Spatially Resolved Recording
of Fluorescence-Lifetime Transients by Line-Scanning TCSPC
Wolfgang Becker, Becker & Hickl GmbH
Abstract: We present a technique that records transient effects in the
fluorescence lifetime of a sample with spatial resolution 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. The technique can be used in all bh FLIM systems based on the bh SPC-830
or SPC-150 TCSPC modules.
Fluorescence lifetime imaging (FLIM) by
multidimensional TCSPC is based on raster-scanning a sample, detecting single
photons of the fluorescence light emitted, and building up a photon
distribution over the coordinates of the scan area, x and y, and the arrival
times, t, of the photons after the laser pulses. The result can be interpreted
as an array of pixels, each containing photon numbers in a large number of time
channels for consecutive times after the excitation pulses [1, 2]. The
advantage of FLIM by multi-dimensional TCSPC is that it delivers near-ideal
recording efficiency, and an extremely high time resolution. Moreover,
multi-dimensional TCSPC solves the problem that the pixel rates in scanning
microscope are often higher than the photon detection rates. No matter of how
fast the scanner runs, the acquisition process simply assigns the photons to
the right pixels and time channels. The accumulation is continued over as many
frames of the scan as needed to obtain the desired signal-to-noise ratio.
Transient effects in the fluorescence
lifetime can be recorded by time-series FLIM. Subsequent FLIM recordings are
performed, and the data saved into consecutive data files [2, 3, 4]. Time-series of FLIM images can be
recorded at surprisingly high rate, especially if readout times are avoided by
dual-memory recording [2, 7]. Nevertheless, each step of a time series requires
at least one complete x-y scan of the sample. With the typical frame rates of
fast galvanometer scanners lifetime changes can be recorded at a maximum
resolution on the order of a few 100 ms.
Lifetime changes faster than that can be
recorded by single-point measurements, as has already been demonstrated in .
However, single-point measurements do not deliver information about the spatial
distribution of the lifetime changes within a sample.
A solution to spatially resolved transient-recording
is provided by combining multi-dimensional TCSPC with line scanning. The
approach is illustrated in Fig. 1. Fig. 1, left, shows the photon distribution
built up by normal TCSPC FLIM. It is a distribution of photon numbers over x,y,
and t. In Fig. 1, right, one spatial coordinate (y) has been replaced with an
experiment time, T. The experiment time, T, is the time after a stimulation
of the sample, or after any other event temporally correlated to a lifetime
change in the sample. X is the distance along a spatially one-dimensional scan.
As the technique aims on recording fluorescence lifetime changes over the
distance of a scan we suggest the name FLITS, Fluorescence Lifetime-Transient
Scanning for it.
Fig. 1: Left: Photon distribution built up by standard FLIM. Right: Photon
distribution built up by fluorescence lifetime-transient scanning.
As long as the stimulation occurs only once
the recording process may appear simple: The sequencer of the TCSPC module 
starts to run with the stimulation, and puts the photons in subsequent data blocks
for consecutive time intervals along the T axis. The result is a time-series of
However, there is an important difference
to a simple time-series recording: The data are still in the memory when the
sequence is completed. Thus, the recording process can be made repetitive: The
sample is stimulated periodically, and the start of T is triggered by the
stimulation. The recording then runs along the T axis periodically, and the
photons from several stimulation periods are accumulated into one and the same
With triggered accumulation, it is no
longer necessary that each T step acquires enough photons to obtain a complete
decay curve in each pixel. No matter when and from where a photon arrives, it
is assigned to the right spatial location x, to the right arrival time, t,
after the laser pulse, and to the right time T, after the stimulation. The
desired signal-to-noise-ratio is obtained by simply running the acquisition
process for a sufficiently large number of stimulation periods. Obviously, the
resolution in T is limited by the period of the line scan only, which is about
1 ms for the commonly used scanners.
FLITS is performed in the FIFO Imaging
mode of the bh TCSPC modules .
The synchronisation with the experiment is accomplished the usual way, via the
pixel clock, line clock, and frame clock pulses. However, in contrast to FLIM the
frame clock for FLITS does not come from the scanner but from the stimulation
of the sample.
The SPC module thus records a photon
distribution n (x, T, t) the coordinates of which are the
distance, x, along the scanned line, the time, t, of the photon after the laser
pulse, and the transient‑time, T, after the stimulation. The transient-time,
T, is given in multiples of the line time. The principle is illustrated in Fig.
Principle of recording transient lifetime effects by line scanning
The time scale of T can be varied either by
varying the scan rate, or by defining a line clock divider value larger than one
in the scan parameter section of the TCSPC system parameters, see .
In early FLITS implementations, the source
of the frame clock had to be switched manually, either by swapping a cable
connector, or be flipping a switch. SPCM Software versions from December 2012
or later have the FLITS integrated: The scanner frame clock and the FLITS
trigger are connected to different inputs, Marker 2 and Marker 3, of the SPC
modules. The source of the frame clock is selected via the SPCM software, see Fig.
3, left. Changing from FLIM to FLITS and vice versa is then only a matter of
selecting the corresponding setup from the Predefined Setups panel, see Fig. 3,
Fig. 3: Left and second left: Clock source selection for FLIM and FLITS.
Second right and right: Selection of FLIM mode and FLITS mode from the
Predefined Setup pane.
The additional input for the FLITS trigger
is provided by a FLITS adapter that is connected between the scan clock cable
and the 15 pin sub-D connector of the SPC module, see Fig. 4.
Fig. 4: Clock
connection diagram. The FLITS adapter provides an input of the FLITS trigger to
To demonstrate fluorescence
lifetime-transient scanning we used the chlorophyll transients that occur
when a live plant is exposed to a sudden increase in light intensity . Upon
illumination, the fluorescence lifetime (and intensity) first increases. The
increase happens within a few milliseconds or tens of milliseconds. It is
attributed to the progressive saturation of photosynthesis channels, and a
corresponding decrease in fluorescence quenching. The corresponding increase in
the fluorescence quantum efficiency (and in the fluorescence lifetime) is
called photochemical transient.
After a few seconds of exposure, the
fluorescence lifetime starts to decrease again. At intensities comparable to
sunlight, it reaches a steady-state level after a few tens of seconds. It is
assumed that this slow decrease in the fluorescence lifetime is a protection
mechanism of the plant. The slow decrease is called non-photochemical
Recording the non-photochemical transient
is relatively easy: It can even be recorded by normal time-series FLIM [2, 3, 4].
To record the transients by FLITS we used a bh DCS‑120 confocal scanning
FLIM system . The sample was a fresh grass blade. An appropriate position of
the line scan in the sample area was selected in the Preview mode of the DCS
system. The excitation intensity during the preview was kept low so that no noticeable
lifetime change was induced.
Then the setup was changed to FLITS. This
changes the frame clock source to the FLITS trigger, and switches the scanner
in the Line mode. The recording was started by generating a single frame
clock pulse. The excitation laser was switched on 180 ms later. The result
for a line time of 60 ms is shown in Fig. 5.
The horizontal coordinate is the distance
along the scanned line, the vertical coordinate is the time, T, after the frame
clock. The line time was 60 ms, the number of lines along the T axis is
256. Thus, the total time along the vertical axis is 15.4 seconds. It can
clearly be seen that both the fluorescence intensity and the fluorescence
lifetime decrease with the time of illumination.
Fig. 5: FLITS image of non-photochemical chlorophyll transient.
Horizontal: Distance along the line scanned. Vertical, bottom to top: Time, T,
after the start of illumination. 256 pixels along the line, line time
60 ms, 256 lines, total time interval recorded 15.36 seconds. Left:
Intensity image. Right Lifetime image. Colour represents lifetime, blue to red corresponds
to 200 ps to 1 ns. Amplitude-weighted lifetime of double-exponential
decay model. Bottom: Decay curve at cursor position.
The effect is shown in detail in Fig. 5. It
shows decay curves for three selected pixel of the line scan for times of
0.5 s, 7.5 s, and 13.4 s after the turn-on of the laser. The
mean lifetimes, tm, obtained from a double-exponential fit are 560 ps,
385 ps, and 311 ps, respectively. The changes in the decay profiles
are clearly visible.
Fig. 6: Decay curves for a selected pixel
within the line for times of 0.5 s, 7.5 s, and 13.4 s after the
turn-on of the laser
For comparison, a normal lifetime image of
the sample is shown in Fig. 7. It was recorded immediately after the FLITS
experiment. It can be seen that the sample had not fully recovered from the
non-photochemical transient yet: The fluorescence lifetime in the vicinity of
the scanned line (horizontal cursor) is still shorter than in the areas nearby.
Fig. 7: Fluorescence lifetime image of grass blade, taken after FLITS
experiment. Horizontal cursor shows position of the FLIS scan. Intensity image
shown left, lifetime image shown right. Coulour represents lifetime, scale from
200 ps to 1 ns. Decay curve at cursor position shown at the bottom.
Recording the photochemical transient
requires periodical stimulation and acquisition of the data over a larger
number of stimulation periods. Both the laser on-off signal  and the frame
clock were therefore generated by a bh DDG-210 pulse generator card . The
on-off period was 1 second, the on time within the period 200 ms.
Each turn-on of the laser initiates a photochemical transient, i.e. a transient
increase in the fluorescence lifetime. In the laser-off period the leaf
partially recovers, so that the next laser-on initiates a new transient. The
total acquisition time was 40 seconds, i.e. 40 on-off periods were accumulated.
The result is shown in Fig. 8. The
intensity image is shown on the left, the lifetime image on the right, and a
decay curve at the cursor position at the bottom. The horizontal axis of the
images is the distance along the line scanned. The vertical axis (bottom to
top) is the time after the start of axis. The line time was 1 ms. 256
lines, i.e. 256 ms in total, were recorded along the T direction. The
Laser was turned on at T=0, and turned off at T=200 ms. The lifetime image
shows the amplitude-weighted lifetime obtained by a double-exponential fit. The
lifetime range is from 450 ps (blue) to 650 ps (red).
Although the lifetime change is less
pronounced than for the non-photochemical transient an increase can clearly be
seen. Changes occur especially in the first 10 to 20 ms after the turn-on
of the laser, see bottom of the FLITS image.
Fig. 8: FLITS image of photochemical chlorophyll transient. Horizontal:
Distance along the line scanned. Vertical, bottom to top: Time, T, after the
start of illumination. Line time 1 ms, 256 lines, total time interval
recorded 256 ms. Laser turned on at T=0, laser-on time 200 ms.
Left: Intensity image. Right Lifetime image. Colour represents lifetime, blue
to red corresponds to 450 ps to 650 ps. Amplitude-weighted lifetime of
double-exponential fit. Bottom: Decay curve at cursor position.
Decay curves and lifetimes for a selected
pixel in the scan and for two points on the T axis are shown in Fig. 9. From
T = 3 ms to T = 191 ms the amplitude-weighted
lifetime of the double-exponential fit increases from 480 ps to
Fig. 9: Decay curves for a selected pixel (x=159) within the line for
T=3ms and T=191ms into the laser-on phases. Double-exponential fit. The
amplitude-weighted lifetime increases from 480 ps to 530 ps
Despite of the small duty cycle of the
laser the illumination necessarily also causes a non-photochemical transient.
When the photochemical transient is complete, activity essentially shuts down,
and no non-photochemical transient is observed any more. Perfect recording of
the photochemical transient therefore requires optimisation of the laser
intensity, the laser duty cycle, and the total accumulation time.
FLITS records fluorescence-lifetime
transients at a time scale down to about one millisecond with spatially
one-dimensional resolution. Technically, FLITS is obtained by line scanning and
replacing the frame clock of a TCSPC FLIM system with a trigger pulse from
the stimulation of the lifetime change in the sample. The technique can thus
easily be implemented in confocal or multiphoton laser scanning microscopes [3,
4], provided these are able to run a fast line scan. The technique works both for
single-shot stimulation, or for periodic stimulation. For a given photon
detection rate, the lifetime accuracy for single-shot stimulation decreases
with decreasing time-channel width along the transient-time axis. This is not
the case for periodic stimulation: Here the accuracy depends on the total
acquisition time. Periodic stimulation is thus the key to high fluorescence-transient
resolution. Potential applications of FLITS are experiments of plant
physiology, electro-physiology, and Ca++ imaging of neuronal tissue.
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picosecond diode lasers, www.becker-hickl.com
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