Single photon counting

Time correlated single photon counting is a well-established technique that has been used to measure fluorescence lifetimes since the mid-1960 s. These early experiments, which used a variety of flashlamps and gaseous gap-discharge arcs as the excitation source, were reviewed by Ware [47, 48] in 1971. The traditional light sources have been replaced by laser sources in recent experiments, thus markedly extending the range of applications of this technique. Particularly well suited excitation sources for this method are the mode-locked lasers and synchronously pumped dye lasers which are capable of operation at MHz repetition rates. 

Although this technique always requires that the pulse rate be kept low enough so that single photons can be detected, there are two alternative methods to correct for pulses missed by the TAG. The first is to make a mathematical correction to the data. Such a correction has been described by Goates [49] and is given by 

The TAG/MCPHA combination must be calibrated to give the time interval per channel. This can be achieved in a number of different ways 

The method of single-photon counting has been applied to measure lifetimes in a lai e number of different systems. An example of its application, the measurement of the subnanosecond decay of Rhodamine B, is provided by Koester and Dowben [54]. The choice of examples to illustrate these techniques is somewhat arbitrary however, this study was chosen since the experimental system constructed by these authors fairly well describes the state of the jurt. Their system utilized a cavity dumped synchronously pumped tunable dye laser producing 35 ps pulses at 

Experimental measurement of fluorescence decay 4.1 SINGLE PHOTON COUNTING 

The TAC/MCPHA combination must be calibrated to give the time interval per channel. This can be achieved in a number of different ways [47] and is a particularly simple process if a mode-locked laser is used as the excitation source. Since the mode-locker frequency should be stable and well known, then the spacing between laser pulses is also well defined. By scattering the laser pulses so that they can be detected by the photon counting system, accurate time markers can be obtained. 

The time resolution of the electronics in a single photon counting system can be better than 50 ps. A problem arises because of the inherent dispersion in electron transit times in the photomultiplier used to detect fluorescence, which are typically 0.1—0.5 ns. Although this does not preclude measurements of sub-nanosecond lifetimes, the lifetimes must be deconvoluted from the decay profile by mathematical methods [50, 51]. The effects of the laser pulsewidth and the instrument resolution combine to give an overall system response, L(f). This can be determined experimentally by observing the profile of scattered light from the excitation source. If the true fluorescence profile is given by F(f) then the 

low-temperature housing A, amplifier S, pulse shaper D, discriminator CT, counter T, timer, (b) Principle of the discriminator Vj and V2 are the low and high signal limits 

The low level comparator will reject pulses of low intensity which are generated by thermoionic emission in the dynodes. It is obvious that an electron emitted by dynode 5 for instance will produce a much smaller avalanche at the anode than an electron emitted by the photocathode but of course thermoionic emission from the photocathode itself would still appear as a genuine pulse, and for this reason PM tubes used in photon counting applications must be cooled down. 

The high level of the discriminator rejects pulses that are much higher than those produced by primary electron emission from the photocathode. Such pulses occur when ionizing radiation passes through the PM tube, because electrons can then be released from several dynodes at (nearly) the same time. 

The distinction between short-lived and long-lived luminescences is not totally arbitrary from the point of view of the experimental conditions required for their observation. It can be set at a lifetime of around 1 ps which corresponds to the quenching time of excited states by molecular 

Horizontal axis, wavelength in nm/100 vertical axis, light intensity in arbitrary units 

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The other coimnon way of measuring nanosecond lifetimes is the time-correlated single-photon counting... 

O Conner D V and Phillips D 1984 Time-Correlated Single Photon Counting (London Academic)... 

Figure C1.5.12.(A) Fluorescence decay of a single molecule of cresyl violet on an indium tin oxide (ITO) surface measured by time-correlated single photon counting. The solid line is tire fitted decay, a single exponential of 480 5 ps convolved witli tire instmment response function of 160 ps fwiim. The decay, which is considerably faster tlian tire natural fluorescence lifetime of cresyl violet, is due to electron transfer from tire excited cresyl violet (D ) to tire conduction band or energetically accessible surface electronic states of ITO. (B) Distribution of lifetimes for 40 different single molecules showing a broad distribution of electron transfer rates. Reprinted witli pennission from Lu andXie [1381. Copyright 1997 American Chemical Society.

Birch D J S and Imhof R E 1977 A single-photon counting fluorescence decay-time spectrometer J. Phys. E Sol. Instrum. 10 1044-9... 

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

Gompf B, Gunther R, Nick G, Pecha R, Eisenmenger W (1997) Resolving sonoluminescence pulse width with time-correlated single photon counting. Phys Rev Lett 79 1405-1408... 

Advanced Time-Correlated Single Photon Counting Techniques... 

The lifetime resolution is the smallest variation in lifetime that can be detected. If external noise sources are ignored, the lifetime resolution depends essentially on the photon-economy of the system. For instance, if a 2 ns lifetime is measured with a 4 gate TG single-photon counting FLIM (F = 1.3) and 1000 photons, variations of about 80 ps can be resolved. However, for reasons discussed earlier, in biological samples these values could be higher. 

Becker, W., Bergmann, A., Hink, M. A., Konig, K., Benndorf, K. and Biskup, C. (2004b). Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc. Res. Tech. 63, 58-66. 

O Connor, D. V. and Phillips, D. (1984). Time-Correlated Single Photon Counting. Academic press, London. 

Becker, W. (2005). Advanced time-correlated single photon counting techniques. Springer, Berlin Heidelberg New York. 

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

Fig. 13.23 (a) Transmission power of microtoroid resonator near the condition of critical coupling (inset shows the MNF/microtoroid sensor), (b) Single photon counting events C(t) as a function of time t after the release of the cold atom cloud at t 0. Reprinted from Ref. 48 with permission. 2008 Nature Publishing Group... 

For fluorescence decay curves of the J-aggregate LB films of [CI-MC] mixed with various matrix agents, measured with a picosecond time-resolved single photon counting system, three components of the the lifetimes fitting to exponential terms in the following equation ... 

The dynamics of the SHL intensity after subpicosecond UV laser excitation of RuC18B LB films is shown in Figure 32[115,116]. The SHL intensity decreased to 70 % of its initial value upon excitation and returned to almost the initial value within several hundred picoseconds as shown by a bold line. The fluorescence decay of RuC18B LB films measured by the single photon-counting... 

For EPy-doped PMMA film, a 308 nm excimer laser (Lumonics TE 430T-2, 6ns) was used as as exposure source. We used a tine-correlated single photon counting systen (18) for measuring fluorescence spectra and rise as well as decay curves of a snail ablated area. The excitation was a frequency-doubled laser pulse (295 nm, lOps) generated from a synchronously punped cavity-dumped dye laser (Spectra Physics 375B) and a CW mode-locked YAG laser (Spectra Physics 3000). Decay curves under a fluorescence microscope were measured by the same systen as used before (19). 

The authors wish to express their sincere thanks to Messrs. A. Kurahashi and S. Eura for their experimental efforts. Thanks are also due to Prof. I. Yamasaki and Dr. N. Tamai who helped us with the single photon counting measurements. The present work is partly... 

Understand the principles of time-correlated single-photon counting. 

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