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Abstract: The DCS-120 system uses excitation by ps diode lasers
or femtosecond titanium-sapphire lasers, fast scanning by galvanometer mirrors,
confocal detection, and FLIM by bh’s multidimensional TCSPC technique to record
fluorescence lifetime images at high temporal resolution, high spatial
resolution, and high sensitivity . The DCS‑120 system is available
with inverted microscopes of Nikon, Zeiss, and
The DCS-120 systems are complete confocal laser scanning microscopes for fluorescence lifetime imaging. The systems use bh’s multi-dimensional TCSPC FLIM technology [22, 28, 30] in combination with fast laser scanning and confocal detection . DCS-120 systems are available with various inverted and upright microscopes, see Fig. 1. The DCS-120 scan head with the control and data acquisition electronics can also be used to upgrade a conventional microscope with FLIM recording. Moreover, a 'DCS-120 MACRO’ system FLIM of centimetre-size objects and a 'DCS-120 MP' multiphoton system are available, 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 (left) and DCS-120MP 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 [11, 57]. 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 [11, 57]. Advanced versions of the DCS-120 system are available for multiphoton excitation and tuneable excitation sources [6, 7].
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, or 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: Optical diagram of the DCS-120 scan head. Simplified, see  for details
The DCS‑120 system is highly modular.
The DCS‑120 scan head is compatible with conventional microscopes of
almost any type and manufacturer. Complete laser scanning systems are available
with microscopes of Zeiss, Nikon, and
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.
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, please see  for details.
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 [11, 28, 57]. The main panel of the SPCM data acquisition software is configurable by the user . Different configurations for FLIM systems are shown in Fig. 6.
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. Please see also ‘Time-Series Recording by Mosaic FLIM’, page 20.
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 [3, 28]. Lifetime images can be displayed at image 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: 256 x 256-pixel images obtained by the online FLIM 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.
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 [22, 28]. Even if one channel overloads the other one is still able to produce correct data.
Fig. 17: 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 DCS-120 WB wideband version can be used with tuneable excitation. Images obtained with a Toptica Ichrome laser are shown in Fig. 18. Additional examples can be found in , , and .
Fig. 18: 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 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.
The DCS‑120 WB version is able to record lifetime images with near-infrared (NIR) fluorophores. NIR fluorophores often display large lifetime variations depending on the binding target. Moreover, because both the excitation and the emission wavelengths are in the near infrared a high penetration depth is obtained. NIR FLIM is therefore a promising technique to obtain metabolic information from biological tissue. An image of a pig skin sample incubated with 3,3’-diethylthiatricarbocyanine is shown in Fig. 20.
Fig. 20: Near-Infrared FLIM. Pig skin sample stained with 3,3’-diethylthiatricarbocyanine, detection wavelength from 780 nm to 900 nm.
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
The large pixel numbers available in SPCM 64 bit allow the full field of view of even the best microscope lenses to be scanned with an oversampling factor of two or more. In other words, extremely large image areas can be scanned without compromising spatial resolution.
Large pixel numbers are especially 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 entire 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 BPAE cell sample. The mosaic has 4x4 elements, each element has 512x512 pixels, each pixel has 256 time channels. DCS-120 MP (multiphoton) system. Data analysis by bh SPCImage. Use Adobe zoom function to see image at full resolution.
With the bh multispectral FLIM detectors the DCS‑120 records FLIM simultaneously in 16 wavelength channels [20, 23, 28]. 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. Two such mosaic data sets are obtained simultaneously through the two channels of the DCS system. 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 data 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.
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. 29. The fluorescence lifetime of the chloroplasts changes due to the Kautski effect induced by the illumination.
Fig. 29: 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 [28, 31]. 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 be analysed in a single SPCImage data analysis run. Fig. 30 shows the change of the lifetime of chlorophyll in plant tissue with the illumination.
Fig. 30: 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.
The DCS‑120 MP is a complete multiphoton laser scanning FLIM microscope. It consists of a Coherent Chameleon or Spectra Physics Mai Tai titanium-sapphire laser, an acousto-optical modulator (AOM), a DCS-120 scan head, a Zeiss Axio Observer or Axio Examiner microscope, two non-descanned detectors, and the associated TCSPC-FLIM data acquisition electronics. A motorised sample stage can be added on demand. All parts are controlled by bh SPCM data acquisition software. A two-photon FLIM image of a convallaria sample recorded by the DCS-120 MP system is shown in Fig. 31.
Fig. 31: Two-photon FLIM image of a convallaria sample
The 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. NDD FLIM images from different depth in a pig skin sample are shown in 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.
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 [15, 28]. 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. 33.
Fig. 33: 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.
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 [13, 25, 28]. FLIM is recorded during the ‘On’ time, PLIM during the ‘Off’ time of the laser, see Fig. 34. The SPCM software delivers separate images for the fluorescence and the phosphorescence which are then analysed with SPCImage FLIM/PLIM analysis software. FLIM/PLIM works both with the ps diode lasers of the DCS-120 and with the Ti:Sa laser of the DCS-120MP.
Fig. 34: Principle of simultaneous FLIM / PLIM
Currently, there is an increasing interest in PLIM for background-free recording and, importantly, for oxygen sensing [1, 2, 37-42, 46, 47, 53, 55, 58, 61, 62]. 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 [48, 52]. An example is shown in Fig. 35.
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 luminophor for 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 [28, 29, 45]. This is enough to record transient changes in the concentration of free Ca2+ in live neurons, as has been demonstrated in [29, 45].
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 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 shows a leaf with a fungus infections.
Fig. 38: Leaf with a fungus infection. ps diode laser excitation, 405nm, scan format 512 x 512 pixels. Right: Decay functions of healthy and infected areas.
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
The DCS-120 Macro can be combined with endoscopes. Optical details are described in . Images of (benign) human skin lesions are shown in Fig. 40
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.
FLIM experiments often have to be supported by precision measurements of the decay functions of special fluorophores. FLIM users then often resort to an additional fluorescence lifetime spectrometer for cuvette-based measurements of decay functions. In most cases, however, such fluorescence decay data can be recorded by the DCS-120 system. An example is shown in Fig. 41.
Fig. 41: Fluorescence decay curve recorded by DCS120, analysed by SPCImage
The use of the DCS-120 system for decay recording has the advantage that the data are recorded under exactly identical conditions as in the FLIM case. Possible anisotropy-decay effects are cancelled by the high NA of the microscope lens, and there is virtually no re-absorption. Moreover, phosphorescence and delayed-fluorescence decay curves can be recorded. Please see DCS-120 handbook  and bh TCSPC Handbook  for further details.
Due to the superior performance of the HPM‑100-40 hybrid detectors theDCS‑120 system delivers highly efficient FCS. There is no afterpulsing peak in autocorrelation data . Thus, accurate diffusion times and molecular-brightness 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 efficiency. The result is a substantial improvement in the SNR of FCS recordings . Gated FCS is possible by hardware gating via the TAC limits of the TCSPC modules, FCCS by cross-correlating the signals of the two DCS channels [3, 28].
Fig. 42: Left: FCS curve recorded by a single HPM-100 detector. The data are free of an afterpulsing peak. Right: Dual-colour FCS, autocorrelation blue and red, cross-correlation green. Online fit with FCS procedures of SPCM software.
Data analysis is performed by the bh SPCImage data analysis package, see Fig. 43. Data analysis can be run over a single FLIM or PLIM image, over several images obtained in parallel TCSPC channels, or over the images recorded in the 16 channels of a multi-wavelength system. SPCImage runs an iterative de-convolution and fit procedure on the decay data in the pixels of the images. Single-double, and triple-exponential decay exponential models are available. Residual fluorescence from previous laser pulses can be accounted for by incomplete-decay models . Multi-exponential decay analysis can be performed with free or fixed lifetimes of the decay components. SPCImage is able to calculate the instrument-response function (IRF) automatically from the decay data. It can, however, also use a recorded IRF, extract an IRF from SHG signals present in the data, or use a manually defined IRF.
Fig. 43: SPCImage data analysis
Lifetime data are displayed as false-colour images of the lifetimes or amplitudes of the decay components, or of ratios of lifetimes or amplitudes of the components. Moreover, SPCImage is able to calculate and display FRET efficiencies from double-exponential decay data obtained in FRET experiments [3, 18, 28]. The data can be exported into ASCII, BMP, and TIF files.
Batch processing of FLIM file series has been introduced in 2012 [3, 28]. A large number of data files can be specified, analysed with identical model and fit control parameters, and displayed with identical colour and display range parameters. For the results of batch processing a batch export routine is implemented.
SPCImage has a histogram function for the decay parameter selected for colour-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. 44.
Fig. 44: 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. 45.
Fig. 45: Main panel of SPCImage showing a FRET cell (left), decay data in selected spot (middle) and 2D Histogram of amplitude ratio of components versus amplitude-weighted lifetime (right)
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 . Independently of their location in the image, pixels with similar decay signature form clusters in this diagram. Different phasor clusters can be selected, and the corresponding pixels back-annotated in the time-domain FLIM images. 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 detectable by normal pixel-by-pixel analysis. Please see Fig. 46 and Fig. 47.
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.
Fig. 47: Selection of different phasor clusters, combination of decay curves, and back-annotation of selected decay signature in the time-domain lifetime image.
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‑830, 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 of a routing system, different channels of a multi-module TCSPC system, or different time windows of a PIE recording are determined, 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
DCS-120 data are compatible with other analysis packages. They can be imported into multi-parameter FLIM analysis [36, 43, 59] and phasor analysis  in the frequency domain. Single-photon parameter-tag data can be analysed by the bh ‘Burst Analyser’ software. This software is able to identify single-molecule photon bursts in the parameter-tag data, analyse fluorescence lifetimes and intensities within the burst, and build up one- and two-dimensional histograms of the parameters. The results can be used to identify different fluorescent species or different FRET states of single molecules. Moreover, the burst data can be used to calculate FCS and cross-FCS, and fit the curves with standard or user-defined model functions.
The advantage of FLIM over other fluorescence imaging techniques is that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on the concentration , see Fig. 49. If fluorescence in a sample is excited (Fig. 49, left) the emission intensity depends both on the concentration of the fluorophore and on possible interaction of the fluorophore with its molecular environment. Changes in the concentration, cannot be distinguished from changes in the molecular environment. Spectral measurements (second right) are able to distinguish between different fluorophores. However, changes in the local environment usually do not cause changes in the shape of the spectrum. The fluorescence lifetime of a fluorophore (Fig. 49, right), within reasonable limits, does not depends on the concentration but systematically changes on interaction with the molecular environment.
Fig. 49: Fluorescence. Left to right: Excitation light is absorbed by a fluorophore, and fluorescence is emitted at a longer wavelength. The fluorescence intensity varies with concentration. The fluorescence spectrum is characteristic of the type of the fluorophore. The fluorescence decay function is an indicator of interaction of the fluorophore with its molecular environment.
By using the fluorescence lifetime, or, more precisely, the shape of the fluorescence decay function, molecular effects can therefore be investigated independently of the unknown and usually variable fluorophore concentration [28, 32, 51]. Common FLIM applications are ion concentration measurements, probing of protein interaction via FRET, and the probing of metabolic activity and cell viability via the fluorescence lifetimes of NADH and FAD. FLIM may also find application in plant physiology because the fluorescence lifetime of chlorophyll changes with the photosynthesis activity.
A particularly efficient energy transfer process is Förster resonance energy transfer, or FRET. The effect was found by Theodor Förster in 1946 . FRET is a dipole-dipole interaction of two molecules in which the emission band of one molecule overlaps the absorption band of the other. In this case the energy from the first molecule, the donor, transfers into the second one, the acceptor, see Fig. 50, left. FRET results in an extremely efficient quenching of the donor fluorescence and, consequently, in a considerable decrease of the donor lifetime, see Fig. 50, right.
The energy transfer rate from the donor to the acceptor increase with the sixth power of the reciprocal distance. Therefore it is noticeable only at distances shorter than 10 nm . FRET is used as a tool to investigate protein-protein interaction. Different proteins are labelled with the donor and the acceptor, and FRET is used as an indicator of the binding between these proteins. Steady-state FRET measurements have the problem that the relative concentration of donor and acceptor varies, that the donor emission spectrally extends into the acceptor emission, and that a fraction of the acceptor is excited directly. FLIM does not have these problems because all it needs is to record a lifetime image at the donor emission wavelength. FRET is the most frequent FLIM application, please see  for references.
Fig. 51 shows FRET in a cultured live HEK cell. The cell is expressing two proteins, one labelled with CFP, the other with YFP. FRET occurs in the places where the proteins interact. The associated changes in the donor lifetime are clearly visible in the lifetime image shown in Fig. 51, left.
FLIM is not only able to detect FRET without interference by donor and acceptor bleedthrough, it even delivers independent images of the donor-acceptor distance and the fraction of interacting donor. Such images can be obtained by double-exponential analysis of the FLIM data: The interacting donor fraction delivers a fast, the non-interacting fraction a slow decay component. The ratio of the two lifetimes is directly related to the donor-acceptor distance, the ratio of the amplitudes of the components is the ratio of interacting and non-interacting donor. Images which resolve these two parameters of the FRET system are shown in Fig. 51, middle and right.
Remarkably, double exponential FRET does not need an external lifetime reference: The reference lifetime is the slow decay component, originating from the non-interaction donor. Please see [3, 22, 28] for details and for further references.
Fig. 51: FRET in HEK cell expressing proteins labelled with CFP and YFP. Left: Lifetime image at donor wavelength, showing lifetime changes by FRET. Middle and right: FRET results obtained by double-exponential lifetime analysis. Ratio of the lifetimes of the decay components, t2/t1 = t0/tfret, and ratio of the interacting and non-interacting donor fractions, a1/a2 = Nfret/N0.
Biological tissue contains a wide variety of endogenous fluorophores . However, the fluorescence spectra of endogenous fluorophores are broad, variable, and poorly defined. Moreover, absorbers present in the tissue may change the apparent fluorescence spectra. It is therefore difficult to disentangle the fluorescence components by their emission spectra alone. Autofluorescence lifetime detection is expected to add an additional separation parameter to the analysis of the data.
More important, the autofluorescence intensities and lifetimes contain information about the binding, the metabolic state and the microenvironment of the fluorophores. Especially interesting are the fluorescence signals from coenzymes, such as flavin adenine nucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). It is known that the fluorescence lifetimes of NADH and FAD depend on the binding . The lifetimes, the ratio of bound and unbound NADH, the ratio of NADH and NADPH, and the NADH / FAD intensity ratio also depend on the metabolic state [33, 35], and on the redox state . The NADH and FAD fluorescence intensities and lifetimes are therefore used to detect precancerous and cancerous alterations . For an overview about the literature please see .
Fig. 52 shows an example of how autofluorescence signals change with the oxygen concentration. Yeast cells were kept in a sugar solution. They produce CO2 which washes out the oxygen from the solution. The left image was recorded under such conditions. Only a few cells are visible Fig. 52, left and middle, the other ones are extremely dim. The image in Fig. 52, right, was recorded after the solution had been saturated with oxygen. The difference in the fluorescence behaviour is striking.
Fig. 52: Autofluorescence of yeast cells. Left and middle: Saturated with CO2, different intensity scale of the same data set. Right: Saturated with O2. Excitation 405 nm, detection at 540 nm.
Fig. 53 shows a pig skin autofluorescence image obtained at 405 nm excitation wavelength. Due to the absence of exogenous fluorophores the fluorescence intensity is low. Nevertheless, the FLIM data contain enough photons for double-exponential decay analysis. The image on the left shows the amplitude-weighted mean lifetime, tm. The image in the middle shows the ratio of the intensities, q1/q2, contained in the fast and the slow decay component. Two typical decay curves are shown on the right.
Fig. 53: Pig skin sample excited at 405 nm, detection from 460 to 500 nm. Double-exponential fit. Left: Amplitude-weighted lifetime. Middle: Intensity ratio of fast and slow decay component. Right: Decay curves in two spots of the image.
In the wavelength interval recorded the emission can be expected to be dominated by NADH fluorescence. The lifetimes of bound and unbound NADH are different. The q1/q2 ratio can therefore be expected to represent the intensity ratio of bound and unbound NADH. It should be noted that accurate NADH analysis, of course, requires spectral unmixing of the NADH signal from contributions of other fluorophores . Due to the variability of the autofluorescence spectra and lifetimes, fluorescence contribution from other fluorophores, and the presence of unknown absorbers the task is extremely complicated. The prospects of unmixing the signals improve considerably with the availability of excitation wavelength multiplexing or tuneable excitation, see Fig. 18, page 12.
Two examples of FLIM of plant tissue are shown in Fig. 54 and Fig. 55 The fluorescence is dominated by the fluorescence of chlorophyll and the fluorescence of flavines. Multi-wavelength FLIM images of a moss leaf recorded with the bh multi-spectral FLIM detector are shown in Fig. 54.
Fig. 54: Multi-spectral FLIM of plant tissue. Moss leaf, excitation at 405 nm, wavelength from 575 nm to 762 nm. DCS-120, MW FLIM detector. Image size 256x256 pixels, 64 time channels, 16 wavelength channels.
The fluorescence of chlorophyll competes with the energy transfer into the photosynthesis channels. Thus, the fluorescence lifetime and its change on illumination is a sensitive indicator of the photosynthesis efficiency. The change in the fluorescence lifetime of the chloroplasts in a moss leaf on exposure to light can recorded by time-series FLIM, see Fig. 55.
Fig. 55: Change of the fluorescence lifetime of chlorophyll with time of exposure. Moss leaf, excitation at 445 nm, 256x256 pixels, 1 image per second.
Faster effects down to the millisecond time scale can be recorded by temporal mosaic FLIM or FLITS, [28, 29] see Fig. 30 and Fig. 37 of this brochure.
The DCS-120 system records lifetime images at high spatial and temporal resolution, extremely high sensitivity, and short acquisition time. Recently introduced 64-bit SPCM operating software has increased the image format of FLIM into the megapixel region. Single-, dual-, multi-wavelength FLIM is now recorded at unprecedented image quality. Moreover, the large memory space available in the 64 bit environment made it possible to implement advanced FLIM techniques, like time series recording and Z stack recording by Mosaic recording. Physiological effects down to the millisecond range can be resolved by triggered mosaic FLIM and by FLITS. Metabolic effects can be recorded by FLIM and correlated with changes in the oxygen concentration simultaneously measured by PLIM. No other FLIM technique and no other FLIM system offers a similar range of advanced capabilities.
Scan head bh DCS-120 scan head
Optical principle confocal, beam scanning by fast galvanometer mirrors
Laser inputs two independent inputs, fibre coupled or free beam
Laser power regulation, optical continuously variable via neutral-density filter wheels
Outputs to detectors two outputs, detectors are directly attached
Main beamsplitter versions multi-band dichroic, wideband, multiphoton
Secondary beamsplitter wheel 3 dichroic beamsplitters, polarising beamsplitter, 100% to channel1, 100% to channel2
Pinholes independent pinhole wheel for each channel
Pinhole size 11 pinholes, from about 0.5 to 10 AU
Emission filters 2 filter sliders per channel
Connection to microscope adapter to left side port or port on top of microscope
Coupling of lasers into scan head (visible) single-mode fibres, Point-Source type, separate for each laser
Coupling of laser into scan head (Ti:Sa) free beam, 1 to 2 mm diameter
Scan Controller bh GVD-120
Principle Digital waveform generation, scan waveforms generated by hardware
Scan waveform linear ramp with cycloid flyback
Scan format line, frame, or single point
Frame size, frame scan 16x16 to 4096x4096 pixels
line scan 16 to 4096 pixels
X scan continuous or pixel-by-pixel
Y scan line by line
Laser power control, electrical via electrical signal to lasers
Laser multiplexing frame by frame, line by line, or within one pixel
Beam blanking during flyback and when scan is stopped
Scan rate automatic selection of fastest rate or manual selection
minimum pixel time for frame size 64x64 128x128 256x256 512x512 1024x1024 2048x2048
Zoom=1 25.6µs 12.8µs 6.4µs 3.2µs 1.6µs 1.2µs
Zoom=8 6.4µs 3.2µs 1.6µs 0.8µs 0.6µs 0.5µs
minimum frame time for frame size 64x64 128x128 256x256 512x512 1024x1024 2048x2048
Zoom=1 0.19s 0.37s 0.64s 1.24s 2.6s 6.5s
Zoom=8 0.037s 0.074s 0.173s 0.320s 1.0s 2.7s
Scan area definition via zoom and offset or interactive via cursors during preview
Fast preview function 1 second per frame, 128 x 128 pixels
Beam park function via cursor in preview image or cursor in FLIM image
Laser control 2 Lasers, on/off, frame, line, pxl multiplexing
Diode lasers bh BDL-SMC or BDL-SMN laser
Number of lasers simultaneously operated 2
Wavelengths 375nm, 405nm, 445nm, 473nm, 488nm, 510nm, 640nm, 685nm, 785nm
Pulse width, typical 30 to 70 ps
Pulse frequency 20MHz, 50MHz, 80MHz, CW
Power in picosecond mode 0.25mW to 1mW injected into fibre. Depends on wavelength version.
Power in CW mode 10 to 40mW injected into fibre. Depends on wavelength version.
Visible and UV range any ps pulsed laser of 20 to 80 MHz repetition rate
Coupling requirements Point-Source Kineflex compatible fibre adapter
Wavelength any wavelength from 400nm to 800nm
fs NIR Lasers for multiphoton operation any fs laser
Coupling requirements free beam, diameter 1 to 2 mm
Wavelength 700 to 1200 nm
Detectors (standard) bh HPM-100-40 hybrid detector
Spectral Range 300 to 710nm
Peak quantum efficiency 40 to 50%
IRF width with bh diode laser 120 to 130 ps
Active area 3mm
Background count rate, thermal 300 to 2000 counts per second
Power supply, gain control, overload shutdown via DCC-100 controller of TCSPC system
Detectors (optional) bh HPM-100-06 and HPM-100-07 hybrid detectors
Spectral Range 290 nm to 600 nm 220 to 850 nm
Peak quantum efficiency 20 % (at 400nm) 26% at 290 nm, 22% at 400nm
System IRF width with fs Ti:Sa laser <20 ps
System IRF width with bh ps diode laser 38 to 90 ps
Active area 3mm
Background count rate, thermal 100 to 1000 counts per second
Power supply, gain control, overload shutdown via DCC-100 controller of TCSPC system
Detectors (optional) bh HPM-100-50 hybrid detector
Spectral Range 400 to 900nm
Peak quantum efficiency 12 to 15%
IRF width with bh diode laser 150 to 200 ps
Active area 3mm
Background count rate, thermal 1000 to 8000 counts per second
Power supply, gain control, overload shutdown via DCC-100 controller of TCSPC system
Detectors (optional) bh MW FLIM GaAsP Multi-Wavelength FLIM detector
Spectral range 380 to 700nm
Number of wavelength channels 16
Spectral width of wavelength channels 12.5 nm
IRF width with bh diode laser 200 to 250 ps
Power supply and overload shutdown via DCC-100 controller of TCSPC system
TCSPC System bh SPC‑150, SPC‑150N, or SPC-160 modules, see  for details
Number of parallel modules (recording channels) 2
Number of detector (routing) channels in each module 16 (for multi-spectral FLIM detector)
Principle Advanced TAC/ADC principle 
Electrical time resolution 2.3 ps rms
Minimum time channel width 813 fs
Dead time 100 ns
Saturated count rate 10 MHz per channel
Dual-time-base operation via micro times from TAC and via macro time clock
Source of macro time clock internal 40MHz clock or from laser
Input from detector constant-fraction discriminator
Reference (SYNC) input constant-fraction discriminator
Synchronisation with scanning via frame clock, line clock and pixel clock pulses
Scan rate any scan rate
Synchronisation with laser multiplexing via routing function
Recording of multi-wavelength data simultaneous, via routing function
Basic acquisition principles on-board-buildup of photon distributions
buildup of photon distributions in computer memory
generation of parameter-tagged single-photon data
online auto or cross correlation and PCH
Operation modes f(t), oscilloscope, f(txy), f(t,T), f(t) continuous flow
FIFO (correlation / FCS / MCS) mode
Scan Sync In imaging, Scan Sync In with continuous flow
FIFO imaging, with MCS imaging, mosaic imaging, time-series imaging
Multi-detector operation, laser multiplexing operation
cycle and repeat function, autosave function
Max. Image size, pixels (SPCM 64 bit software) 2048x2048 1024x1024 512x512
No of time channels, see  256 1024 4096
Data Acquisition Software, please see  for details
Operating system Windows 7 or Windows 10, 64 bit
Loading of system configuration single click in predefined setup panel
Start / stop of measurement by operator or by timer, starts with start of scan, stops with end of frame
Online calculation and display, FLIM, PLIM in intervals of Display Time, min. 1 second
Online calculation and display, FCS, PCH in intervals of Display Time, min. 1 second
Number of images diplayed simultaneously max 8
Number of curves (Decay, FCS, PCH, Multiscaler) 16 in one curve window
Cycle, repeat, autosave functions user-defined, used for
for time-series recording, Z stack FLIM,
microscope-controlled time series
Saving of measurement data User command or autosave function
Optional saving of parameter-tagged single-photon data
Link to SPCImage data analysis automatically after end of measurement or by user command
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