DCS-120 Confocal and Multiphoton FLIM Systems
SPCImage NG FLIM Data Analysis
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 bhs 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 Olympus. It can also be used to convert an existing
conventional microscope into a fully functional confocal or multiphoton laser
scanning microscope with TCSPC detection. Due to its fast beam scanning and its
high sensitivity the DCS-120 system is compatible with live-cell imaging.
DCS-120 functions include simultaneous recording of FLIM or steady-state
fluorescence images simultaneously in two fully parallel wavelength channels,
laser wavelength multiplexing, time-series FLIM, time-series recording, Z stack
FLIM, phosphorescence lifetime imaging (PLIM), fluorescence lifetime-transient
scanning (FLITS) and FCS recording. Applications focus on lifetime variations
by interactions of fluorophores with their molecular environment. Typical
applications are ion concentration measurement, FRET experiments, metabolic
imaging, and plant physiology.
systems are complete laser scanning microscopes for fluorescence lifetime imaging.
The systems use bhs multi-dimensional TCSPC FLIM technology [25, 31, 33] 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 and Fig. 2. A DCS-120
MACRO system is available for FLIM of centimetre-size objects, see Fig. 2,
second row, right. Advanced versions of the DCS-120 system are available for
multiphoton excitation with Ti:Sa lasers and femtosecond fibre lasers (Fig. 2,
bottom). The system also works with tuneable excitation sources [7, 9, 10]. Moreover, the DCS-120 scan head
with the associated control and data acquisition electronics can be used to
upgrade a conventional microscope with scanning and FLIM recording.
Fig. 1: The DCS‑120 system with a Zeiss Axio Observer microscope
Fig. 2: Upper
row: DCS-120 Axio Observer system, DCS‑120 MACRO system. Lower row:
DCS-120 MP multiphoton system with Ti:Sa laser, DCS-120 MP
multiphoton with femtosecond fibre laser
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, 79]. 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, 79]. Advanced
versions of the DCS-120 system are available for multiphoton excitation and
tuneable excitation sources [6, 7].
Principle of Data Acquisition
The bh FLIM systems use a combination of
bhs 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.
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
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.
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 Olympus. The DCS‑120 MACRO system
scans macroscopic objects directly in the image plane of the scan head. The DCS
system can be used with a variety of different lasers and detectors. It can be
operated with ps diode lasers of various wavelength, with tuneable excitation
sources, and with fs lasers for multiphoton excitation.
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
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.
excitation, two-photon excitation and descanned and non-descanned detectors can
be combined in one system. In that case, a ps diode laser is injected via the
second laser port, an the one-photon images are detected by confocal detectors.
By enabling either the non-descanned detectors or the confocal detectors the
system can be switched from one-photon and multiphoton operation and vice
64-bit SPCM Data Acquisition Software
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, 31, 79]. The main panel of the SPCM data acquisition software is configurable
by the user . Different configurations for FLIM systems are shown in 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
Megapixel FLIM Images in Two Parallel Channels
With 64 bit SPCM software pixel numbers can
be increased to 2048 x 2048 pixels, with a temporal resolution of 256
time channels. Images are shown in Fig. 7 and Fig. 8 (facing page).
Fig. 7: BPAE sample (Invitrogen) scanned
with 2048 x 2048 pixels. Green channel, 485 to 560 nm
The DCS-120 system is able to
simultaneously record two high-resolution images in different wavelength or polarisation
channels, see Fig. 7 and Fig. 8. Recording is performed in two fully parallel
TCSPC channels, avoiding any electronic lifetime or intensity crosstalk. Even
if one channel should saturate the other is still producing correct data.
The capability to record images of large
pixel numbers is beneficial for a wide range of FLIM applications. One example
is tissue imaging where the samples are large, and the images are containing a
wealth of detail. It is also useful 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 exactly 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. 8: BPAE sample (Invitrogen),
scanned with 2048 x 2048 pixels. Red channel, 560 to 650 nm
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. 9. 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
Fig. 9: 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.
Interactive Scanner Control
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.
Interactive scanner control
Automatic Scanner Speed
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. 11: Automatic selection of scan
Easy Change Between Instrument Configurations
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. 12.
Fig. 12: Changing between different instrument configurations: The DCS-120
system switches from a FLIM configuration into an FCS configuration by a simple
Fast Preview Function
When FLIM is applied to live samples the
time and excitation dose 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. 13.
Fig. 13: SPCM software in fast preview
mode, display rate one image per second.
Online Lifetime Display
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
Fast Beam Scanning - Fast Acquisition
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 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. 15.
Fig. 15: 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. 16.
Fig. 16: Bacteria in motion. Autofluorescence, acquisition speed 2 images
per second, scan speed 6 frames per second
The DCS-120 system can be combined with the
bh FASTAC Fast-Acquisition FLIM system. The FASTAC system uses four parallel TCSPC
channels and a device that distributes the photon pulses of a single detector
into the four recording channels. For details please see [17, 18] and .
Fig. 17: Left: FASTAC 1024x1024-pixel image recorded with a DCS-120 system.
Acquisition time 10 seconds. Right: 512x512-pixel image recorded with a DCS-120
MACRO system. Acquisition time 2 seconds.
The bh HPM‑100‑40 GaAsP hybrid
detectors of the DCS‑120 combine ultra-high 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 and from the non-descanned beam path of the
DCS-120 MP system . In contrast to conventional PMTs or SPADs there is no
secondary peak or diffusion tail in the temporal response. Importantly, 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. The combination of
these features makes it easy to detect fluorescence from endogenous
fluorophores in single cells and split the decay curves into several decay
components, see Fig. 18.
Fig. 18: Autofluorescence lifetime image of NADH in single cells. Lifetime
image of mean lifetime of double exponential decay (left) and image of
amplitude of fast decay component, a1 ( right).
Sub-20 ps Time Resolution
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 .
Fig. 19 shows NAD(P)H FLIM recorded with a
DCS-120 MP multiphoton system. It shows an image of the mean lifetime, tm, an
image of the amplitude ratio, a1/a2, the system IRF, and a decay curve from the
cursor position in the images.
Fig. 19: NAD(P)H imaging of live cells. Image of
the mean lifetime, tm, image of the amplitude ratio, a1/a2, system IRF and
decay curve at the cursor position.
High Time Resolution, Low Background
The pinhole of a confocal system not only
suppresses out-of-focus fluorescence but also roomlight background and optical
reflections. The decay data are therefore extraordinarily clean. Moreover, with
the fast bh HPM-100-06 and -07 hybrid detectors the temporal instrument
response function is essentially given by the laser pulse width. With the
375 nm, the 405 nm diode lasers the IRF width is less than 40 ps
FWHM. A system IRF and a decay curve recorded with the HPM-100-06 and a diode
laser are shown in Fig. 20. Please note the smooth residuals of the fit, an
indication that the data are free of optical reflections.
Fig. 20: Left: IRF with HPM-100-06 and bh 405 nm ps diode laser.
Right: Fluorescence decay recorded with HPM-100-06 detector and bh 375 nm
Wide Range of Excitation Wavelengths
The DCS-120 confocal system can be used
with a wide range of excitation wavelengths. Available diode-laser wavelengths
range from 375 nm for excitation of NADH to 785 nm for excitation of
NIR dyes. An NADH (autofluorescence) image is shown in Fig. 21, an image of a
pig skin sample incubated with 3,3-diethylthiatricarbocyanine in Fig. 22.
UV-Excitation FLIM. NADH image of cells, excitation 370 nm, detection 420
to 475 nm.
Fig. 22: Near-Infrared FLIM. Pig skin sample stained with
3,3-diethylthiatricarbocyanine, detection wavelength, excitation 690 nm,
detection wavelength from 780 nm to 900 nm.
In the DCS-120 WB wideband version
lasers can be swapped without the need of re-alignment. The wideband system can
even be used with tuneable excitation. Images obtained with a Toptica Ichrome
laser  are shown in Fig. 23.
Fig. 23: 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.
Autofluorescence FLIM of Small Organisms
The wide range of excitation and detection
wavelengths and the high sensitivity makes the DCS-120 an excellent system for
autofluorescence FLIM of small organisms. Fig. 24 shows an autofluorescence
image of Artemia salinas, a small shrimp living in briny water. The excitation
wavelength was 405 nm, the detection wavelength from 420 nm to
Fig. 24: Autofluorescence FLIM of Artemia salinas, DCS-120 confocal
system with HPM-100-40 hybrid detectors and SPC-180 TCSPC modules
Laser Wavelength Multiplexing
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 lasers and 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. 25.
Fig. 25: 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
The most frequent application of excitation
wavelength multiplexing is metabolic FLIM, where NAD(P)H and FAD data have to
be acquired simultaneously, see [3, 31]. NADH and FAD Images recorded by
multiplexed excitation is shown in Fig. 26.
Fig. 26: Laser-multiplexed FLIM of NADH and FAD
DCS‑120 MP: Multiphoton FLIM with Ti:Sa or
Femtosecond Fibre Laser
Multiphoton FLIM is used when images from
deep layers of biological tissue have to be imaged. A two-photon FLIM image of Artemia
salinas (a small shrimp) recorded by the DCS‑120 MP system is
shown in Fig. 27. The image was recorded with the Femto-Fibre-Pro (fibre-laser
version) of the DCS-120 MP .
Fig. 27: Autofluorescence FLIM image of Artemia salinas, a brine
shrimp. Mean (amplitude-weighted) lifetime of double-exponential decay. Decay
functions of selected areas shown on the right.
FLIM images of pig skin in different depth
of the tissue are shown in Fig. 28. The images were recorded by a DCS-120 MP
with a Ti:Sa-Laser.
Fig. 28: Pig skin, autofluorescence, image in different depth in the
sample. Amplitude-weighted lifetime of triple-exponential decay model
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.
Multiphoton NADH FLIM with Ultra-Fast Detectors
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, 31]. 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. 29.
Fig. 29: 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.
FLIM of Ultra-Fast Fluorescence-Decay Processes in
Due to the short pulse width of the
femtosecond lasers the DCS-120 MP system delivers extremely high temporal
resolution. An example is shown in Fig. 30. The image shows mushroom spores of Boletus
edulis . The image was recorded by a DCS-120 MP with a Femto-Fibre-Pro
laser (Toptica), ultra-fast HPM-100-06 detectors, and SPC-150NX TCSPC modules.
The data show clearly a decay component of 20 ps lifetime.
Fig. 30: Spores of Boletus edulis. Left to right: Image of fast
decay component, t1, of triple-exponential decay model, histogram of
t1, and decay curve in selected spot. Data from .
Integrated Control of Ti:Sa Laser and AOM
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.
Fig. 31: Ti:Sa Laser and AOM control panel
The DCS-120 Metabolic FLIM system is based
on simultaneous recording of lifetime images of NAD(P)H (nicotinamide adenine
(pyridine) dinucleotide) and FAD (flavin adenine dinucleotide). The metabolic
FLIM system uses laser wavelength multiplexing to simultaneously record lifetime
images of the two fluorophores. By multi-exponential decay analysis of the NADH
and FAD signals, the system delivers information on the metabolic state of the
cells or the tissue investigated. Please see [3, 31].
Fig. 32: NADH and FAD images, recorded
simultaneously by the DCS-120 metabolic-FLIM system
In metabolic FLIM, the primary information
is not in the mean lifetime, tm, but in the amplitudes of the decay components,
a1 and a2. Due to its superior photon efficiency and time
resolution the DCS-120 metabolic FLIM system provides such images at
unprecedented accuracy. An example is shown in Fig. 33.
Fig. 33: NADH and FAD images, showing the amplitude of the fast decay
With the bh multispectral FLIM detectors
the DCS‑120 records FLIM simultaneously in 16 wavelength channels [23, 26, 31]. 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, 16 images in 16 wavelength channels can be recorded at a
resolution of 512x512 pixels and 256 time channels. An example is shown in Fig.
Fig. 34: 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. 35 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. 34, 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. 36.
Fig. 35: Two images from the array shown in Fig. 34, 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. 36: Decay curves at selected pixel position in the images shown above.
Blue dots: Photon numbers in the time channels. Red curve: Fit with a
Z Stack Recording by Record-and-Save Procedure
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. 37.
Fig. 37: Z stack recording, part of a
water flee, autofluorescence. 14 steps in Z, step width 4 um.
Z-Stack recording by Mosaic FLIM
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. 38: 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
Time-Series FLIM by Record-and-Save Procedure
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. 39. The fluorescence lifetime of the chloroplasts changes due to
the Kautski effect induced by the illumination.
Fig. 39: Time-series FLIM, 2 images per second. Chloroplasts in a leaf, the
fluorescence lifetime of the chlorophyll decreases with the time of exposure.
Recording of Dynamic Effects by Mosaic FLIM
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 [31, 34]. 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. 40 shows the change of the lifetime of chlorophyll in
plant tissue with the time of illumination.
Fig. 40: 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.
FLITS: Fluorescence Lifetime-Transient Scanning
FLITS records dynamic 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 [31, 32].
Fig. 41: 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
The technique has been used to record Ca++
transients in live neurons at a resolution of 2 ms, see [, 54].
Phosphorescence and fluorescence lifetime
images are recorded simultaneously by bhs proprietary FLIM/PLIM technique. The
technique is based on modulating a ps diode laser synchronously with the pixel
clock of the scanner [28, 31].
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 
Currently, there is increasing interest in
PLIM for background-free recording and for oxygen sensing [1, 2, 44, 45, 56, 67,
80]. 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. 42.
Fig. 42: 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, see Fig. 43. A typical
example is the study of migration, possible dissolution, and chemical reaction
of nanoparticles from sunscreens in skin. The results of such studies are
highly important to skin cancer research.
Fig. 43: Phosphorescence lifetime imaging of nanoparticles, here of the
phosphor of a cathode-ray tube. Left: Lifetime image. Right: Phosphorescence
decay curve at selected position within the image
FLIM of cm-Size Objects
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. Please see [3, 31, 75].
Fig. 44: Lifetime Images of tumor in
a mouse. NADH image, recorded by the DCS-120 Macro system.
DCS Macro with Motor Stage
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. 45.
Fig. 45: 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 . Images of (benign)
human skin lesions are shown in Fig. 46.
Fig. 46: 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. 47: 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
SPCImage NG is a
new generation of bh's TCSPC-FLIM data analysis software. It combines
time-domain and frequency-domain analysis, uses a maximum-likelihood (MLE)
algorithm to calculate the parameters of the decay functions in the individual
pixels, and accelerates the analysis procedure by GPU processing. 1D and 2D
parameter histograms are available to display the distribution of the decay
parameters over the pixels of the image or over selectable ROIs. Image
segmentation can be performed via the phasor plot. Pixels with similar phasor
signature can be combined for high-accuracy time-domain analysis. SPCImage NG
provides decay models with one, two, or three exponential components,
incomplete-decay models, and shifted-component models. Another important
feature is advanced IRF modelling, making it unnecessary to record IRFs for the
individual FLIM data sets. For detailed description please see [3, 31] and
SPCImage NG Overview Brochure . A typical main panel of SPCImage NG is
shown in Fig. 48.
Fig. 48: Example of SPCImage NG main panel. Combination of time-domain analysis (left and lower
right) and phasor plot (upper right)
Deconvolution and Fit Procedure
SPCImage NG runs
an iterative fit and de-convolution procedure on the decay data of the individual
pixels of the FLIM images. In the simplest case, the result is the lifetime of
the decay functions in the individual pixels. For complex decay functions the
fit procedure delivers the lifetimes and amplitudes of the decay components.
SPCImage then creates colour-coded images of the amplitude- or
intensity-weighted lifetimes in the pixels, images of the lifetimes or
amplitudes of the decay components, images of lifetime or amplitude ratios, and
images of other combinations of decay parameters, such as FRET intensities,
FRET distances, bound-unbound ratios, or the fluorescence-lifetime redox ratio,
FLIRR. A few examples are shown in Fig. 49 and Fig. 50. For details please see [3, 31, 20].
Fig. 49: Cell with interacting proteins, labelled with a FRET donor and a
FRET acceptor. Left to right: Classic FRET efficiency, fraction of interacting
donor, FRET distance
Fig. 50: Metabolic FLIM. Bound-unbound ratio of NADH, Bound/unbound ratio
of FAD, Fluorescence-Lifetime Redox Ratio, FLIRR.
SPCImage NG uses GPU (Graphics Processor
Unit) processing. GPU processing is running on NVIDIA cards and a number of
other NVIDIA-compatible devices. The TCSPC data are transferred into the GPU,
which then runs the de-convolution and fit procedure for a large number of
pixels in parallel. This way, data processing times for large images are
reduced from formerly more than 10 minutes to a few seconds.
SPCImage versions, SPCImage NG uses a maximum-likelihood algorithm (or
maximum-likelihood estimation, MLE) for fitting the data. In contrast to the
usual least-square fit, the MLE algorithm takes into account the Poissonian
distribution of the photon numbers. Compared to the least-square method, the
fit accuracy is improved especially for low photon numbers, and there is no
bias toward shorter lifetime as it is often observed for the least-square fit.
For comparison with older data sets the weighted least-square fit and the
first-moment algorithms of the previous SPCImage versions are still available
in SPCImage NG.
avoids troublesome recording of an instrument response function (IRF) for each
FLIM measurement. This is achieved by modelling the IRF with a generic function.
The parameters of this function are determined by fitting it to the FLIM data
together with the selected decay model. The results of this procedure are so
good that an accurate IRF is obtained even for decay functions containing
ultra-fast components, see Fig. 51. For details please see SPCImage chapter in
Fig. 51: Synthetic IRF. Left: Autofluorescence of cells, ps diode laser,
HPM-100-40. Right: Sample with extremely fast decay component, femtosecond
fibre laser, HPM-100-06
SPCImage NG combines time-domain multi-exponential
decay analysis with a 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 the phasor plot. Different phasor clusters can
be selected, and the corresponding pixels back-annotated in the time-domain
FLIM images. 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 . An example is shown in Fig. 52.
Fig. 52: Combination of time-domain analysis with phasor plot. Left to
right: Lifetime image, phasor plot, decay curve of combined pixels within
selected phasor range
SPCImage NG therefore provides automatic
image segmentation functions via the phasor plot and 2D histograms of the decay
parameters. Areas with different decay signature form separate clusters in
these presentations. Interesting clusters can be selected and back-annotated in
the images. The decay data of the corresponding pixels are combined into a
single decay curve with extremely high photon number. Multi-exponential decay
analysis on the combined data delivers precision decay parameters even if the
photon numbers in the individual pixels are low. An example is shown in Fig. 53.
Fig. 53: Image segmentation on temporal-mosaic FLIM data of a live water
SPCImage can also be used to analyse single
decay curves, The data can come from traditional cuvette experiments, or from
the combined pixels of a FLIM recording. An example is shown in Fig. 54.
Fig. 54: SPCImage used for fluorescence decay analysis of single curves.
NADH dissolved in water, recorded with bh DCS-120 MP FLIM system.
FLIM uses the fluorescence decay function
of a fluorophore as an indicator of its molecular environment. The fluorescence
decay function, within reasonable limits, neither depends on the fluorophore
concentration nor on the excitation power, or other instrumental details. This
is a striking advantage over intensity-based imaging techniques. If fluorescence
in a sample is excited (Fig. 55, left) the emission intensity (second left) depends
not only on possible interaction of the fluorophore with the molecular
environment but also on the fluorophore concentration, on possible absorption
in the sample, on the excitation power, and on the light collection efficiency
of the optics. Changes in the molecular environment can thus not be
distinguished from changes in these parameters. 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 however (Fig. 55, right), only
depends on the interaction of the fluorophore with the molecular environment.
Fig. 55: 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
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 [31, 37,
64]. Common FLIM applications are ion concentration measurements, probing of
protein interaction via FRET, and the probing of the metabolic state and the cell
viability via the fluorescence decay parameters 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 molecular interaction 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. 56, left. FRET results in an extremely efficient quenching
of the donor fluorescence and, consequently, in a considerable decrease of the
donor lifetime, see Fig. 56, right.
Fig. 56: Fluorescence Resonance Energy
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 [64 Lakowicz Principles 1999]. FRET is therefore 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. There are hundreds of publications using FLIM FRET, please see 
Fig. 57 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. 57, 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. 57,
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, 25, 31] for details and for further
Fig. 57: 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 [69König Riemann JBO
2003; Marcu in Mycek; Richards-Kortum in Mycek; Schweitzer Hammer Schweitzer
Anders JBO 2004; Urayama & Mycek in Mycek; Wagnieres in Mycek; Zheng
in Mycek;]. Fluorescence lifetime imaging improves the
contrast of separation of the different fluorophores. Moreover, TCSPC FLIM is
more and more introduced into clinical applications. In these applications
label-free imaging is needed because staining the tissue with exogenous
fluorophores is either not possible or not permitted. FLIM delivers a wealth of
additional image information which often makes the use of exogenous
fluorophores unnecessary [42, 60, 70, 73, 72, 76].
The most important capability of
autofluorescence FLIM is, however, that it delivers information on the
metabolic state of the cells or the tissue under investigation. The most promising
signals come from NAD(P)H (nicotinamide adenine (pyridine) dinucleotide) and
FAD (flavin adenine dinucleotide). NAD(P)H and FAD are coenzymes involved in
the cell metabolism. NAD(P)H and FAD are unique in the sense that their
fluorescence intensities and fluorescence decay functions bears direct
information on the metabolic state of the cells.
The fluorescence lifetimes of NAD(P)H and
FAD depend on the binding to proteins [63, 64, 68]. Unbound NAD(P)H has a
fluorescence lifetime of about 0.3 to 0.5 ns. When NAD(P)H binds to
proteins the lifetime typically increases to a few ns . For FAD the effect
of binding is opposite: Bound FAD has a lifetime of a few 100 ps, unbound
FAD of a few ns. The resulting decay functions are thus double exponential.
Typical decay functions of NAD(P)H and FAD in cells are shown in Fig. 58.
Fig. 58: Typical decay functions of NAD(P)H (left) and FAD (right). The
blue dots are the data points, the red curve is a fit with a double-exponential
decay model. Recorded from human epithelium cells by bh metabolic FLIM system .
The ratio of the amplitudes of the decay
components, a1/a2, often called amplitude ratio, directly represents the
concentration ratio of unbound/bound NADH or bound/unbound FAD, see Fig. 59.
The ratios of bound to unbound NAD(P)H and unbound to bound FAD depends on the
type of the metabolism. A cell can run both a oxidative metabolism (oxidative
phosphorylation) and a reductive one (glycolysis). A shift from oxidative
phosphorylation to glycolysis is accompanied by a decrease of the bound
fractions of NAD(P)H and FAD. The resulting change in the a1/a2 ratio is shown
schematically in Fig. 59, compare upper row and lower row.
Fig. 59: The composition of the decay functions of NAD(P)H and FAD and
changes with the metabolic state. Effect of metabolic state on the decay curves
The effects shown in Fig. 59, left are seen
in many NAD(P)H FLIM recordings. A few examples are shown in Fig. 60. The
figure shows FLIM images of the amplitude-weighted lifetimes, tm, (upper row)
and amplitudes of the fast decay component, a1, of various cells. The left
three cells are tumor cells, the right two are normal ones. In agreement with Fig.
59, there is a clear trend in the tm and the a1 from the
tumor cells to the normal ones.
Fig. 60: Images of amplitude-weighted lifetimes, tm, (upper row) and
amplitudes, a1, of various cells. The left three cells are tumor cells, the
right two are normal.
FAD FLIM images comparing tumor cells and
normal cells are still relatively rare. As shown in Fig. 59, the change in the
lifetime and in the amplitudes for the FAD should go into the opposite
direction as for the NAD(P)H. Although there is still some controversy about
the direction of the effect, the opposite lifetime change is confirmed by
results of the group of Melissa Skala at Vanderbilt University  see Fig. 61.
Fig. 61: Change in the mean (amplitude-weighted) lifetime of NADH and FAD
with the state of the cells, from normal (oxidative phosphorylation) to
high-grade tumor (glycolysis). With permission, from .
The opposite changes in the decay curves of
NAD(P)H and FAD are a possible explanation of numerous discrepancies in
fluorescence lifetimes measured in tumours. There have been as many reports for
decreased lifetime as for increased ones. The source of the discrepancies is
probably that the measurements did not cleanly separate the signals from NADH
and FAD. This can easily happen because both the excitation and the emission
spectra are strongly overlapping. Depending on which fluorophore dominates the
net decay function the result can indeed be a decrease or an increase of the
lifetime in the tumor compared to normal tissue. The problem can be solved by using
the correct excitation and emission wavelengths. The DCS system provides laser
multiplexing functions to perform the recordings quasi-simultaneously, thus
avoiding errors by photobleaching or metabolism-induced dynamic effects during
the measurement. For examples see [3, 31. There is an increasing number of papers
about NADH / FAD FLIM [38, 39, 51, 64, 65, 66, 73, 77, 78, 81, 83 Lakowicz 1992, Lakowicz 1999; Paul &
Schneckenburger, Naturwissenschaften 1996]. Please see  or  for
more references and for a detailed discussion of the technique.
Oxygen sensing is based on the quenching of
the phosphorescence decay of (endogenous) phosphorescent dyes by oxygen. Two
examples are shown in Fig. 62. The figure shows PLIM images of cultured human
embryonic kidney cells incubated with a palladium-based phosphorescence dye. Fig.
62, left was recorded under atmospheric oxygen partial pressure. The maximum of
the lifetime distribution over the pixels (upper right) is at 75 µs. Fig. 62,
right, was recorded under decreased oxygen partial pressure. As can be seen,
the maximum of the lifetime distribution has shifted to 144 µs.
Fig. 62: HEK cells incubated with a palladium dye imaged under different
oxygen partial pressure. Left: Atmospheric O2 pressure. Right: reduced oxygen
partial pressure. Recorded by bh DCS‑120 confocal scanning system, data
analysis by bh SPCImage. Lifetime scale 0 (red) to 300 µs (blue).
Phosphorescence lifetime at the Cursor-Position 65 µs. The maximum of the
lifetime distribution over the pixels is at 75 µs.
The DCS-120 system is able to record
phosphorescence and fluorescence lifetime images simultaneously . The
function can be used to obtain metabolic information from the NAD(P)H and
compare the results with the oxygen concentration in the cells [57, 62, 74].
Simultaneously recorded fluorescence and phosphorescence lifetime images of
live cultured human embryonic kidney cells stained with
5,10,15,20-tatrakis(4-carboxyphenyl)porphyrin-Pd(II) are shown in Fig. 63. The
FLIM and PLIM images on the left were recorded under atmospheric oxygen
concentration. The images on the right were recorded after adding sodium sulphite
(Na2SO3). Na2SO3 binds oxygen. The
oxygen concentration in therefore low. It can clearly be seen that the phosphorescence
lifetime became longer, indicating there is indeed less oxygen (please note
different colour scales). In the FLIM image, it can be seen that the
amplitude-weighted lifetime, tm, of the NADH has decreased.
Fig. 63: Simultaneous FLIM/PLIM of human embryonic kidney cells. Left: High
(atmospheric oxygen concentration). Right: Low oxygen concentration. Upper row
FLIM and PLIM images, lower row decay curves in indicated position.
Lifetime-colour scale given underneath the images. bh DCS-120 confocal
scanning FLIM system, excitation wavelength 375 nm. Adapted from
Shcheslavskiy et al. .
Two examples of FLIM of plant tissue are shown in Fig. 64 and Fig. 65
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. 64.
Fig. 64: 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. 65.
Fig. 65: Change of the fluorescence lifetime of chlorophyll with time of
exposure. Moss leaf, excitation at 445 nm, 256x256 pixels, 1 image per
Faster effects down to the millisecond time
scale can be recorded by temporal mosaic FLIM or FLITS, [31, 32] see Fig. 40 and
Fig. 41 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
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
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
Y scan 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
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 from 400nm to 800nm
fs NIR Lasers for multiphoton operation any
Coupling requirements free
beam, diameter 1 to 2 mm
to 1200 nm
Detectors (standard) bh
HPM-100-40 hybrid detector
Spectral Range 300
Peak quantum efficiency 40
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
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
Peak quantum efficiency 12
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
Number of wavelength channels 16
Spectral width of wavelength channels 12.5
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
Number of detector (routing) channels in
each module 16 (for multi-spectral FLIM detector)
TAC/ADC principle 
Electrical time resolution 2.3 ps
Minimum time channel width 813
Dead time 100 ns
Saturated count rate 10 MHz
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
Reference (SYNC) input constant-fraction
Synchronisation with scanning via
frame clock, line clock and pixel clock pulses
Scan rate any
Synchronisation with laser multiplexing via
Recording of multi-wavelength data simultaneous,
via routing function
Basic acquisition principles on-board-buildup
of photon distributions
of photon distributions in computer memory
of parameter-tagged single-photon data
auto or cross correlation and PCH
Operation modes f(t),
oscilloscope, f(txy), f(t,T), f(t) continuous flow
(correlation / FCS / MCS) mode
Sync In imaging, Scan Sync In with continuous flow
imaging, with MCS imaging, mosaic imaging, time-series imaging
operation, laser multiplexing operation
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 
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,
time-series recording, Z stack FLIM,
Saving of measurement data User
command or autosave function
saving of parameter-tagged single-photon data
Link to SPCImage data analysis automatically
after end of measurement or by user command
1. E. Baggaley, S. W. Botchway, J. W. Haycock, H. Morris, I. V.
Sazanovich, J. A. G. Williams, J. A. Weinstein, Long-lived metal complexes open
up microsecond lifetime imaging microscopy under multiphoton excitation: from
FLIM to PLIM and beyond. Chem. Sci. 5, 879-886 (2014)
2. E. Baggaley, M. R. Gill, N. H. Green, D. Turton, I. V. Sazanovich,
S. W. Botchway, C. Smythe, J. W. Haycock, J. A. Weinstein, J. A. Thomas,
Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission
Microscopy Probes for Cellular DNA. Angew. Chem. Int. Ed. Engl. 53, 3367-3371
Becker & Hickl GmbH, DCS-120 Confocal and
Multiphoton FLIM Systems, user handbook, 9th edition 2021. www.becker-hickl.com, printed copies available
4. Becker & Hickl GmbH, The HPM‑100-40 hybrid detector.
Application note, available on www.becker-hickl.com
5. Becker & Hickl GmbH, Spatially resolved recording of
fluorescence-lifetime transients by line-scanning TCSPC. Application note,
available on www.becker-hickl.com
6. Becker & Hickl GmbH, DCS-120 Confocal Scanning FLIM System:
Two-Photon Excitation with Non-Descanned Detection. Application note, available
7. Becker & Hickl GmbH, DCS-120 Confocal FLIM system with wideband
beamsplitter. Application note, available on www.becker-hickl.com
8. Becker & Hickl GmbH, Non-Descanned FLIM Detection in
Multiphoton Microscopes. Application note, available on www.becker-hickl.com
9. Becker & Hickl GmbH, DCS-120 Confocal Scanning FLIM System with
User-Specific Lasers: FIANIUM SC400 Laser. Application note, available on
10. Becker & Hickl GmbH, DCS-120 Confocal Scanning FLIM System with
User-Specific Lasers: NKT SuperK EXTREME Laser. Application note, available on
11. Becker & Hickl GmbH, Megapixel FLIM with bh TCSPC Modules -
The New SPCM 64-bit Software. Application note, available on
12. TCSPC at Wavelengths from 900 nm to 1700 nm. Application
note, available on www.becker-hickl.com
13. Becker & Hickl GmbH, Simultaneous Phosphorescence and
Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse
Excitation. Application note, available on www.becker-hickl.com
14. Becker & Hickl GmbH, SPCM software runs online-FLIM at 10
images per second. Application note, available on www.becker-hickl.com
15. Becker & Hickl GmbH, Sub-20ps IRF Width from Hybrid
Detectors and MCP-PMTs. Application note, available on www.becker-hickl.com
16. Becker & Hickl GmbH, Ultra-fast HPM Detectors Improve
NAD(P)H FLIM. Application note, available on www.becker-hickl.com
17. Becker & Hickl GmbH, Fast-Acquisition TCSPC FLIM System
with sub-25 ps IRF width. Application note, available from www.becker-hickl.com
18. Becker & Hickl GmbH, Fast-Acquisition TCSPC FLIM: What are
the Options? Application note, available from www.becker-hickl.com
19. Becker & Hickl GmbH, New SPCImage Version Combines Time-Domain
Analysis with Phasor Plot. Application note, available on www.becker-hickl.com
20. Becker & Hickl GmbH, SPCImage next generation FLIM data
analysis software. Overview brochure, available on www.becker-hickl.com
21. Becker & Hickl GmbH, Two-Photon FLIM with a Femtosecond Fibre
Laser. Application note, available on www.becker-hickl.com
22. Becker & Hickl GmbH, Modular FLIM systems for Zeiss
LSM 510 and LSM 710 family laser scanning microscopes. User handbook.
Available on www.becker-hickl.com
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36. W. Becker, C. Junghans, A. Bergmann, Two-Photon FLIM of Mushroom
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