Photon counting histograms

Reviews listed in Further Reading provide excellent introductions to PCS. Related techniques have been developed to detect other molecular properties. These properties include fluorescence cross-correlation spectroscopy (FCCS) (28) to detect codiffusing fluorophores and photon-counting histograms (PCH) (29), or fluorescence intensity distribution analysis (FIDA) (29) to distinguish fluorescent species according to their brightness. 

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

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...

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)... 

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.

Perroud, TD, Bo, HA, Wallace, MI, and Zare, RN, Photon counting histogram for one-photon excitation. Chemphyschem i 2003) 1121-1123. 

Muller, JD, Chen, Y, Gratton, E, in R Rigler and ES Elson (Eds), Photon counting Histogram Statistics. Fluorescence Correlation Spectroscopy Theory and Applications. Springer, Berlin, 2001, pp. 410 37. 

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. 

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.

FRET fluorescence resonance energy transfer FCS fluorescence correlation spectroscopy TIRF total internal reflection fiuorescence PCFI photon counting histogram ICCD intensified charge coupled device EMCCD eiectron muitipiying charge coupled device CMOS complimentary metal oxide semiconductor AFM atomic force microscope. 

FCS fluorescence correlation spectroscopy PCH photon counting histogram TCSPC time correlated single photon counting MCS muiti-channei scaiar APDiavaianche photodiode PMT photo-mutipiiertube PCi peripherai component interconnect. 

Perroud, TD, Huang, B, Zare, RN, Effect of Bin Time on the Photon Counting Histogram for One-Photon Excitation. Chemphyschem 6 (2005) 905 912. 

Chirico, G, Olivini, F, and Beretta, S, Fluorescence excitation volume in two-photon microscopy by autocorrelation spectroscopy and photon counting histogram. Applied Spectroscopy 54 (2000) 1084—1090. 

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. 

Huang, B., Perroud, T.D., Zare, R.N. Photon counting histogram one-photon excitation. Chemphyschem. 5, 1523-1531 (2004)... 

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