The Photon Counting Histogram

The PCH/FIDA technique can be extended for two-dimensional histograms of the intensity recorded by two detectors in different wavelength intervals or under different polarisation. 2D-FIDA delivers a substantially improved resolution of different fluorophores [257, 258]. Further improvement is achieved by using 

2D-FIDA and FILDA do not directly deliver information about the diffusion times of the fluoreseent species. Diffusion times are, however, obtained by calculating the PCHs in different sampling time intervals and fitting the result by a model that contains both the moleeular brightness and the diffusion time [258]. 

Although TCSPC is used successfully for BIFL experiments, little has been published about applications for FID A. The reason is probably that the dead time of TCSPC is considered a drawback. However, a simple consideration shows that the detectable burst rate is not substantially reduced by the dead time of the TCSPC device. The commonly used detectors lose 50% of the photons at an input rate of about 1610 s [408], fast TCSPC modules at 10-10 s (see Fig. 5.94, page 162). This means the dead time of the TCSPC module is only slightly longer than the dead time of the detector. The use of TCSPC for PCH experiments therefore does not result in a considerable increase of dead-time-related errors. Moreover, BIFL applications have shown that the burst count rates are well within the counting capability of TCSPC (see below). 

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Chen Y, Muller JD, So PTC, Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys. J. 

J.D. Muller, Y. Chen, E. Gratton, Resolving Heterogeneity on the single molecular level with the photon-counting histogram, Biophys. J. 78, 474-586 (2000)... 

Perroud, TD, Huang, B, and Zare, RN, Effect of bin time on the photon counting histogram for one-photon excitation. ChemPhysChem 6 (2005) 905-912. 

Muller, JD, Chen, Y, and Gratton, E, Resolving heterogeneity on the single molecular level with the photon- counting histogram Biophysical Journal 78 (2000) 474—486. 

Perroud, T.D., Bokoch, M.P., Zare, R.N. Cytochrome c conformations resolved by the photon counting histogram watching the alkaline transition with single-molecule sensitivity. Proc. Natl. Acad. Sci U.S.A. 102, 17570-17575 (2005)... 

Fig. 5.122 Photon counting histogram. Left Photons counted in snccessive sampling time intervals. Right Histogram of the number of time intervals contarning N photons...

Photon-Counting Histogram. Contains the distribution of the fluorescence intensity of a small number of molecules measured within consecutive time bins. The PCH is the basis of Fluorescence Intensity Distribution Analysis (FIDA). 

A higher level of sophistication in analysis is achieved by using photon counting histograms (PCH). PCH are formed by a thorough statistical analysis of the distribution of the number of detected photons in each burst (or the distribution of the fluorescence intensity measured in each counting interval). PCH is mainly... 

Figure 2.2 Schematic illustration of the conceptual stages in the development of a model to fit photon counting histograms, (a) The case of a non-fluctuating fluorescent particle fixed at the centre of a closed excitation/detection volume (I/q). (b) The case when fluctuations are created by diffusion of the fluorescent molecule around a closed volume with spatially varying excitation/detection efficiency. (c)The case of multiple diffusing molecules in the closed volume, (d) The case when molecules can enter and leave the volume, (e) The case when molecules with different molecular brightness can enter and leave the volume.

Figure 3.17 Photon count histogram of an ideal scatterer placed at the laser focus. The collected photon count distribution (circles, normalized) is fitted exactly by a Poissonian function (line) indicating that there are no fluctuations in the detected signal arising from instability of the light source or other instrumentation.

Figure 5.11 Photon Count histogram of 5 nM unbound conjugate (estradiol-Tamra) (a) in the presence of 1 (xM competitor (estradiol) or (b) in the absence of competitor at full complex formation with nanoparticles. Total number of photons counted within the measurement time was 1,848,127 (a) and 1,504,390 (b). Reprinted with permission from SchaertI ef a/., A novel and robust homogeneous florescence-based assay using nano particles from pharmaceutical screening and diagnostics. Journal of Biomolecular Screening 5 (2000) 227-237. Copyright 2000 Sage Publications, Inc.

In the related photon counting histogram technique, a histogram of the intensity of fluorescence from individual molecules is collected as molecules diffuse through the focal volume of a confocal microscope [278-280]. If the sample contains a mixture of molecules with different fluorescence properties, the histogram can reveal the relative amplitude of the fluorescence from a single molecule of each class, as well as the number of molecules in each class. 

The measurement of the time histogram of a sequence of photons with respect to a periodic event. This technique requires a time-to-ampUtude converter. See also Photon Counting... 

Time-correlated single photon counting A technique for the measurement of the time histogram of a sequence of photons with respect to a periodic event, e.g. a flash from a repetitive nanosecond lamp or a CW operated laser mode-locked laser). The essential part is a time-to-amplitude-converter (TAG) which transforms the arrival time between a start and a stop pulse into a voltage. Sometimes called single photon timing. 

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. 

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