Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC
and Multi-Pulse Excitation
Wolfgang Becker, Stefan
Smietana, Becker & Hickl GmbH, Berlin, Germany
Abstract. We present a fluorescence and
phosphorescence lifetime imaging (FLIM / PLIM) technique that
simultaneously records FLIM and PLIM in confocal or multiphoton laser scanning
systems. Different than other techniques, it uses not only one, but multiple
laser pulses for every phosphorescence excitation cycle. The sensitivity is
thus orders of magnitude higher. Our technique is based on on-off modulating a
high-frequency pulsed laser synchronously with the pixel clock of the scanner,
and recording the fluorescence and phosphorescence signals by multi-dimensional
TCSPC. 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. The technique does not require a reduction
of the laser pulse repetition rate by a pulse picker, and eliminates the need
of high pulse energy for phosphorescence excitation.
Motivation of Using Phosphorescence Lifetime Imaging
Phosphorescence occurs when an excited
molecule transits from the first excited singlet state, S1, into the first
triplet state, T1, and returns from there to the ground state by emitting a photon
. Both the S1-T1 transition and the T1-S0 transition are forbidden
processes. The transition rates are therefore much smaller than for the S1-S0
transition. That means that phosphorescence is a slow process, with lifetimes
on the order of microseconds or even milliseconds. Phosphorescence of organic
dyes or endogenous fluorophores is extremely weak or even not detectable at
room temperature. However, strong phosphorescence with lifetimes from the
microsecond up to the millisecond range is obtained for lanthanide complexes  and organic complexes of ruthenium [24, 25],
platinum [20, 24, 27, 30], terbium, and palladium . Of special interest for
live-cell imaging is that the phosphorescence of these complexes is strongly
quenched by oxygen. The dyes are therefore excellent oxygen sensors [21, 24, 27, 28, 29, 30, 32]. Applications are aiming at the
measurement of oxygen partial pressure in biological objects, and its effect on
the metabolism of the cells. To reach this target it is desirable that PLIM and
FLIM measurements are performed simultaneously. The oxygen concentration is
then derived from the PLIM data, the metabolic information from the FLIM data, preferably
from the NAD(P)H fluorescence. To obtain clean FLIM and PLIM data from within
cells and tissue the imaging technique must provide depth resolution, and
should be able to deliver data from deep tissue layers. The best optical technique
to obtain these data is confocal and multiphoton laser scanning, and the best electronic
technique to obtain time-resolved data with scanning is multi-dimensional TCSPC
Excitation Pulse Period and Laser Power
The obvious problem of PLIM is that the
excitation pulse period must be a few times longer than the phosphorescence
decay time. For ruthenium dyes with phosphorescence lifetimes below 1 us
the reduction in laser repetition rate may still be feasible, see Hosny et al.
. However, the lifetimes for platinum and palladium-based dyes are on the
order of 50 to 100 µs, and the lifetimes of europium and terbium dyes can
be in the millisecond range. PLIM with these dyes would require a laser
repetition rate of less than 10 kHz. Reducing the repetition rate - if
possible at all - results in a substantial reduction in the average excitation
power, and, consequently, low phosphorescence intensity. Attempts to compensate
for the drop in average power by higher peak power are limited by the
capabilities of the laser, by saturation and other nonlinear effects in the
sample, or, in multiphoton systems, unwanted excitation of higher energy levels
or even ionisation. In other words, the effect of reducing the excitation pulse
rate is poor sensitivity. Low sensitivity can partially be compensated by high
phosphor concentration. However, the commonly used phosphorescence dyes are
potentially toxic, and using them in high concentration is not desirable.
Simply reducing the laser repetition rate
causes a significant problem for recording FLIM simultaneously with PLIM. In
principle, it would be possible to derive FLIM and PLIM data from a one and the
same decay curve that is excited by low-repetition rate laser pulses and
simultaneously recorded at two different time scales. One channel would record
a photon distribution over the FLIM time scale, the other over the PLIM time
scale. However, this would unavoidably create a pile-up problem for the FLIM
channel. Typical fluorescence lifetimes are on the order of a few nanoseconds. Neither
the detector nor the TCSPC electronics of the FLIM channel are able to detect
several photons within this time and determine their arrival times at
picosecond accuracy. Detection of several photons per excitation pulse must
therefore be avoided. That means the detection rate must be kept at a level no
higher than 10% of the excitation rate [10, 12, 13]. With excitation rates on the
order of 100 kHz (for Ruthenium) and 10 kHz (for Platinum and
Palladium) the available detection rates become extremely low, and,
consequently, the acquisition times unacceptably long.
Another problem is that any sample that
emits phosphorescence necessarily also emits fluorescence. The fluorescence
both comes from endogenous fluorophores of the sample, and from singlet
emission of the phosphorescence probe. At high laser peak power the peak power
of fluorescence becomes extremely high. This causes transient overload and
extreme afterpulsing in the detectors. It is then impossible to detect a
correct phosphorescence decay in the first few microseconds after the laser
pulse. In principle, the overload problem can be solved by using laser pulses
with a duration in the microsecond range. However, apart from the fact this is
not simply feasible with most lasers it would make simultaneous FLIM
impossible. More importantly, microsecond pulse duration is not an option for
Interference with Scanning
PLIM in scanning systems has also another
problem. The time the scanner stays within the excited sample volume must be
longer than the phosphorescence lifetime. If the scanner runs off the excited
volume within the phosphorescence decay time photons in the tail of the decay
function are lost, and the recorded decay profile gets distorted. Reasonable
recording, even of pure intensity images, can thus be obtained only by sufficiently
slow scanning. However, if both the pixel time and the pulse repetition period
are long there are only a few excitation pulses within the pixel time. Unless
the laser pulse sequence is synchronised with the pixel sequence the number of
excitation pulses in the pixels varies systematically. This induces Moiré
effects in the images. The problem can be solved by synchronising the laser
pulses with the pixel frequency, but there is usually no provision for this in
normal laser scanning microscopes. Without synchronisation, the pixel time had
to be at least 100 times longer than the laser period. This leads to extremely
long frame times, and to a further increase of the acquisition time.
Fig. 1: Challenges of PLIM. Left: Low
laser repetition rate results in low average excitation intensity. Second left:
High peak-to-average power ratio causes high peak intensity of fluorescence, detector
overload and afterpulsing, and pile-up in parallel FLIM recording. The
phosphorescence intensity remains low due to low average power. Second right:
Scanning must be slow enough to stay in the excited pixel over the time of the phosphorescence
decay. Right: Low scan rate interferes with low laser pulse repetition rate. This
induces Moiré effects in the images.
FLIM - PLIM by Multipulse Excitation
The problems described above are avoided by
a FLIM / PLIM technique developed by bh in 2010 [4, 11]. The technique is based on the idea
that, if a single short laser pulse is not efficient in exciting phosphorescence,
a burst of multiple laser pulses will perform much better. As long as the burst
duration is shorter than the phosphorescence lifetime the excitation efficiency
will increase in proportion to the number of pulses within the burst. Multi-pulse
excitation has been used for multiphoton phosphorescence imaging earlier  but bh were first to apply it to TCSPC
The principle is shown in Fig. 2. The
sample is excited by a pulsed laser running at a repetition rate in the 50 to
80 MHz range, i.e. at a repetition rate as it is typically used for TCSPC FLIM.
However, the laser does not run continuously. Instead, it is turned on only for
a given period of time, Ton, at the beginning of each pixel. Within the
on-time, Ton, the laser pulses excite fluorescence, and, pulse by
pulse, build up phosphorescence. The phosphorescence intensity at the end of
the laser-on time is far higher than for a single laser pulse.
For the rest of the pixel time the laser is
turned off. After the last laser pulse, the fluorescence decays quickly, and
for the rest of the pixel dwell time, Toff, pure phosphorescence is detected.
Principle of Microsecond FLIM. A high-frequency pulsed laser is on-off
modulated synchronously with the pixels. FLIM is recorded in the Laser ON
phases, PLIM in the Laser OFF phases.
The buildup of TCSPC FLIM and PLIM images
with this excitation sequence is straightforward. For each photon, the TCSPC module determines the time, t, within the
laser pulse period, and the time, T, after the start of the modulation pulse.
The TCSPC process builds up photon distributions over these times and the scan coordinates
13, 15, 16]
The TCSPC principle is shown in Fig. 3. A
fluorescence lifetime image is obtained by building up a photon distribution
over the times, t, of the photons in the laser pulse period, and the scanner
position, x, y, during the Ton periods. The phosphorescence lifetime
image is obtained by building up a similar distribution over the times, T, within
the laser modulation period and the beam position, x, y. Thus, fluorescence and
phosphorescence lifetime images are obtained simultaneously, in the same scan,
and from photons excited by the same laser pulses.
Fig. 3: Simultaneous fluorescence and phosphorescence lifetime imaging
The procedure can be further refined by
using the laser on/off information as a routing signal to better separate the
fluorescence in laser-on phases from the phosphorescence in the laser-off
phases, please see [6, 7, 12].
The principle solves all the problems
discussed in the previous section. The excitation pulse rate of FLIM gets
de-coupled from the excitation rate of PLIM: The FLIM excitation rate is the
laser pulse period, the PLIM excitation period is the period of the on/off
modulation. The average excitation intensity drops only by the duty cycle of
the laser modulation, and the FLIM excitation rate remains high. High
phosphorescence intensity is obtained, and there is no problem with pile-up.
The peak intensity of the laser pulses need not be higher than for a normal
TCSPC FLIM measurement. The principle thus remains compatible with multiphoton
excitation. Moreover, there is no excessively high fluorescence peak intensity,
and no detector overload problem. Also the Moiré problem is solved: The laser
modulation is automatically synchronised with the pixels of the scan. Every
pixel thus gets the same number of excitation pulses.
Implementation in the bh FLIM Systems
All SPC-150, SPC-150N, and SPC‑160
TCSPC module as well as SPC‑830 modules later than serial number 3D0178
(May 2007)  have the hardware functions to record simultaneous FLIM / PLIM.
The only system requirement is that there is a way to on/off modulate the
excitation laser according to the principle shown in Fig. 2. Modulation is
performed in different ways in the bh FLIM systems for different laser scanning
DCS-120 Confocal Scanning FLIM System
Laser on/off modulation in the DCS-120
system is achieved via the laser multiplexing function of the GVD‑120
scan controller . The system
normally has two lasers which can be multiplexed within one pixel. PLIM operation
for one laser is obtained by enabling the pixel multiplexing function, and
turning the other laser off optically. The laser then turns on at the beginning
of each pixel, runs for a fraction of the pixel time, and then turns off.
The parameter definitions are shown in Fig.
4. Both lasers are turned on. The second laser is disabled optically by turning
the laser attenuator wheel at the scanner fully down. Laser multiplexing is set
to Pixel. The fraction of the pixel time in which Laser 1 is on is defined
in the field left of % for 1st laser. This is the time when the laser is
running, and fluorescence is measured. For the rest of the pixel time the laser
is off, and phosphorescence is measured.
For PLIM, a scan speed must be selected
that keeps the scanner within the same pixel for a period of time a few times
longer than the phosphorescence decay time. The automatic selection of the scan
speed (normally used for FLIM recording) must therefore be turned off, and an
appropriate scan speed be selected. This is achieved by turning off the Auto
button for the scan rate, and selecting a pixel time, Tpxl, a few
times longer than the expected phosphorescence decay time.
To avoid that the scanner moves during the
pixel time the DCS-120 scanner has an option to run along the lines in steps of
the individual pixels (scanners normally run continuously to achieve fast
scanning). Stepping along the lines is defined by setting Line Type to
Scan format and scan area definitions in
the scanner control panel are the same as for standard FLIM. Please see  for details.
Fig. 4: DCS‑120 scanner setup for simultaneous FLIM/PLIM. Left: Scan
and laser control parameters. Right: PLIM timing parameters
The time range of PLIM is defined in the
Configure sub-menu of the TCSPC system parameters, see Fig. 4, right. For
efficient PLIM recording, Tpxl should be a about the same as the Time Range selected in the Configure panel. Please see  and  for details.
DCS-120 MP Multiphoton FLIM System
The DCS-120 MP is the multiphoton version
of the DCS‑120 confocal FLIM system. It uses a Ti:Sa laser for excitation
. Since November 2015 the
DCS‑120 MP is available with a AOM (acousto-optical modulator) for laser
power control and modulation. Laser modulation is controlled the same way as
for the ps diode lasers of the confocal system. The parameter settings are the
same as shown in Fig. 4.
PZ-FLIM-110 Piezo Scanning FLIM System
The PZ-FLIM-110 system uses sample scanning
by a piezo stage . Since the stage is driven by the bh GVD-120 scan
controller FLIM / PLIM is available the same way as in the DCS-120 system. Please
see Fig. 4 for the setup of the scan control parameters.
Zeiss LSM 710, 780, 880 Systems
For the FLIM systems for the Zeiss LSM 710 / 780 / 880
microscope family a bh DDG-210 pulse generator card is added to the FLIM system.
The DDG card triggers on the pixel clock of the LSM, and sends a Laser On
signal to the laser controller of the microscope. The principle is shown in Fig.
5. The pixel clock is split off from the scan synchronisation cable and
connected into the trigger input of the DDG card. The Laser On signal is
connected into the laser control module of the Zeiss LSM via a PLIM input.
Please note that this input is optional; it has to be ordered from Zeiss via an
INDIMO (individual modification) request. A PLIM macro has to be installed to
activate and de-activate the PLIM input. PLIM Laser control via the DDG‑210
card is integrated in the SPCM software, see Fig. 5, right. The laser-on time
is defined on the left. The times on the right define a routing signal that is
used to separate the photon from the laser-on and the laser-off times in the
SPC module. The routing signal can be delayed with respect to the
laser-modulation pulse to compensate for the delay in the AOM of the
microscope. Please see  for
Fig. 5: Left: Principle of laser on/off control for the Zeiss LSMs. Right:
Laser control panel of bh SPCM software.
Leica SP5, SP8, SP11 Multiphoton Systems
Since October 2015 FLIM / PLIM is available
also for the bh FLIM systems for the Leica SP5, SP8, and SP11 multiphoton
microscopes. Laser on-off is controlled by a bh DDG-100 pulse generator module
that is added to the FLIM system. The card is triggered by the pixel clock of
the microscope. The on-off signal from the DDG is fed into the beam blanking control
of the microscope via a logic gate.
Laser power control in the Leica
multiphoton systems is performed by an EOM (electro-optical modulator). The EOM
is fast enough for PLIM on-off modulation. However, we often found that it does
not turn the laser entirely off. This is no problem in standard imaging applications
but it can be a problem for PLIM. Spurious excitation during the laser-off phases
causes a large background in the phosphorescence decay or even makes it
impossible to record phosphorescence at all. The solution is an ND filter in
the excitation beam path. FLIM / PLIM is performed at no more than 5% of the
available laser power. A filter that transmits about 20% shifts the power range
from 0 to 5% to 0 to 25%, and reduces the laser power in the off phases
sufficiently to avoid spurious excitation. Please use a reflective filter (an
absorptive filter may crack), and tilt it by a few degrees to avoid
back-reflection into the laser.
Leica systems use a sinusoidal scan in x
direction. The nonlinearity of the scan is compensated by a non-uniform pixel
time. This is not a problem for the bh FLIM systems: The bh systems use the
pixel clock from the Leica scanner and thus avoid distortion of the images [5, 14]. For PLIM, however, the variable pixel
time along the lines results in a variable laser on/off period and a variable
effective PLIM excitation rate. Also this is not normally a problem. However, the
scan rate should be selected slow enough to let the phosphorescence completely decay
within the pixel time. Normally, incomplete decay can be taken into account by
a suitable model in the SPCImage data analysis . However, this requires
that the excitation period is constant over the entire image. This is not the
case for PLIM with the Leica microscopes.
Oxygen sensing by PLIM has become a hot
topic in biomedical microscopy, see [21, 24, 27, 28, 29, 30, 32]. Until
recently, phosphorescence imaging has mainly been performed by gated camera
techniques. The disadvantage of these techniques is that they neither yield
images from deeper tissue layers nor images with optical sectioning. PLIM by
the technique described here solves these problems by confocal and two-photon
laser scanning microscopy, and, additionally, yields FLIM and PLIM simultaneously.
An increasing number of publications therefore aims at the use of PLIM for
oxygen sensing in cells and tissue. Toncelly et al. used the technique to
characterize the sensor dyes . The penetration into cells and the behaviour
of the dyes in the biological environment was investigated by Dmitriev et al. . The response of the cells and
cell clusters on variations in the oxygen concentration in physiological
conditions has been investigated by [18, 22, 23, 29]. An overview on the FLIM /
PLIM technique and an introduction into the use of an oxygen-sensitive solid matrix
for cells has been given by Jenkins et al. .
Examples are shown in the figures below. Fig.
6 and Fig. 7 show cultured human embryonic kidney cells incubated with a palladium-based
phosphorescence dye. Fig. 6 was recorded under atmospheric oxygen partial
pressure. The maximum of the lifetime distribution over the pixels is at
75 s. Fig. 7 was recorded under decreased oxygen partial pressure. As can
be seen, the maximum of the lifetime distribution has shifted to 144 µs.
Fig. 6: HEK cells incubated with a palladium dye under atmospheric 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.
Fig. 7: HEK cells incubated with a palladium dye under 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 212 µs. The maximum of the
lifetime distribution over the pixels is at 144 µs.
Simultaneous Recording of PO2 and NAD(P)H Images
Simultaneously recorded fluorescence and
phosphorescence lifetime images of live cultured human squamous carcinoma
(SCC-4) cells stained with tris (2,2-bipyridyl) dichlororuthenium (II)
hexahydrate are shown in Fig. 8, left and right. The data were acquired on a
Zeiss LSM 780 NLO microscope with a bh Simple-Tau 152 system. The
excitation wavelength was 750 nm. The image on the left was recorded in a
wavelength interval from 440 to 480 nm. It contains mainly fluorescence of
NAD(P)H. The data were analysed with a double-exponential decay model. The
image shows the ratio of the amplitudes, a1 and a2, of the decay components. The
a1/a2 ratio directly represents the ratio of unbound (a1) and bound (a2)
NAD(P)H. The image on the right is the PLIM image. It shows the phosphorescence
lifetime of the Ruthenium dye. The lifetime is reciprocally related to the
Fig. 8: FLIM and PLIM images of SCC-4 cells stained with (2,2-bipyridyl)
dichlororuthenium (II) hexahydrate. FLIM shown left, PLIM shown right. Zeiss
LSM 780 NLO with PLIM option, Simple-Tau 152 FLIM/PLIM system, 2-photon excitation
at 750 nm.
Although the results obtained so far look
promising caution appears indicated when PLIM data are interpreted in terms of
absolute O2 concentration measurement. As can be seen from Fig. 8
the ruthenium dye binds to the constituents of the cells. The phosphorescence
lifetime of bound and unbound dye can be different. Moreover, quenching
phenomena are at least in part diffusion-controlled. The quenching rate - and
thus the sensitivity to oxygen - more or less depends on the oxygen diffusion
constant. The diffusion constant may be different inside the cells and outside,
and in different compartments of the cells. pO2 results derived from
PLIM decay times may therefore not necessarily be comparable for different
sub-structures of the cells.
Detection of Zinc Oxide Nanoparticles
There are also FLIM / PLIM applications
that use phosphorescence to identify nanoparticles in biological tissue, and
follow their migration or possible dissolution. The principle is used to track
ZnO nanoparticles from sunscreens or cosmetical products in human skin, and
investigate possible influence on the viability via the fluorescence of NAD(P)H
. Fig. 9 shows zinc oxide nanoparticles can easily be detected by PLIM. The
decay function is multi-exponential, with average (intensity-weighted)
lifetimes up to 20 µs.
Fig. 9: PLIM of zinc oxide nanoparticles. Left: Lifetime image, intensity
weighted lifetime of double-exponential fit. Right: Decay curve at cursor
position. Zeiss LSM 710, two-photon excitation at 750 nm,
PLIM of Inorganic Materials
Fig. 10 was obtained from an Autumit crystal
(a uranium mineral). The phosphorescence lifetimes vary from about 100 us
to 400 us. The lifetime image is shown on the left, decay curves of two
selected spots on the right. The pixel time was 3.6 ms, the laser-on time
200 µs. The excitation wavelength was 405 nm, a 435 nm long pass
filter was used in the emission path.
Fig. 10: PLIM image of a uranium
mineral. Decay curves if two arbitrary selected spots are shown on the right.
256x256 pixels, 256 time channels, pixel time 3.6 ms, excitation
405 nm, emission filter long pass 435 nm.
Suppression of Autofluorescence
Other applications are using PLIM for
suppressing of autofluorescence by using the long lifetime of PLIM as a
discrimination parameter [1, 2].
The SPCM software offers this option online, without the need of special data
analysis, see [12, 6, 7].
Compared with PLIM techniques that use a
single excitation pulse for every phosphorescence decay cycle our techniques
has a number of significant advantages. The first one is that excitation with
multiple pulses obtains a significantly higher triplet population than excitation
with a single pulse. The sensitivity is therefore much higher. The technique
can thus be used at correspondingly lower concentration of the phosphorescence
probe, which, in turn, helps reduce possible toxicity. The second advantage is
that it is compatible with multiphoton excitation. Due to the excitation with
multiple laser pulses it does not require higher laser power or laser pulse
energy than normal confocal or multiphoton FLIM. A third advantage is related
to the TCSPC technique itself. TCSPC FLIM can record no more than one photon
per laser pulse. The photon rate thus has to be limited to no more than 10% of
the excitation pulse rate. This is no problem for the 80 MHz or 50 MHz pulse
rates of Ti:Sapphire or picosecond diode lasers but it would be a problem if
the pulse repetition rate was reduced to the kHz range. Our technique avoids
this limitation because it works at the full laser repetition rate. The acquisition
times is therefore on the order of 10 to 100 seconds, depending on the
expectations to the signal-to-noise ratio of the lifetimes [10, 12]. The only
remaining limitation is in the scan rate. The pixel time must not be shorter
than about 5 times the phosphorescence decay time. This leads to minimum frame
times in the range of 1 second for ruthenium dyes and about 10 seconds for
platinum dyes. This no longer than the acquisition time required to obtain the
desired signal-to-noise ratio. It thus has no influence on the total acquisition
time of the FLIM / PLIM process.
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 (2014)
3. Becker & Hickl GmbH, FLIM in the FIFO Imaging Mode: Large
Images with Small TCSPC Modules. Application note, available on
4. Becker & Hickl GmbH, Microsecond Decay FLIM: Combined
Fluorescence and Phosphorescence Lifetime Imaging. Application note, available
5. Becker & Hickl GmbH, Multiphoton FLIM with the Leica HyD RLD
Detectors. Application note, available on www.becker-hickl.com
6. Becker & Hickl GmbH, DCS-120 Confocal Scanning FLIM Systems, 6th
ed. (2015), user handbook. www.becker-hickl.com
7. Becker & Hickl GmbH, Modular FLIM systems for Zeiss
LSM 710 / 780 / 880 family laser scanning microscopes. 6th ed. (2015), user
handbook. available on www.becker-hickl.com
8. Becker & Hickl GmbH, DCS-120 MP system records multiphoton
FLIM and PLIM. Application note (2015), available on www.becker-hickl.com
9. Becker & Hickl GmbH, PZ-FLIM-110 piezo scanning FLIM
system. Data sheet (2015), www.becker-hickl.com
10. W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005
W. Becker, B. Su, A. Bergmann, K. Weisshart, O. Holub,
Simultaneous Fluorescence and Phosphorescence Lifetime Imaging. Proc. SPIE 7903, 790320
12. W. Becker, The bh TCSPC handbook. 9th edition, Becker & Hickl
GmbH (2021), available on www.becker-hickl.com
13. W. Becker, Introduction to Multi-Dimensional TCSPC.
In W. Becker (ed.) Advanced time-correlated single photon counting
applications. Springer, Berlin, Heidelberg, New York (2015)
14. 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 (2015)
15. W. Becker, Fluorescence Lifetime Imaging
Techniques: Time-correlated single-photon counting. In: L. Marcu. P.M.W.
French, D.S. Elson, (eds.), Fluorecence lifetime spectroscopy and imaging.
Principles and applications in biomedical diagnostics. CRC Press, Taylor &
Francis Group, Boca Raton, London, New York (2015)
16. W. Becker, Fluorescence lifetime imaging by multi-dimensional
time correlated single photon counting. Medical
Photonics 27, 41-61 (2015)
17. L.J. Charbonniere, N. Hildebrandt, Lanthanide complexes and quantum
dots: A bright wedding for resonance energy transfer. Eur. J. Inorg. Chem.
2008, 3241-3251 (2008)
18. R. I. Dmitriev, A. V. Zhdanov, Y. M. Nolan, D. B. Papkovsky, Imaging
of neurosphere oxygenation with phosphorescent probes. Biomaterials 34,
19. R. I. Dmitriev, A. V.
Kondrashina, K. Koren, I. Klimant, A. V. Zhdanov, J. M. P. Pakan, K. W. McDermott,
D. B. Papkovsky, Small molecule phosphorescent probes for O2 imaging in 3D
tissue models. Biomater. Sci. 2, 853-866 (2014)
20. A. Ferchner, S.M. Borisov, A. V. Zhdanov, I. Klimant, D.B.
Papkovsky, Intracellular O2 sensing probe based on cell-penetrating
phosphorescent nanoparticles. ACS Nano 5 5499-5508 (2011)
21. H.C. Gerritsen, R. Sanders, A. Draaijer, Y.K. Levine, Fluorescence
lifetime imaging of oxygen in cells, J. Fluoresc. 7, 11-16 (1997)
22. 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)
23. 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)
24. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edn.,
25. N. A. Hosny, D. A. Lee, M. M. Knight, Single photon counting
fluorescence lifetime detection of pericellular oxygen concentrations. J.
Biomed. Opt. 17(1), 016007-1 to -12 (2012)
26. 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 (2015)
27. A. Y. Lebedev, A. V. Cheprakov, S.
Sakadzic, D. A. Boas, D. F. Wilson, Sergei A. Vinogradov, Dendritic
Phosphorescent Probes for Oxygen Imaging in Biological Systems. Applied
Materials & Interfaces 1, 1292-1304 (2009)
28. D. Papkovsky, A. V. Zhdanov, A.
Fercher, R. I. Dmitriev, and J. Hynes, Phosphorescent oxygen-sensitive probes
29. D. B. Papkovsky, and R. I. Dmitriev, Biological detection by optical
oxygen sensing, Chem Soc Rev 42, 8700-8732 (2013)
30. S. Sakadic, E. Roussakis, M. A.
Yaseen, E. T. Mandeville, V. J. Srinivasan1, K. Arai, S. Ruvinskaya, A. Devor,
E. H. Lo, S. A. Vinogradov, D. A. Boas, Two-photon high-resolution measurement
of partial pressure of oxygen in cerebral vasculature and tissue. Nature
Methods 7(9) 755-759
31. W. Y. Sanchez, M. Pastore, I. Haridass, K. König, W. Becker, M. S. Roberts, Fluorescence
Lifetime Imaging of the Skin. In: W. Becker (ed.) Advanced time-correlated
single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)
32. M. Shibata, S. Ichioka, J. Ando, A. Kamiya, Microvascular and interstitial
PO2 measurement in rat skeletal muscle by phosphorescence quenching. J. Appl.
Physiol. 91, 321-327 (2001)
33. C. Toncelli, O. V. Arzhakova, A. Dolgova, A. L.
Volynskii, N. F. Bakeev, J. P. Kerry, D. B. Papkovsky, Oxygen-sensitive
phosphorescent nanomaterials produced from high density polyethylene films by
local solvent-crazing. Anal. Chem. 86(3), 1917-23 (2014)
Becker & Hickl GmbH