Single photon counting time-correlated

It consists of exciting the sample with package of short 20 pulses at maximum. After each excitation, only the first emitted photon is detected and counted. The excitation pulses could have slightly different intensities but they reach the sample after a defined time interval. The time that elapses between excitation and the detection of the first emitted photon is recorded. The distribution of these times gives a statistical picture of the fluorescence decay. 

Insirumant Theoretical Kinetic Model Fitted Function 

Equation 2.37 describes tile mathematical descrqition of a reconvolution ana sis 

Composed of two a and p chains (MW = 5710 and 20572, respectively), Lens culinaris agglutinin (LCA) is a tetramer p2 with a molecular weight equal to 52570 (Loris et al. 1993). LCA contains five Trp residues, three embedded in the protein matrix (Trp 152p, Trp 19a and Trp 40a) and two near the protein surface ( Trp 53p and Trp 128p). 

In the single photon counting, the detection system measures the time between the excited pulse and the aiTival of the first photon. The distribution of arrival times represents the decay curve. 

Th e are many subtleties in TCSPC which are not obvious at fust examination. Why is the photem counting rate limited to one photon per 100 las pulses Present electronics feu TCSPC only allow detection of the first arriving photon. Once the first photon is det ted, the dead time in the electronics prevents detection of anoth photon resulting from the same excitation pulse. Recall that onis-slon is a random event. Following the excitation pulse, mc e photons are emitted at early times than at late times. If all could be measured, then the histogram of arrival times would represent the intensity decay. However, if many arrive, and only the first is counted, then the intensity decay is distorted to shorter times. This effect is described in more detail in Section 4.5.F. 

Another important feature of TCSPC is the use of the rising edge of the photoelectron pulse for timing. This allows phototubes with nanosecond pulse widths to provide subnanosecond resolution. This is possible because the rising edge of the single photon pulses are usually steeper than one would expect from the time response of the PMT. Also, the use of a constant fraction discriminator provides improved time resolution by removing the variability due to the amplitude of each pulse. 

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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.

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... 

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. 

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

The technique of time-correlated single-photon counting (Figure 3.4) is used to measure an excited singlet-state lifetime, x. The sample is irradiated with a very short-duration light pulse ( lns) to ensure any given molecule will only be excited once during the pulse. As soon as... 

Figure 3.4 Schematic diagram of the principal components of a time-correlated single-photon counting apparatus...

D. J. S. Birch and R. E. Imhof, Time-domain fluorescence spectroscopy using time-correlated single-photon counting, in Topics in Fluorescence Spectroscopy (J. R. Lakowicz, ed.), Vol. 1, pp. 1-95, Plenum Press, New York (1991). 

From the standpoint of time domain (e.g., time-correlated single photon counting) experiments the method of modelocking is not too crucial as long as the pulse jitter is modest (some picoseconds), and the pulse intensity doesn t vary too much if the time-to-amplitude converter is being started instead of stopped by the excitation pulse, it may be immaterial. From the standpoint of the frequency domain, however, the... 

Figure 8.9. Time-resolved fluorescence spectra of 9,10-diphenylanthracene, recorded wi th time-correlated single photon counting, Aa = 360 nm. Parameters gate width and delay time relative to the intensity maximum of the excitation pulse.

The transient response of luminescent substances to pulsed excitation can be captured in the time domain by sampling enough data points within the time span of the decay. For example, fast digitizers are commonly employed to store phosphorescence decays. If fast digitizers are unavailable, time-correlated single-photon counting can be used to monitor fluorescence decays. 

S. Yu. Egorov, V. F. Kamalov, N. I. Koroteev, A. A. Krasnovsky Jr, B. N.Toleutaev,andS. V. Zinukov, Rise and decay kinetics of photosensitized singlet oxygen luminescence in water measurements with nanosecond time-correlated single photon counting technique, Chem. Phys. Lett. 421 —424 (1989). 

D. J. S. Birch, D. McLoskey, A. Sanderson, K. Suhling and A. S. Holmes, Multiplexed time-correlated single-photon counting, J. Fluorescence 4, 91-102 (1994). 

S. Kinoshita and T. Kushida, High-performance time-correlated single photon counting apparatus using a side-on type photomultiplier, Rev. Sci. Instrum. 53, 469-472(1982). 

D. J. S. Birch, R. E. Imhof and C. Guo, Fluorescence decay studies using multiplexed time-correlated single-photon counting application to aminotetraphenylporphyrins, /. Photochem. Photobiol. A Chem. 42, 223-231 (1988). 

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