The bh TCSPC Technique
Principles and Applications
Becker & Hickl
GmbH, Berlin, Germany
from a discussion of the peculiarities of high-resolution low-level optical
signal recording, this article describes the recording process of classic TCSPC
and its extension, multi-dimensional TCSPC. It shows why TCSPC reaches a time
resolution, sensitivity, and photon efficiency beyond the reach of any other
optical signal recording technique. The article then passes to the general
features of the bh TCSPC technique: Outstanding time resolution, outstanding
timing stability, and a unsurpassed variety of multi-dimensional recording
principles. These features are demonstrated on examples of high-resolution
fluorescence decay recording, multi-detector operation and laser multiplexing,
simultaneous fluorescence and phosphorescence recording, parameter-tag
recording, FLIM, multi-wavelength FLIM, spatial and temporal mosaic FLIM, and
Time-correlated single photon counting
(TCSPC) is an amazingly sensitive technique for recording low-level light
signals with extremely high time resolution and extremely high precision. It is
based on the detection of single photons, the measurement of the times of the
photons after the reference (usually excitation) pulses, and the construction
of the waveform of the optical signal from the photon times [12, 44].
TCSPC has been derived from the delayed
coincidence method for the measurement of excited nuclear state lifetimes .
The technique has been used since the 60s of the last century. For many years
TCSPC was used primarily to record fluorescence decay curves of organic dyes in
solution [31, 39, 40, 50, 53]. Due to the low intensity and low repetition rate
of the light sources and the limited speed of the electronics of the 70s and
80s the acquisition times were extremely long. More important, classic TCSPC was
intrinsically one-dimensional, i.e. limited solely to the recording of the
waveform of the light signal.
Light sources ceased to be a limitation
when the first mode-locked Argon lasers and synchronously pumped dye lasers
were introduced. For the recording electronics, the situation changed with the
introduction of the SPC-300 modules of Becker & Hickl in 1993. Due to a new
Time-to-Amplitude and Analog-to-Digital Conversion (TAC/ADC) principle these
modules worked at photon count rates almost 100 times higher than previous
TCSPC devices. Time resolution (or IRF width) improved from about 50 ps
for classic devices to 15 ps for the SPC-300. Currently, the fastest bh
TCSPC modules reach <3 ps (FWHM) internal IRF width, and a time-channel
width down to 203 femtoseconds.
Another novelty introduced by bh was the extension
of the classic TCSPC process to multi-dimensional recording. Already the SPC-300
and SPC-330 modules recorded photon distributions not only over the time in a
fluorescence decay but simultaneously over the wavelength of the photons or
over a spatial coordinate. Within a few years, bh added more and more dimensions
to multidimensional TCSPC. Fast sequential recording was introduced with the
SPC-430 in 1995, fast scanning with the SPC-535 in 1996. Time-tag recording was
introduced with the SPC-431 in 1996. FLIM for laser scanning microscopy was
introduced in 1999. Since then, the bh TCSPC systems became bigger, faster and
more complex. Recent TCSPC modules can be configured for sequential recording,
imaging, or time-tag recording by a simple software command. They can run
classic TCSPC experiments, FLIM, multi-wavelength FLIM, spatial and temporal
mosaic FLIM, FLITS, and simultaneous FLIM/PLIM. Multi-module systems, like the bh
SPC‑154, the bh Max‑Tau 12‑channel system, or the bh FASTAC
FLIM system, can be used for recording at unprecedented count rates and acquisition
speeds without compromise in time resolution.
Fig. 1: SPC-150NX TCSPC/FLIM module, SPC-180
TCSPC/FLIM Module, Simple-Tau 152 dual-channel TCSPC/FLIM system
Fig. 2: SPC-154 four-channel TCSPC/FLIM
module, Power-Tau 4-channel TCSPC system, MAX-Tau 12 Channel TCSPC system
The TCSPC Recording Process
Detection of Low-Level Light Signals
Time-correlated single photon counting, or
TCSPC, is based on the detection of single photons of a periodic light signal,
the measurement of the detection times of the photons, and the reconstruction
of the waveform from the individual time measurements [12, 44]. TCSPC makes use of the fact that for low-level, high-repetition rate
signals the light intensity is low enough that the probability to detect more
than one photon in one excitation pulse period is negligible. The situation is
illustrated in Fig. 3.
Fig. 3: Detector signal for fluorescence
detection at a pulse repetition rate of 80 MHz
Fluorescence of a sample is excited by a
laser of 80 MHz pulse repetition rate (a). The expected fluorescence
waveform is (b). However, the detector signal, (as measured by an oscilloscope)
has no similarity with the expected fluorescence waveform. Instead, it is a sequence
of extremely narrow pulses randomly spread over the time axis (c). A signal
like this often looks confusing to users not familiar with photon counting.
However, there is a simple explanation: The pulses represent single photons of
the light signal arriving at the detector. The shape of the pulses has nothing
to do with the waveform of the light signal. It is the response of the detector
to the detection of a single photon.
The Classic TCSPC Process
There are two conclusions from the signal
shape in Fig. 3 (c). First, the waveform of the optical signal is not the
detector signal. Instead, it is the distribution of the detector pulses over
the time in the excitation pulse periods. Second, the detection of a photon
within a particular excitation pulse period is a relatively unlikely event. The
photon detection rate of (c) was about 107 s-1. This
is close to the maximum permissible count rate of most single-photon detectors.
A detection rate of 107 s-1 means that the
probability to detect a photon in one 80 MHz period is 0.125. The probability
to detect two photons is 0.0156, the detection of more photons is even less
likely. Therefore, only the first photon within a particular pulse period has
to be considered. The build-up of the photon distribution over the pulse period then
becomes a relatively straightforward process.
The principle is illustrated in Fig. 4,
left. When a photon is detected, the arrival time of the corresponding detector
pulse in the signal period is measured. The detection events are collected in a
memory by adding a 1 at an address proportional to the detection time. After
many signal periods a large number of photons has been detected, and the
distribution of the photons over the time in the signal period has been built
up. The result represents the waveform of the optical pulse.
All bh TCSPC modules are able to work by
the classic TCSPC process. However, the bh TCSPC devices are able to do much
more than that. Unlike classic TCSPC devices, they can record photon
distributions not only over the time in the excitation pulse period, but also
over additional parameters that are associated to the individual photons. This
can be the wavelength of a photon, the spatial location where it came from, the
time from the start of an experiment, the time within the period of a
stimulation of the sample, the time within the period of a modulation of the
excitation laser, or any other parameters that are determined or actively
controlled during the recording process [12, 16, 17]. The multi-dimensional
TCSPC process is illustrated in Fig. 4, right.
Fig. 4: Left: Classic TCSPC records a
distribution over the times of the photons after the excitation pulses. Right:
Multi-dimensional TCSPC records a photon distribution over the photon times and
one or several other parameters, here the wavelength of the photons.
By having both classic and
multi-dimensional TCSPC implemented, the bh SPC devices work for the classic
fluorescence decay applications as well as for anti-bunching experiments,
simultaneous fluorescence and phosphorescence decay measurement,
multi-wavelength fluorescence decay measurement, fluorescence lifetime imaging
(FLIM), multi-wavelength FLIM, simultaneous FLIM and PLIM, ultra-fast
time-series fluorescence decay recording, fast time-series FLIM, fluorescence
correlation (FCS), single-molecules experiments, and other multi-dimensional
photon recording tasks. Please see  for examples and applications.
Key Parameters of a TCSPC System
High Photon Efficiency - High Lifetime Accuracy
In a TCSPC device operated at reasonable
count rate all detected photons contribute to the result. There is no suppression
of photons due to gating as in Boxcar devices or gated image intensifiers,
and no variable weight as in sine-wave modulation techniques. TCSPC therefore
reaches a near-ideal photon efficiency.
The high photon efficiency of TCSPC
translates directly into the accuracy for fluorescence lifetime recording.
Under ideal conditions a single-exponential fluorescence lifetime can be determined
at a signal-to-noise ratio, SNR, of
from a number of detected photons, N. TCSPC
comes very close to the theoretical SNR [1, 37]. In combination with the fact
that TCSPC delivers the shortest possible IRF for a given detector (see below)
it yields the best possible lifetime accuracy (or Photon Efficiency) for a
given number of detected photons [1, 37].
Different than for an analog-recording
technique, the time resolution of TCSPC (both classic and multi-dimensional) is
not limited by the single-photon response of the detector. Instead, it is given
by the transit time jitter, or transit-time spread, of the detector-TCSPC
combination, see Fig. 5. The transit time spread is much shorter than the width
of the single-photon response. The difference can be enormous, as shown in Fig.
6 for a fast hybrid detector. The single-photon response of the detector is
about 1 ns wide (Fig. 6, left). However, the transit time jitter is less
than 20 ps, resulting in a correspondingly short TCSPC response (Fig. 6,
right, note different time scale).
Fig. 5: Single-photon response of detector and instrument response
function (IRF) of a TCSPC system. The IRF width is given by the transit-time
spread, not by the width of the single-photon response of the detector.
Fig. 6: Single electron response (SER, left) and TCSPC Instrument response
function (IRF, right) for a bh HPM-100‑06 hybrid detector. The TCSPC IRF
is more than 50 times faster than the SER. Note the different time scale.
The distribution of the recording events
over the time after a reference pulse for an infinitely short light pulse is
called the Instrument Response Function, or IRF. Please note that there are
different parameters to describe the time resolution of a TCSPC system: The
FWHM (Full Width at Half Maximum) describes the width of the IRF, the RMS (Root
Mean Square) describes the average timing jitter, or the standard deviation of
the photon times. For near-Gaussian IRF shapes the RMS jitter value is 2.5 to 3
times smaller than the FWHM of the IRF.
Signal theory demands the IRF to be sampled
with a density of data points that yields at least 10 data points on the IRF.
Only then the full information can be extracted from the recorded signals. Small
time-channel widths (or high effective sampling rates) can easily be obtained
by TCSPC, but not by analog-recording techniques. Please note that the
time-channel width is sometimes specified as time resolution. This is wrong -
the true time resolution is given by the FWHM of the IRF or the RMS timing
jitter. Oversampling a broad IRF with a large number time channels does not
result in real time resolution.
In many instances, timing stability (including
low-frequency timing wobble) is even more important than time resolution.
Examples are distance measurements and diffuse-optical imaging applications,
where changes in the mean time-of-flight of the photons on the order of a few
ps are recorded over tens of minutes. Even in standard fluorescence-decay
measurements timing drift matters: Timing shift between the fluorescence
recording and a related IRF recording transfers directly into the measured
fluorescence lifetime. Moreover, there are setups where an exact IRF is
difficult to record. Typical examples are confocal microscopes where dichroic
mirrors and filters block the detection path for the excitation wavelength. In
these cases, it is important that a stable IRF is maintained over a long period
of time. The bh TCSPC technique addresses these issues by extraordinarily low
timing drift and timing wobble, please see section below.
Maximum Count Rate
Both the classic and the multidimensional
TCSPC process require that the photon rate is lower than the excitation pulse
rate. This requirement and possible Pile-Up errors induced by high detection
rate are constant subject of controversy. We have, however, shown that
detection rates of up to 10% of the excitation pulse rate can be used without
inducing noticeable errors. In practice, the count rate is rather limited by
the photostability of the sample than by the pile-up limit of the TCSPC
Another controversial parameter is the
Dead Time of a TCSPC device. Dead time is the time the device needs to
process a single photon. If a new photon is detected within this time it is not
recorded. Consequently, there is a Counting Loss which increases with the
detector count rate. However, dead time (within reasonable limits) has also
positive effects. It helps suppress afterpulses of the detectors, and it avoids
an influence of the recording of a photon on the timing of the next one. The
dead time prevents the recording of photons that are too close to each other and
thus helps maintain a high time resolution and a high IRF stability over a wide
range of count rates. Please see  and  for details.
TCSPC techniques of the 1960s and 1970s
determined the photon times from the excitation pulse to the photon. With the
introduction of excitation sources with pulse periods of 8 to 12 ns the
timing was reversed . The reason is that it is technically difficult, if
not impossible, to start a time-measurement cycle every 8 ns, reset the
circuitry if no photon was detected, and start it again. Recent TCSPC devices
therefore start the time measurement with the photon, and measure the time to
the next excitation pulse or to the delayed excitation pulse [12, 16].
The bh SPC-150N, SPC-160N, and SPC-180N
series devices use a patented high-speed high-resolution TAC/ADC principle [12,
16]. The internal timing jitter is on the order of 2 to 3 ps (rms) for the
standard modules, 1.6 ps for the SPC-150NX modules, and 1.1 ps (rms)
for the SPC-150NXX boards, see Fig. 7. The FWHM IRF widths are 6.8 ps,
3.5 ps, and <3 ps, respectively. This is much better than for any
TCSPC device based on direct time-to-digital conversion (TDC) , and significantly
smaller than the timing jitter of the commonly used detectors. Moreover, the signals
are sampled with a sufficient number of sufficiently small time channels: The
minimum time-channel width for the standard SPC-N boards is 810 fs, 405 fs
for the SPC-NX board, and 202 fs for the SPC-NXXs board. It is thus
possible to resolve extrely fast decay processes . Please note that a 202-fs
time-channel width is equivalent to a sampling rate of 5 THz!
Fig. 7: Electrical IRF of an SPC-150NX (left) and SPC-150NXX (right)
IRF with Fast Detectors
Due to their short electrical IRF and small
time-channel width the bh TCSPC modules deliver unprecedented system IRF widths
with fast photon detectors. The FWHM instrument response width for fast hybrid
detectors, MCP PMTs and superconducting NbN detectors is in the sub-20 ps
range [2, 3]. With
single-nanowire NbN detectors less than 5 ps IRF width have been achieved
. Examples are shown in Fig.
Fig. 8: Instrument response functions for a fast hybrid detector, a superconducting
NbN detector, and a superconducting single-nanowire detector.
Different than TCSPC devices based on TDCs,
the bh devices are virtually free of IRF drift or low-frequency timing wobble. IRF
drift, if perceptible at all, remains below the electrical IRF width over
minutes or even hours. That indirectly means that the IRF neither broadens in
an experiment of long acquisition time nor shifts over a longer series of
recordings. Fig. 9, left and right, shows a series of 100 IRF recordings over
100 seconds. The horizontal axis is the TCSPC time axis, the vertical axis is
the time into the series. The variance in the time of the IRF centroid is less
than 0.4 ps. Overall timing drift of a real measurement system is shown in
Fig. 10. It shows two IRFs of an SPC-150NX system with a superconducting NbN
detector, recorded 5 minutes apart. The timing drift is less than the
time-channel width (405 fs for this measurement).
Fig. 9: Series of 100 (electrical) IRF recordings over 100 seconds. Left:
Result displayed as series of curves. Right: Colour-Intensity display. The
horizontal axis is the TCSPC time axis (bar indicates 8 ps), the vertical
axis is the time into the recording sequence. The variance in the centroid of
the IRF is less than 0.4 ps rms.
Fig. 10: Two IRFs of a TCSPC system with a superconducting NbN detector,
recorded 5 minutes apart (black and red dots). The drift is less than one
time-channel (405 fs).
Application: High-Resolution Fluorescence-Decay Recording
The most frequent application of classic
TCSPC is fluorescence-decay recording. A sample is excited with a
high-frequency pulsed laser, and the fluorescence decay functions are recorded
With their short IRF functions and high
timing stability, bh TCSPC devices record high-quality decay curves at
extremely high time resolution . Fig. 11 shows the fluorescence decay of a
rhodamine dye. The fluorescence was excited by a bh BDS-405 picosecond
diode laser, the photons were detected by a HPM-100-06 ultra-fast hybrid
detector. When the fluorescence decay is recorded over an interval of 0 to
50 ns (Fig. 11, left) the IRF width is almost invisible. A recording at a
faster time scale (Fig. 11, right, 0 to 5 ns) shows that the IRF is about
40 ps wide. It is essentially determined by the laser pulse width, which was
approximately 35 ps. In the 0 to 5 ns interval, the fluorescence
almost looks like phosphorescence.
The fluorescence decay of an infrared dye
recorded with a superconducting single-nanowire detector  is shown in Fig. 12.
At first glance, the curve may look like a decay curve of fluorescein recorded
in a standard lifetime spectrometer. In fact, the IRF width is 5 ps FWHM,
and the decay time is 43.7 ps, i.e. almost 100 times shorter than that of
Fig. 11: Left: Fluorescence decay of a rhodamine dye recorded with a bh ps
diode laser, a fast hybrid detector, and a bh SPC-150N TCSPC module. Logarithmic
scale, time axis 0 to 50 ns. Right: Same signal recorded in a time range of
0 to 5ns.
Fig. 12: Fluorescence decay of an infrared dye, recorded with a femtosecond
laser, a single-nanowire superconducting NbN detector, and an SPC-150NXX TCSPC
module. The IRF width is 5 ps, a fit of the data with a single-exponential
model function delivers a fluorescence decay time 43.7 ps.
The term Routing refers to the ability of
the TCSPC device to route photons into different measurement data blocks
depending on an external control signal. The routing function can be used to
record signals from several detectors by a single TCSPC device, to separate
photons excited by several multiplexed lasers, or to record photons from
spatially different positions of an object by fast optical switches . Routing
has already been introduced in the classic-TCSPC era. It is, however, an
important element of multi-dimensional TCSPC: The photons are assigned one or more
additional parameters (detection channel, excitation wavelength, spatial
position), and the result is a photon distribution over the time in the optical
waveform and these parameters.
Application: Dual-excitation and dual-emission wavelength
recording of decay curves
An example of dual-detector and dual-laser
operation is shown in Fig. 13. Two lasers were multiplexed at a rate of 20 Hz,
and the photons detected by two detectors through different filters. The result
contains three decay curves for different excitation/detection wavelength
combinations. The fourth combination does not deliver data because the
detection wavelength is shorter than the excitation wavelength. Please see 
for further details.
Fig. 13: Multiplexed measurement of the fluorescence of a leaf. Multiplexed
excitation at 405 nm and 650 nm, simultaneous detection at
510 nm and 700 nm. Multiplexing rate 20 Hz.
Dual Time-Base Recording
The bh TCSPC modules can associate two
times to every individual photon. The first one, the micro time is the time
in the excitation pulse period. This is the usual TCSPC time, and it is
available at an accuracy in the ps range. The second time, the macro time, is
a time from the start of the experiment or from an external event. The
capability to associate two times to the photons results in recording
principles which are beyond the reach of classic TCSPC. Two examples are
described below, for details and more applications please see .
Application: Simultaneous Fluorescence and Phosphorescence
The technique is based on on-off modulating
a high-frequency pulsed laser and recording the fluorescence and
phosphorescence signals by dual time-base TCSPC. The fluorescence decay is
obtained by building up a photon distribution over the times of the photons in
the laser pulse period (the micro times), PLIM by building up the distribution
over the times of the photons in the laser modulation period (the macro times)
. The modulation and photon timing principle is illustrated in Fig. 14. An
example is shown in Fig. 15. Please see also Simultaneous FLIM / PLIM, page 24.
Fig. 14: Modulation and photon timing for simultaneous fluorescence and phosphorescence
Fig. 15: Simultaneous recording of fluorescence (left) and phosphorescence
decay (right). Mixture of fluorescein and a ruthenium dye.
Fluorescence Correlation Spectroscopy (FCS)
is based on the recording of fluorescence from a limited number of fluorescing
molecules in a small sample volume, and correlating intensity fluctuations
caused by the motion of the molecules [13, 45]. The correlation curves are
where G(t) is the autocorrelation function of a single signal, I(t),
and G12(t) the cross-correlation function of
two signals, I1(t) and I2(t). N(t)
is the photon number in a given macro-time interval. A correlation curve of a
fluorescein solution is shown in Fig. 16.
FCS can be combined with fluorescence decay
recording. The decay curves are obtained by building up the photon distribution
over the micro times of the photons. Please see  for more information.
Fig. 16: FCS curve recorded with bh SPC-150 module and HPM‑100-40
hybrid detector. FCS curve calculated online by SPCM software. The red curve is
a fit with a two-component diffusion model.
Recording of Parameter-Tagged Photons
The TCSPC processes described above build
up photon distributions in the memory of the TCSPC device or transfer the
single-photon data into the system computer which immediately builds up the photon
distributions. Once a photon has been put in the photon distribution the
information associated to it is no longer needed and, normally, discarded.
Parameter-tagged photon data may, however,
be used to build up other results than multi-dimensional photon distributions.
At the time of the experiment it may even not be clear how exactly the photon
data are to be processed. User-interaction during the data processing may be
required, or the processing may require so much computation power that it
cannot be performed online. In these cases the single-photon data (micro time,
macro time, routing information, markers for external events) can be saved for
later off-line processing [12, 16, 17]. Most of the applications of
parameter-tag recording are in the field of single-molecule spectroscopy. An
example is described below.
Application: Single-Molecule Burst Analysis
Consider a solution of fluorescent molecules, excited by a focused
laser beam through a microscope lens, with the emitted photons being detected
through a confocal pinhole that transmits light only from a volume of
diffraction limited size. When the concentration of fluorescent molecules is
low enough only one molecule will be in the detection volume at a time. As the
molecule diffuses through the excitation/detection volume it emits photons.
Thus, the detection signal consist of bursts of photons caused by individual
molecules, see Fig. 17.
Fig. 17: Photon bursts from single molecules travelling through a
femtoliter-size detection volume
For single-molecule analysis, the
single-photon data stream from the TCSPC module is stored, the bursts from the
individual molecules are identified in the data, and multi-dimensional histograms of the fluorescence parameter values
are built up. It is even possible to record changes in the fluorescence
parameters within the individual bursts, derive FRET efficiencies, and conclude
on conformational changes of the molecules [42, 52]. Please see , chapter Multi-Parameter
Single-Molecule Burst Analysis.
Multi-Dimensional TCSPC Techniques
Multi-wavelength TCSPC is based on splitting
the light spectrally into a number of detector channels (or channels of a
multi-anode PMT), and using the number of the channel the photon arrived at as
a second coordinate of the photon distribution [7, 14]. The principle is shown
in Fig. 18. For each photon, the detector delivers a single-photon pulse which
indicates the detection time, and a Channel signal which indicates in which
channel of the multi-anode PMT the photon arrived. The TCSPC module builds up a
photon distribution over the photon time and the channel number. The result is
identical with a set of decay curves (in this case 16) for different wavelengths.
Fig. 18: Principle of multi-wavelength TCSPC
Application: Multi-Wavelength Fluorescence Decay Recording
Please note that multi-wavelength TCSPC does not use any wavelength
scanning, detector switching, or multiplexing. Every photon is put into a place
in the photon distribution according to its detection time and wavelength.
Compared to scanning the spectrum with a monochromator and recording individual
decay curves, the efficiency is much higher. Multi-detector TCSPC, especially
multi-wavelength detection, has therefore become a commonly used technique of autofluorescence
lifetime imaging of biological samples [30, 41, 46]. An example is shown in Fig.
Fig. 19: Multi-wavelength fluorescence-decay recording. PML-16 GaAsP
multi-wavelength detector with SPC‑150 TCSPC module. The peak on the
lower left is the excitation light.
Ultra-Fast Time Series Recording
Multi-dimensional TCSPC is able to record
ultra-fast time series of fluorescence decay curves. The process is based on a periodic
induction of a dynamic effect in the sample and recording a two-dimensional photon
distribution over the times of the photon after the excitation pulses and after
the stimulation of the sample .
Application: Recording of Chlorophyll Transients
As an example, Fig. 29 shows the
photochemical transient of chlorophyll in a plant. Stimulation was performed by
periodically switching on and off the excitation laser. Time per curve is 100
microseconds - a speed impossibly to be obtained by classic TCSPC.
Fig. 20: Ultra-high speed time-series
recording. Photochemical chlorophyll transient in a leaf, sequence of 128 decay
curves, 100 µs per curve.
Fluorescence Lifetime Imaging (FLIM)
FLIM by multi-dimensional TCSPC is based on
scanning a sample by the focused beam of a high-repetition rate laser and
detecting single photons of the fluorescence signal. Each photon is
characterised by its time in the laser period and the x-y position of the laser
spot in the moment of the photon detection. The recording process builds up a
photon distribution over these parameters [12, 15, 16, 17]. The principle is illustrated in Fig. 21.
The result is an array of pixels, each containing a full fluorescence decay
curve with a (typically large) number of time channels. The process works at
any scan rate, and delivers near-ideal photon efficiency and extremely high
Fig. 21: Fluorescence lifetime imaging
An example of a FLIM image is shown in Fig.
22. A convallaria sample was scanned with a bh DCS-120 confocal scanner .
The excitation source was a BDL-SMN 473-nm ps diode laser, the photons were detected
by a bh HPM-100-40 hybrid detector and processed by an SPC-150 TCSPC/FLIM
module. The data were recorded into 2048 x 2048 pixels and 256 time channels
per pixel. The brightness of the image represents the photon number, the colour
the fluorescence decay time. Decay curves for selected pixels shown on the
Fig. 22: FLIM image of a Convallaria Sample, 2048x2048 pixels, 256
time-channels per pixel. Decay curves for selected pixels shown on the right.
The bh devices record FLIM images with
different scanning techniques and optical systems, and with image scales from
nanometers (STED ),
micrometers (confocal and multiphoton microscopy ) or millimeters and centimetres
(bh macro scanner , clinical systems ). For more examples and
references please see [5, 6, 16]
FRET, or Foerster Resonance Energy
Transfer, is used in cell biology to study the interactions between proteins .
The proteins are labelled with a donor and an acceptor. When the donor is
excited, the energy can be emitted via fluorescence or transferred to the
acceptor. The energy transfer rate sensitively depends on the distance to the
acceptor. In practice, FRET occurs only when the donor-labelled protein is
chemically linked to the acceptor-labelled one. The energy transfer rate is a
measure of the distance. Compared to intensity-based FRET techniques FLIM-FRET
is much more reliable. All that is needed is a lifetime image at the donor
emission wavelength. The decrease in the donor fluorescence lifetime then
indicates the rate of the energy transfer. Another advantage is that
interacting and non-interacting donor (or interacting and non-interacting
proteins) can be separated by double-exponential decay analysis. It is thus
possible to measure the fraction of interacting proteins. This is biological
information not accessible by intensity-based FRET techniques. Please note that
double-exponential analysis of FRET decays requires high time resolution.
Therefore, the technique benefits considerable from the high time resolution of
the bh TCSPC / FLIM devices. Please see FRET chapter in The bh TCSPC Handbook .
An example of double-exponential FLIM-FRET is shown in Fig. 23.
Fig. 23: FRET result obtained by double
exponential lifetime analysis. Left: t0/tfret, indicating distance variation,
Right: Nfret/N0, indicating variation in amount of interacting proteins.
Application: Metabolic Imaging
The composition of the decay curves of NADH
(nicotimamide adanine (pyridine) dicucleotide) is an indicator of the metabolic
state of cells and tissues. A high amplitude of the fast component (from free
NADH) indicates the cells are running glycolysis, a low amplitude indicates
that oxidative phosphorylation dominates . It turns out that the amplitude
of the fast component (or the amplitude ration of the fast and slow component)
is a much better indicator of the metabolic state than the fluorescence
lifetime itself . However, the determination of the amplitudes requires
double-exponential decay analysis, and, consequently, high resolution of the
decay data. Metabolic FLIM therefore benefits from the high time resolution of
the bh FLIM devices. An example is shown in Fig. 24 and Fig. 25. The sample was
excited by two-photon excitation with a femtosecond Ti:Sa laser, the photons
were detected by a HPM-100-06 ultra-fast hybrid detector. The result shows the
best separation of the NADH decay components ever obtained. The data not only
yield an excellent image of the amplitude-weighted mean lifetime, but also
high-quality images of the amplitude ratio and the component lifetimes . For clinical application please see [22,
Fig. 24: Left: NADH
Lifetime image, amplitude-weighted lifetime of double-exponential fit. Right:
Decay curve in selected spot, 9x9 pixel area. FLIM data format 512x512 pixels,
1024 time channels. Time-channel width 10ps.
Fig. 25: Left to right: Images of the amplitude ratio, a1/a2
(unbound/bound ratio), and of the fast (t1, unbound NADH) and the slow decay
component (t2, bound NADH). FLIM data format 512x512 pixels, 1024 time channels.
Time-channel width 10ps.
Application: Ultra-Fast Fluorescence Decay in Biological
With their short IRF bh FLIM systems are
able to detect ultra-fast decay components which have never been seen before [24,
25, 26, 27]. An example is shown in Fig. 26.
Fig. 26: Ultra-fast
decay in mushroom spores, t1 image and decay curve. The lifetime of
the fast decay component is 12 ps.
Application: FLIM in Ophthalmology
TCSPC FLIM is so sensitive that it can be
used to record fluorescence-lifetime images of the human retina. Examples are
shown in Fig. 27. The images were recorded with the Heidelberg-Engineering FLIO
system, containing bh TCSPC FLIM modules, bh ps diode lasers, and bh HPM hybrid
detectors. Also here, time resolution and timing stability are extremely
important: Fluorescence decay times range from 200 to 600 ps, with component
lifetimes down to less than 80 ps. The problem is not only that the lifetimes
are very short but also that the optical path length is not constant and that
the retina signal is contaminated by fluorescence from the front part of the
eye . Technical details are described in  and . Ophthalmic FLIM is
currently under clinical trial. The results show that FLIM is able to detect
early changes in the metabolism of the retina before these have caused
irreversible damage. Please see ,  and  for references.
Fig. 27: Lifetime images of the human retina. Left: Healthy eye. Right: Eye
of an AMD (age-related macula degeneration) patient.
Multi-Dimensional FLIM Techniques
The photon distribution of TCSPC FLIM can
be extended by additional parameters. These can be the wavelength of the
photons, the time from the start of the experiment or from a stimulation of the
sample, the excitation wavelength, or the time in the period of an additional
modulation of the excitation laser. The resulting photon distributions are
four- or five-dimensional, the data representing multi-spectral FLIM,
ultra-fast time-series FLIM, multi-excitation-wavelength FLIM, and simultaneous
FLIM/PLIM. A few examples are shown in the sections below. Please see [16, 17, 18,
48] for more information.
Multi-wavelength (or multi-spectral) FLIM
uses a combination of the FLIM architecture shown in Fig. 21 with
multi-wavelength detection principle described in Fig. 18. In addition to the
times of the photons and the positions, x, and y, of the scanner, the TCSPC
module determines the detector channel that detected the photon. These pieces
of information are used to build up a photon distribution over the time of the
photons in the fluorescence decay, the wavelength, and the coordinates of the
image [7, 14, 16, 17]. The result is an image that contains several decay
curves for different wavelength in each pixel. An example of a multi-wavelength
FLIM image is shown in Fig. 29.
Fig. 28: Multi-wavelength FLIM. The recording process builds up a photon
distribution over x,y,t, and λ.
Fig. 29: Multi-wavelength FLIM of a convallaria sample
FLIM with Excitation-Wavelength Multiplexing
The routing function of the bh TCSPC
modules can be used to record FLIM quasi-simultaneously at several excitation
wavelengths. Several lasers are multiplexed synchronously with either the
pixels, the lines, or the frames of the scan, and the photons excited by
different lasers are routed into different FLIM data blocks. The result
represents separate lifetime images for the individual laser wavelengths .
The principle is shown for two lasers in Fig. 30.
Fig. 30: Principle of TCSPC FLIM with laser wavelength multiplexing
Excitation-wavelength multiplexing is often
combined with detection in several emission wavelength intervals via several
parallel TCSPC modules or a router. The result is then a data set which
contains FLIM images for all combinations of excitation and detection
Application: Metabolic FLIM with NADH and FAD
Although the fluorescence of NADH is the
best metabolic indicator also FAD (flavin adenine dinucleotide) exhibits
changes in its decay behaviour with the metabolic state of a cell. Lifetime
images of FAD are therefore often used to support the metabolic information
obtained from NADH. The problem of this approach is that NADH and FAD data are
desirably to be recorded simultaneously. Unfortunately, NADH and FAD signals
can only be separated if both different excitation and detection wavelengths
are used. The task can be solved by the excitation multiplexing principles
shown above. A result is shown in Fig. 31. The data were recorded by a bh
DCS-120 system with two lasers, 375 nm and 410 nm, and two TCSPC /
FLIM channels, detecting from 420 nm to 470 nm and 490 nm to 600 nm,
respectively . Technical details and the discrimination of healthy cells and
tumor cells are described in [22, 23, 49].
Fig. 31: a1 Images of human bladder cells, recorded with two
multiplexed lasers and two TCSPC channels.
Originally, bh introduced Mosaic FLIM to record
large images with the Tile Imaging function of laser scanning microscopes [6,
16]. The microscope scans the sample, and performs a raster stepping (Tile
stepping) of the sample. For every step the sample is scanned for a defined
number of frames. The TCSPC device records the data by its normal FLIM
procedure. However, the memory is configured to provide space not only for a
single image of the defined frame format but for the entire mosaic of images of
the tile stepping. The TCSPC FLIM process starts in the first mosaic element.
After a defined number of frames the recording proceeds to the next mosaic element.
Provided the number of frames per tile of the microscope stepping and the
number of frames per mosaic elements are the same the TCSPC module records the
entire tile array into a single photon distribution. The recorded photon distribution
represents a FLIM image of the entire array. The TCSPC FLIM process of Mosaic
FLIM is illustrated in Fig. 32. An example is shown in Fig. 33.
Fig. 32: Mosaic FLIM, recording of a X-Y mosaic
Fig. 33: Mosaic FLIM of a BPAE cell sample
Temporal Mosaic FLIM
The idea that Mosaic FLIM records several
images into one photon distribution leads to a more general concept of Mosaic
FLIM: The transition from one mosaic element in the FLIM data to the next can
be associated also to a change in another parameter of the experiment. An
example is temporal mosaic FLIM. The sample is repeatedly scanned around the
same spatial position, but subsequent images are recorded in consecutive elements
of the FLIM mosaic. The result is a time series, the time step of which is a
multiple of the frame time .
Compared to conventional time-laps
recording the temporal mosaic FLIM has several advantages: No time has to be
reserved for the save operations, and the data can be better analysed with
global-parameter fitting. The biggest advantage is, however, that mosaic time
series data can be accumulated: A sample would be stimulated repeatedly by an
external event, and the start of the mosaic recording be triggered with the
stimulation. With every new stimulation the recording procedure runs through
all elements of the mosaic, and accumulates the photons. Accumulation allows
data to be recorded without the need of trading photon number and lifetime
accuracy against the speed of the time series. Consequently, the time per step
(or mosaic element) is only limited by the minimum frame time of the scanner.
Application: Recording of Calcium Transients in Neurons
Temporal Mosaic FLIM is thus an excellent
way to investigate fast physiological processes in live systems [20, 33]. An
example for recording Ca2+ transients in live neurons is shown in Fig.
34. Please see  for more information.
Fig. 34: Ca2+ transient in cultured neurons, incubated with
Oregon Green Bapta. Electrical stimulation, stimulation period 3s, data
accumulated over 100 stimulation periods. Time per mosaic element is 38 ms.
Simultaneous FLIM / PLIM (fluorescence and
phosphorescence lifetime imaging) is based on the dual time-base capability of
the bh TCSPC modules. A high-frequency pulsed laser is periodically switched on
and off in the microsecond range. For every photon, two times are determined.
One is the time in the laser pulse period, the other the time in the laser
modulation period. FLIM is obtained by building up a photon distribution over
the times of the photons in the laser pulse period and the scan coordinates,
PLIM by building up the distribution over the times of the photons in the laser
modulation period and the scan coordinates [9, 16, 19]. To avoid aliasing of the modulation
frequency with the pixel frequency the modulation is synchronised with the
pixels of the scan. The principle of laser modulation and photon timing is
shown in Fig. 35, a typical result in Fig. 36.
Fig. 35: Laser modulation and photon
timing for simultaneous FLIM / PLIM
Application: Metabolic FLIM with Oxygen Sensing
The most frequent application of the bh
FLIM/PLIM technique is oxygen sensing (pO2 sensing) in biological systems.
Applications benefit from the fact that the technique is able to record FLIM
and FLIM simultaneously. Therefore, it delivers the current metabolic state of
the cells together with the current oxygen concentration. An example is shown
in Fig. 36. Yeast cells were stained with a Ruthenium
dye, and imaged at the NADH and at the Ruthenium emission wavelengths. The FLIM
images is shown on the left, the PLIM image on the right. For references, further
applications, and technical details please see [16, 34, 35, 38].
Fig. 36: Simultaneous FLIM and PLIM of yeast cells. Autofluorescence of the
cells (left) and phosphorescence of a ruthenium compound (right)
Classic TCSPC records the waveform of a
periodic optical signal by detecting single photons of the signal and building
up of a photon distribution over the time in the signal period. The technique yields
high sensitivity, near-ideal photon efficiency and extraordinarily high time
resolution. With bh TCSPC devices an electrical IRF width of less than
3 ps FWHM is achieved. The system IRF with fast hybrid detectors is
shorter than 20 ps FWHM; with superconduction nanowire detectors down to
4.4 ps FWHM have been achieved.
The disadvantage of the classic TCSPC
method is that it is one-dimensional - it just delivers the waveform of the
optical signal. In 1993, bh therefore introduced a multi-dimensional TCSPC
technique. The technique is based on the idea that TCSPC records a photon
distribution. Classic TCSPC records a photon distribution over the times of the
photons in the excitation pulse period; multi-dimensional TCSPC records a
photon distribution over the time in the excitation pulse period and one or
more other parameters that can be associated to the individual photons. These
may be the wavelength, the location in a sample where the photon came from, the
time from a stimulation of the sample, or the time within the period of an
additional modulation of the excitation light source. The result are techniques
like multi-wavelength fluorescence decay recording, ultra-fast time series
fluorescence decay recording, FLIM, multi-wavelength FLIM, ultra-fast
time-series FLIM, spatial and temporal mosaic FLIM, and simultaneous FLIM /
PLIM. The bh technique is open to the use of other parameters, such as temperature
of the sample, oxygen partial pressure, or voltage applied to or measured at
the sample. Thus, there may be possibilities which have even not been
considered yet. Please see The bh TCSPC Handbook  for further suggestions
R.M. Ballew, J.N. Demas, An error analysis of
the rapid lifetime determination method for the evaluation of single
exponential decays, Anal. Chem. 61, 30 (1989)
Becker & Hickl GmbH, World Record in TCSPC
Time Resolution: Combination of bh SPC-150NX with SCONTEL NbN Detector yields
17.8 ps FWHM. Application note, available on www.becker-hickl.com
Becker & Hickl GmbH, Sub-20ps IRF Width from
Hybrid Detectors and MCP-PMTs. Application note, available on
Becker & Hickl GmbH, SPC-QC-104 3/4 Channel
TCSPC/FLIM Module, User Manual. Available on www.becker-hickl.com.
Becker & Hickl GmbH, DCS-120 Confocal and
Multiphoton Scanning FLIM Systems, user handbook 9th ed. (2021). Available on
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
Becker & Hickl GmbH, PML-16-C 16 and
PML-16 GaAsP 16-channel channel TCSPC / Detectors, PML-SPEC and MW FLIM
Multi-Wavelength detectors. User handbook (20016). Available on www.becker‑hickl.com
Becker & Hickl GmbH, bh - Abberior
Combination Records STED FLIM at Megapixel Resolution. Application note,
available on www.becker-hickl.com
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
Ultra-fast HPM Detectors Improve NAD(P)H FLIM.
Application note, available on www.becker-hickl.com
Becker & Hickl GmbH, FLIM Systems for Laser
Scanning Microscopes. Overview brochure, available on www.becker-hickl.com
W. Becker, Advanced time-correlated single-photon counting techniques. Springer,
Berlin, Heidelberg, New York, 2005
W. Becker, A. Bergmann, E. Haustein, Z.
Petrasek, P. Schwille, C. Biskup, L. Kelbauskas, K. Benndorf, N. Klöcker,
T. Anhut, I. Riemann, K. König, Fluorescence lifetime images and
correlation spectra obtained by multi-dimensional TCSPC, Micr. Res. Tech. 69,
W. Becker, A. Bergmann, C. Biskup, Multi-Spectral
Fluorescence Lifetime Imaging by TCSPC. Micr. Res. Tech. 70, 403-409 (2007)
W. Becker, Fluorescence Lifetime Imaging -
Techniques and Applications. J. Microsc. 247 (2) (2012)
W. Becker, The bh TCSPC handbook. 8th edition,
Becker & Hickl GmbH (2019), available on www.becker-hickl.com
W. Becker, Introduction to Multi-Dimensional TCSPC.
In W. Becker (ed.) Advanced time-correlated single photon counting
applications. Springer, Berlin, Heidelberg, New York (2015)
W. Becker, V. Shcheslavskiy, H. Studier, TCSPC FLIM with Different
Optical Scanning Techniques, in W. Becker (ed.) Advanced time-correlated
single photon counting applications. Springer, Berlin, Heidelberg, New York
W. Becker, V. Shcheslavskiy, A. Rück,
Simultaneous phosphorescence and fluorescence lifetime imaging by
multi-dimensional TCSPC and multi-pulse excitation. In: R. I. Dmitriev (ed.),
Multi-parameteric live cell microscopy of 3D tissue models. Springer (2017)
W. Becker, S. Frere, I. Slutsky, Recording Ca++
Transients in Neurons by TCSPC FLIM. In:F.-J. Kao, G. Keiser, A. Gogoi, (eds.),
Advanced optical methods of brain imaging. Springer (2019)
W. Becker, J. Breffke, B. Korzh, M. Shaw, Q.-Y.
Zhao, K. Berggren, 4.4 ps IRF width of TCSPC with an NbN Superconducting
Nanowire Single Photon Detector. Application note, available on
W. Becker, A. Bergmann, L. Braun, Metabolic
Imaging with the DCS-120 Confocal FLIM System: Simultaneous FLIM of NAD(P)H and
FAD, Application note, Becker & Hickl GmbH (2019)
Becker Wolfgang, Suarez-Ibarrola Rodrigo,
Miernik Arkadiusz, Braun Lukas, Metabolic Imaging by Simultaneous FLIM of
NAD(P)H and FAD. Current Directions in Biomedical Engineering 5(1), 1-3 (2019)
W. Becker, C. Junghans, A. Bergmann, Two-Photon
FLIM of Mushroom Spores Reveals Ultra-Fast Decay Component. Application note,
available on www.becker-hickl.com.
W. Becker, T. Saeb-Gilani, C. Junghans, Two-Photon
FLIM of Pollen Grains Reveals Ultra-Fast Decay Component. Application note, available
Wolfgang Becker, V. Shcheslavskiy, Axel Bergmann,
FLIM at a Time-Channel Width of 300 Femtoseconds. Application note, available
W. Becker, V. Shcheslavskiy, V. Elagin
Ultra-Fast Fluorescence Decay in Malignant Melanoma. Application note,
available on www.becker-hickl.com.
W. Becker, A. Bergmann, L. Sauer,
Shifted-Component Model Improves FLIO Data Analysis. Application note, Becker
& Hickl GmbH (2019)
L. M. Bollinger, G. E. Thomas, Measurement of
the time dependence of scintillation intensity by a delayed coincidence method.
Rev. .Sci. Instrum. 32, 1044-1050 (1961)
D. Chorvat, A. Chorvatova, Multi-wavelength
fluorescence lifetime spectroscopy: a new approach to the study of endogenous
fluorescence in living cells and tissues. Laser Phys. Lett. 6 175-193 (2009)
S. Cova, M. Bertolaccini, C. Bussolati, The
measurement of luminescence waveforms by single-photon techniques, Phys. Stat.
Sol. 18, 11-61 (1973)
Dysli, C., Wolf, S., Berezin, M.Y., Sauer, L.,
Hammer, M., Zinkernagel, M.S., Fluorescence lifetime imaging ophthalmoscopy,
Progress in Retinal and Eye Research (2017), doi:
S. Frere, I. Slutsky,
Calcium imaging using Transient Fluorescence-Lifetime Imaging
by Line-Scanning TCSPC. In: W. Becker (ed.) Advanced time-correlated single
photon counting applications. Springer, Berlin, Heidelberg, New York (2015)
J. Jenkins, R. I. Dmitriev, D. B. Papkovsky, Imaging Cell and Tissue O2 by TCSPC-PLIM. In: W. Becker (ed.) Advanced time-correlated
single photon counting applications. Springer, Berlin, Heidelberg, New York
S. Kalinina, V. Shcheslavskiy, W. Becker, J.
Breymayer, P. Schäfer, A. Rück, Correlative NAD(P)H-FLIM and oxygen
sensing-PLIM for metabolic mapping. J. Biophotonics 9(8):800-811 (2016)
S. Kinoshita, T. Kushida, Subnanosecond
fluorescence-lifetime measuring system using single photon counting method with
mode-locked laser excitation, Rev. Sci. Instrum. 52, 572-575 (1981)
M. Köllner, J. Wolfrum, How many photons are
necessary for fluorescence-lifetime measurements?, Phys. Chem. Lett. 200,
H. Kurokawa, H. Ito, M. Inoue, K. Tabata, Y.
Sato, K. Yamagata, S. Kizaka-Kondoh, T. Kadonosono, S. Yano, M. Inoue & T.
Kamachi, High resolution imaging of intracellular oxygen concentration by
phosphorescence lifetime, Scientific Reports 5, 1-13 (2015)
C. Lewis, W.R. Ware, The Measurement of
Short-Lived Fluorescence Decay Using the Single Photon Counting Method, Rev.
Sci. Instrum. 44, 107-114 (1973)
B. Leskovar, C.C. Lo, Photon counting system for
subnanosecond fluorescence lifetime measurements, Rev. Sci. Instrum. 47,
A. Marcek Chorvatova,
Time-Resolved Spectroscopy of NAD(P)H in Live Cardiac
Myocytes. In: W. Becker (ed.) Advanced time-correlated single photon
counting applications. Springer, Berlin, Heidelberg, New York (2015)
S. Pallikkuth, D. J. Blackwell, Z. Hu, Z. Hou,
D. T. Zieman, B. Svensson, D. D. Thomas, S. L. Robia, Phosphorylated
Phospholamban Stabilizes a Compact Conformation of the Cardiac Calcium-ATPase.
Biophys. J. 105, 18121821 (2013)
A. Periasamy, N. Mazumder, Y. Sun, K. G.
Christopher, R. N. Day, FRET Microscopy: Basics,
Issues and Advantages of FLIM-FRET Imaging. In: W. Becker (ed.) Advanced
time-correlated single photon counting applications. Springer, Berlin,
Heidelberg, New York (2015)
D.V. OConnor, D. Phillips, Time-correlated
single photon counting, Academic Press, London (1984)
R. Rigler, E.S. Elson (eds), Fluorescence
Correlation Spectroscopy, Springer Verlag Berlin, Heidelberg, New York (2001)
A. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, R.
Steiner, SLIM: A New Method for Molecular Imaging. Micr. Res. Tech. 70, 403-409
V. I. Shcheslavskiy, M. V. Shirmanova, V. V.
Dudenkova, K. A. Lukyanov, A. I. Gavrina, A. V. Shumilova, E. Zagaynova, W.
Becker, Fluorescence time-resolved macroimaging. Opt. Lett. 43, No. 13,
V. I. Shcheslavskiy, M. V. Shirmanova, A.
Jelzow, W. Becker, Multiparametric Time-Correlated Single Photon Counting
Luminescence Microscopy. Biochemistry (Moscow), 84, Suppl. 1, pp. S51-S68
R. Suarez-Ibarrola, L. Braun, P. F. Pohlmann, W.
Becker, A. Bergmann, C. Gratzke, A. Miernik, K. Wilhelm, Metabolic Imaging of
Urothelial Carcinoma by Simultaneous Autofluorescence Lifetime Imaging (FLIM)
of NAD(P)H and FAD. Clinical Genitourinary Cancer (2020)
R. Schuyler, I. Isenberg, A Monophoton
Fluorometer with Energy Discrimination, Rev. Sci. Instrum. 42 813-817 (1971)
D. Schweitzer, M. Hammer, Fluorescence Lifetime
Imaging in Ophthalmology. In: W. Becker (ed.) Advanced time-correlated single
photon counting applications. Springer, Berlin, Heidelberg, New York (2015)
D. Singh, H. Sielaff, M. Börsch, G. Grüber,
Conformational dynamics of the rotary subunit F in the A3B3DF
complex of Methanosarcina mazei Gö1 A-ATP synthase monitored by single-molecule
FRET.FEBS Letters 591, 854-862 (2017)
J. Yguerabide, Nanosecond fluorescence
spectroscopy of macromolecules, Meth. Enzymol. 26, 498-578 (1972)
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