below) must fit to the selected secondary dichroic. Using filters that are contradicting the beamsplitter characteristics can result in a complete loss of the signal.
The pinhole wheels contain 11 pinholes from 0.25 to 5 mm in diameter, and a ‘closed’ position. For the commonly used objective lenses these diameters correspond to approximately 0.5 to 10 Airy disc diameters in the focal plane. The effect of the pinhole size on the recorded image is shown in Fig. 87. In a thick sample (upper row), the image intensity increases dramatically with the pinhole size. Simultaneously, the image definition decreases. The reason is that a large pinhole transmits photons from a larger depth interval. In a thin sample (lower row) the effect is less pronounced. Once the recording depth interval exceeds the thickness of the sample there is almost no further increase in image intensity. For normal FLIM experiments we recommend to start with a pinhole size of 1. If there is enough image intensity and you want to squeeze out the last bit of resolution, decrease the pinhole to 0.5. If the intensity is low and cannot be increased by more laser power, increase the pinhole size.
Fig. 87: Effect of the pinhole size on the recorded images. Left to right: Pinhole = 1, 2, 4. Thick sample (upper row) and thin sample (lower row).
Each detection channel has two positions to insert emission filter. The filters are contained in sliders which fit into corresponding slots of the scan head, see Fig. 88. Both bandpass and long-pass filters are delivered with the DCS‑120 scanner. For general characteristics of the standard filters please see Fig. 62. For detailed filter curves please see ‘Filter Characteristics’, page 357. Other filters and empty filter sliders are available on request.
Fig. 88: Emission filters. Left: Filter sliders. Right: Filter positions in DCS-120 scan head
Please note that filter sliders must be inserted in both filter slots of each detection channel to keep daylight out of the light path. If you want to remove a filter from the beam path without putting a filter in you can either pull the slider halfway out or insert an empty filter slider.
FLIM data acquisition is controlled by SPCM software, please see chapter ‘SPCM Software’, page 67 for a detailed description. The SPCM main panel in the FLIM configuration is shown in Fig. 89. It shows images of the two detection channels of the DCS system. In the lower part it displays status information, such as count rates, number of photons collected, and indicates whether a measurement is running and the laser is working.
Fig. 89: Main panel of the SPCM software
Setup parameters and user interface configurations for different FLIM data formats and different FLIM modes are selected from the ‘Predefined Setup’ panel in the upper right. For details, please see ‘User Interface’, page 68, and ‘Creating Predefined Setups’, page 119.
The SPCM software also controls the scanner, the lasers, the detectors, and other hardware components of the system. The control panels for the system components of the basic DCS-120 system are open in the lower right of Fig. 89.
The detector control panel is shown in Fig. 90. After system start-up the detectors are disabled (Fig. 90, left). They are activated by a click on the ‘Enable Outputs’ button. The panel with the detectors active is shown in Fig. 90, middle. The detector control panel after an overload shutdown is shown in Fig. 90, right. When an overload shutdown has occurred, remove the reason of the overload (usually too-high laser power or a turned-on microscope lamp), and click on the ‘Reset’ button. The detector then resumes normal operation, as shown in Fig. 90, middle.
Fig. 90: Detector control panel. Left: After system start, detectors are disabled. Middle: Detectors enabled. Right: After overload shutdown of Detector 1.
Under normal circumstances, there is no need to change the other detector parameters. Especially, do not attempt to change the sensitivity of the system by changing the detector gain. The DCS system records signals by photon counting. A change in the detector gain changes the amplitude of the single-photon pulses, not the amplitude of the recorded decay curves. Normally, the detector gain settings are locked to the values determined during the setup of the system. Please see ‘Detector Control’, page 113.
The scanner of the DCS-120 system is controlled via the panel shown in Fig. 91. The panel allows you to start and to stop the scanning and the measurement, run a fast preview for focusing and sample positioning, select the laser, define the pixel number of the scan, select the scan speed, and define the scan area.
A click on the ‘Preview’ button starts a fast series of preview images. The preview function is used to adjust the focus and the sample position in the microscope, adjust the laser power, and select the desired scan area. The scan area within the maximum field of view is shown as the white rectangle in the lower right. You can change it by changing the Zoom factor, by moving the ‘Offset’ sliders in the lower left, or by shifting the white rectangle with the mouse cursor. The effect on the images becomes immediately visible in the preview images.
Fig. 91: Scanner control panel. Left: Standard FLIM Mode, 512x512 pixels, scanning not started. Right: Preview mode running
When the preview is running the ‘Preview button is marked by yellow colour, see Fig. 91, right. The preview is stopped by clicking on the yellow preview button.
The scan for the final FLIM measurement is started by clicking on the ‘Start Scan’ button. This starts the scanning with the defined number of pixels, and also starts the acquisition of the FLIM data. The scanner panel in the FLIM acquisition state is shown in Fig. 92.
With the display parameter setup suggested in section ‘System Parameters for Basic FLIM Experiments’ the buildup of the FLIM data is displayed in intervals of a few seconds. The images are displayed with the autoscale function of the display parameters. As the accumulation proceeds, the images displayed do not change in brightness, but the signal-to-noise ratio increases. The measurement is stopped by clicking on the Stop Scan button. After clicking the button the scanner completes its current frame before the measurement is really stopped. If you want to abort a measurement immediately, click the stop button twice.
Fig. 92: Scanner panel in FLIM acquisition state
The pixels number of the final FLIM scan is normally taken from the setup data loaded from the predefined setups. You can, however, change it by clicking in the ‘Frame Format’ field and selecting one of the suggested formats. When have you changed the frame format in the scanner panel, please click on the symbol to transfer the new pixel number into the SPCM system parameters.
The scanner panel controls a number of other functions, such as beam blanking, laser multiplexing, laser on/off modulation for PLIM, scan modes and scan speed, and the beam park position for single-curve of FCS measurement. These functions are used for advanced DCS-120 applications. Please see ‘Scanner Control’, page 106.
The synchronisation and count rates are displayed in the lower left part of the SPCM main panel, see Fig. 93.
Fig. 93: Count rates (left) status information (upper middle)
The SYNC rate is the effective laser repetition rate, i.e. the number of laser pulses per second. It is a bit lower than the repetition rate of the laser itself because the laser is turned off during beam flyback. The lasers is also turned off when the system is not scanning. Therefore you can see the Sync rate only when the system is in the Preview mode or when a measurement is running. If there is no sync rate during a measurement you probably forgot to turn on the laser in the scanner control panel, see Fig. 91.
The CFD, TAC and ADC count rates indicate the rate of the detected, converted, and stored photons, respectively . The rates may fluctuate due to inhomogeneous intensity in the scan area, and due to the beam blanking during the beam flyback. The count rates are direct indicators of the progress of the data acquisition:
- The acquisition time needed to obtain a given signal-to-noise ratio (or to record a given number of photons, see ‘Acquisition Time of FLIM’, page 222) depends on the count rate. Higher count rate thus leads to shorter acquisition time. A good count rate is 105 to 106 photons per second. If you cannot achieve a reasonable count rate check whether you are using the right laser wavelength and laser power, the right filters, the right secondary beam splitter, and a reasonable pinhole size. Consider using a microscope lens with higher NA, or a sample that contains higher fluorophore concentration.
- The maximum count rate the TCSPC system can reasonably work with is about 5×106 photons per second, see ‘Counting Loss and Pile-Up Effects’, page 223. If it is higher than 5×106 the laser power or the pinhole size should be decreased.
- A slow decrease in the count rates indicates that there is photobleaching. As long as the total decrease does not exceed 20 % the effect on the lifetimes may still be tolerable. However, you should try to run the experiment at lower power. To compensate for the drop in count rate use a larger pinhole, or use a microscope lens of higher NA. Check that you are using the correct secondary beamsplitter and the correct filters.
- the CFD, TAC, and ADC rates should be approximately the same. An ADC rate substantially lower than the CFD and TAC rate indicates that most of the photons are outside the time interval in which the decay functions are recorded. The reason is usually that the system is recording more daylight than fluorescence. Make sure that the microscope lamp is turned off!
The signal-to-noise ratio in a lifetime image is the square root of the number of photons, N1/2, in the pixels [193, 259], see ‘Signal-to-Noise Ratio of FLIM’, page 219. Therefore the acquisition time required to record useful data depends on your requirements to the lifetime accuracy, on the count rate at which the measurement is run, and on the number of pixels in the image. 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. A general answer on how long the acquisition should be continued therefore cannot be given. For bright samples you can get reasonably good 256 x 256 or 512 x 512 pixel images within 5 seconds, see Fig. 12, page 8. For large images and a lifetime accuracy in the 1% range you may need several minutes, see ‘Acquisition Time of FLIM’, page 222. The software cannot answer these questions for you, it can only assist you in making the right decisions. One way the software can help is to display the photon number in the brightest pixel of an image, and the pixel number at location selected by an image cursor, please see Fig. 152and Fig. 153, page 100.
When you start into a new experimental area we therefore recommend to record a few test images at first, analyse the data with SPCImage, and check whether the expected lifetime differences are resolved. In other words, you have to develop a feeling of how good the raw images should be for your special application. The software cannot decide this, it can only support you in deciding whether to stop or to continue. Therefore, the general answer to the acquisition time question is: Record as long as the photostability of sample allows you to do so.
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 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 Techniques and Procedures’.
1. Turn on the FLIM system: First the extension box, then the laptop computer. Turn on the GDA-120 scan amplifier. Turn on the DCS switch box. Start the SPCM.
2. Start the SPCM Software. Load an instrument setup from the ‘predefined setup’ panel.
3. Put the sample under the microscope, switch the microscope beam path to ‘eyepiece’ and adjust the focus and the sample position.
5. Turn the secondary beamsplitter wheel of the scanner into the desired position, insert the right filters, and set a reasonable pinhole size. Switch the microscope beam path to the port where the scanner is attached.
6. If not already active, open the scanner control panel of SPCM. Activate a laser. Enable the detectors. Click on the yellow ‘Preview’ button to start a preview scan. Make sure the microscope lamp is turned off.
7. Adjust the laser power to obtain a CFD count rate between 105 and 106. In case a detector shuts down by overload (right panel) decrease the laser power or reduce the pinhole size.
8. By looking at the preview images, adjust the focus, the sample position, and the zoom area. Stop the preview by clicking on the yellow ‘Preview’ button.
9. Click on ‘Start Scan’. Let the measurement run until you are satisfied by the signal-to-noise ratio.
10. Stop the scanning. Send the data to the SPCImage lifetime data analysis.
From 2012 on the DCS‑120 system has a piezo-driven pinhole alignment. The alignment is done manually via the hand controller shown in Fig. 94.
Fig. 94: Hand controller for pinhole alignment
The two outer knobs shift the beam in the pinhole in laterally (X and Y), the centre knob moves the beam focus longitudinally (Z). Alignment is done in the preview mode. Start with a pinhole size of 1 in both channels. Put a sample in, start the Preview mode, and focus correctly by the microscope focus drive (this is important!). Open the display parameters, and turn off ‘Autoscale’. Then maximise the intensity by turning the X and Y controls. If the display gets saturated click ‘Autoscale’ on and off, if the count rate exceeds 106 reduce the laser power. When you have obtained maximum intensity optimise the Z axis (the focus into the pinhole). For best results, repeat the procedure with a pinhole size of 0.5 or 0.25, and with a smaller piezo step width, see below. When the system is aligned it stays in the selected position, even if the hand controller or the entire DCS-120 system are switched off.
The piezo hand controller has variable step width. You can change it by pushing the control knobs. We recommend to start with a step width of 10 in X and Y, and 40 in Z, see Fig. 95, left. In the final alignment steps the piezo step width can be reduced to 2 in X and Y, and 10 in Z. The hand controller has also options to change the voltage at the actuators, and the speed of the individual steps. You get into these modes by pushing a button and rotating it. We recommend not to change these settings. Please use the parameters shown in Fig. 95, middle and right. The current mode of the controller is indicated by small triangles on the left of the settings that are going to be changed. To get back to the pinhole adjust mode push the buttons until the triangles have disappeared.
Fig. 95: Piezo hand controller in step width selection mode (left) and voltage selection mode (middle), and speed selection mode (right)
Even minor misalignment has a noticeable effect on the sensitivity and the image quality. Fig. 96 shows three examples.
Fig. 96: Effect of pinhole alignment on the image. Left: 24 steps (12 clicks with step width 2) off in Y. Middle: 240 steps (24 clicks with step width 10) off in Z. Right: Perfectly aligned in all three axis. Pinhole size 0.5.
The BDL-SMC lasers are class 3B laser products. The laser safety regulations dictate that the lasers be labelled with the stickers shown in Fig. 97, and that the labels and the location of the labels on the lasers be described in the manual. The laser class is indicated on the laser by an ‘explanatory label’, Fig. 97, left. The laser aperture is marked with the aperture labels, Fig. 97, middle and right.
Fig. 97. Left to right: Explanatory label, aperture labels.
Moreover, each laser has a manufacturer identification, as shown in Fig. 98. The position of the labels on the laser modules is shown Fig. 99.
Fig. 98: Manufacturer identification label
Fig. 99: Location of the labels on the lasers
Laser safety regulations forbid the user to open the housing of the laser, or to do any maintenance or service operations at or inside the laser. Use of controls or adjustments or performance of procedures other than specified herein may result in hazardous radiation exposure or damage to the laser module.
It is required that the lasers have a ‘remote interlock connector’ that can be pulled to turn off the laser reliably. For DCS-120 systems that have the lasers connected via the laser switch box shown in Fig. 100, left, the interlock connector is the 15‑pin connector at the laser side of the laser switch box. For DCS systems with the DCS connection box the interlock connectors are the 15 pin connectors shown in Fig. 100, right. The connector can be pulled off or plugged in at any time without causing damage to the laser.
Fig. 100: Remote interlock connector. Left: At laser switch box. Right: At DCS connection box
The operation and maintenance of the DCS‑120 system requires a few additional precautions. Harmful situations may especially arise if a collimated laser beam becomes accessible. This may happen particularly
- when the fibre is removed from the laser
- when the fibre is attached to the laser but removed from the scan head
- when no microscope lens is in the lens turret
These situations are illustrated in Fig. 101.
Fig. 101: Situations potentially harmful to the eye. Left: Emission of a collimated beam from the laser when no fibre is plugged in. Middle: Emission of a collimated beam from the end of the fibre. Right: Emission of a collimated beam from a free microscope lens position
Especially the last situation is often not taken into regard. Therefore, please cover empty lens positions reliably. (This is not only a matter of laser safety, but also to keep the dust out!)
Some microscopes have beamsplitters that allow you to operate the scanner and simultaneously see the image in the eyepieces. For alignment purpose it is, of course, convenient if you can watch the laser spot scanning the sample. In practice looking at the laser spot in a sample is rarely harmful. Unless you put a mirror under the microscope the intensity is far too low to do any damage. However, it is a clear violation of the laser safety rules. Therefore, either block such beamsplitter positions or use a long-pass filter in the eyepiece pass that blocks all the laser wavelengths used. For the Nikon TE2000U bh deliver a filter that blocks the 405, 445, and 473 nm lasers.
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 10 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, and 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 different 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 97, and ‘Creating Predefined Setups’, page 119.
The SPCM software also controls the DCC-100 module that runs the detectors, the DB‑32 delay switch box, the bh GVD-120 scan controller, the Ti:Sa laser and AOM of the DCS-120 MP system, and a motorised sample stage . The parameters for these devices 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 DCS-120 confocal and multiphoton FLIM systems are complex multi-modal systems which acquire 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. 102.
Fig. 102: Examples of user interface configurations of the bh SPCM software
TCSPC-FLIM data are multi-dimensional distributions. In the simplest case, a FLIM image is a three-dimensional data array. The data represent an array of pixels, each of which contains fluorescence decay data in form of photon numbers for consecutive time channels in the laser pulse period. However, FLIM data can contain additional dimensions. These may be the wavelength of the photons, the time within the period of a stimulation of the sample, or the Z coordinate of a FLIM Z stack. The display of such data is controlled by the ‘3D Trace Parameters’, the ‘Window Parameters’ and the ‘Display Parameters’. The function of these parameters is described in the section below.
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. 103 and Fig. 105.
Fig. 103 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.
Fig. 103: 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
For all definitions shown in Fig. 103 the ‘Display mode’ is F(x,y). That means, the data shown in the individual display windows are intensity distributions over the two spatial coordinates, x and y.
However, this is not the only way the data can be displayed. FLIM data are three-dimensional x-y-t arrays. That means, when the FLIM results are displayed different projections of the data can be used. The way the data are projected is selected by ‘Display Mode’. The principle is shown in Fig. 104.
Fig. 104: Different projections of FLIM data
F(x,y) projects the intensities (photon numbers) of a selected time window in the x-y plane. The result is a time-gated image, or, if the time window covers the entire t axis, an intensity image of the sample.
F(t,x) projects the photon numbers of a selected y window into a t-x plane. The result displays the intensity decay along the t axis versus the location in x for a selected window in y. F(t,y) projects the photon numbers of a selected x window into the t-y plane. The result displays the intensity along the t axis versus the location in x for a selected window in y. In both cases, the spatial window can also cover the entire x or y coordinate. Examples of trace parameters with different projection are shown in Fig. 105.
A special case is the direct display of the fluorescence lifetime. In this case, the display routine calculates the first moment of the decay data along the t axis of the individual pixels, converts it into the fluorescence lifetime. When ‘Lifetime Display’ is selected (W3 and W4 in Fig. 103) the colour of the image represents the lifetime, the brightness the total or gated intensity.
So far, things are still relatively straightforward. However, TCSPC data can easily become four-dimensional. This happens especially when the routing function is used. Data in the individual routing channels then represent different detection wavelengths, different excitation wavelengths, or different states of an external signal applied to the sample. The ‘Routing Window’ defines a range of routing channels the data of which are projected into individual x‑y, t‑x, or t‑y planes and displayed as described above. Trace-parameter examples for these situations are shown in Fig. 105. 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.
Fig. 105: 3D Trace parameters for the display of decay functions over one spatial and one temporal 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. 106 shows definitions for 8 windows along each coordinate.
Fig. 106: 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 is 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. 107.
Fig. 107: 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. 108.
Fig. 108: 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 the definitions for the lifetime display. These include the lifetime range, a reference moment of the IRF, and contrast and brightness of the image displayed. An example is shown in Fig. 109.
Fig. 109: 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. 110 to Fig. 112 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. 110, one single display window (W1) is enabled (green button). 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. 111, 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. 112, 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 the window parameters. It contains the photons of all time channels (from 1 to 256, see Fig. 111). The SPC Main panel with an image defined by these parameters is shown in Fig. 113.
Fig. 110: 3D Trace parameters for display of a single image
Fig. 111: Window Parameters for display of a single image
Fig. 112: Display parameters
Fig. 113: SPCM Main panel with the image defined by the parameters shown above
Fig. 114 shows the parameters for the display of four time-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. 114: 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. 115. The SPCM main panel with the four gated images is shown in Fig. 116.
Fig. 115: Display parameters for the four gated images
Fig. 116: 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. 117. 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. 117: 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. 118. The SPCM main panel with the two images is shown in Fig. 119.
Fig. 118: Display parameters for the two images defined by the parameters in Fig. 117
Fig. 119 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. 120 trough Fig. 122. 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. 120: 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. 121. 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. 122.
Fig. 121: Display parameters in the first three display channels of a multi-wavelength measurement
Fig. 122: SPCM Main panel for multi-wavelength FLIM
Fig. 123 and Fig. 124 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. 123: 3D trace parameters for run-time display of lifetime images
The display parameters are shown in Fig. 124. 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 283) 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. 125.
Fig. 124: Display parameters for run-time lifetime display
Fig. 125: 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 167. 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. 126 shows the trace and window parameter setup. The 3D Trace parameters define four display windows. The first one is the FLIM window. It displays the photons from the laser-on phases, recorded in routing channel 1. The second one is a pure PLIM window. It contains the photons from the Laser-off phases, recorded in routing channel 2. The third and the fourth window show the total luminescence. Data type for the first window is ps FLIM, for the other 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. 126: 3D Trace parameters and window parameters for simultaneous FLIM/PLIM
The display parameters for the four windows are shown in Fig. 127. 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 display 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 an image of the total luminescence, i.e. entire signal in both phases of the laser modulation therefore ‘Routing window’ is 3.
W4 does not displays the same data as W3. However, instead of an image 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.
Fig. 127: Display parameters for fluorescence image, phosphorescence image, total luminescence, and phosphorescence decay curves
The SPCM main panel with these setup parameters is shown in Fig. 128. The fluorescence image is displayed on the left, the phosphorescence image in the middle, and the image of the total luminescence on the right. The decay curves of the total luminescence are displayed middle left.
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. 128: 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 ‘2D Trace Parameters’ define which information the traces contain and in which style and colour they are displayed. An example is shown in Fig. 129.
Fig. 129: Left: Display of several decay curves recorded by a single SPC module. Right: 2D 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. 130. 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 section ‘System Parameters for Basic FLIM Experiments’ page 121.
Fig. 130: 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 right. 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 laser scanning microscopes. 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 and SPC-150N 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 galvanometer scanners. 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 [58, 59, 160, 374]. 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 DCS-120 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 [42, 446]. 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 167.
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. 143, page 97.
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’ . The trigger function is used to record Z stacks or microscope-controlled time series in combination with laser scanning systems of other manufacturers, e.g. with the Zeiss LSM 710/780/88 systems.
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. 131. 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. 131: 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. 132. The parameters shown in Fig. 132, left, result in a FLIM images of the same pixel number as the LSM scan. The setup shown Fig. 132, right, bins the FLIM image down to 1/4 of the LSM scan pixels.
Fig. 132: 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 (active edge) of X Sync Pulses
YSync Polarity: Polarity (active edge) of Y Sync Pulses
Pixel Clock Polarity: Polarity (active edge) of Pixel Clock Pulses
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 the DCS-120 system.
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. 133, right. It opens by a click on the ‘Configure’ button in the measurement control part of the system parameter panel, see Fig. 133, left.
Fig. 133: 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 167. All three functions can be activated simultaneously, as shown in Fig. 133, right.
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 75.