Fluorescence photon counting histogram

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. 

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.

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. 

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. 

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. 

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 voltage pulse produced by the TAC is fed to the multichannel analyzer (MCA), and is stored in a specific channel according to its amplitude, and hence time, post-excitation. The probability of a single photon event being counted is high soon after excitation and decreases with time. Repetitive operation of the TAC produces a probability histogram for the detection of fluorescence photons, which is identical to the fluorescence decay curve. 

To obtain fluorescence lifetimes time correlated single photon counting (TCSPC) was used. In TCSPC, the elapsed time is measured between an excitation pulse from a pulsed laser and a detected photon. A histogram of the elapsed times provides a fluorescence decay curve, from which the fluorescence lifetime, Zf, is extracted. Examples of decay curves for bare silica and single R6G molecules on silica taken with a near-field probe are shown in Fig. 11 [21]. 

Figure 11. Time-correlated single-photon counting rate histograms. Top Panel Plots obtained from a single Rhodamine-6G molecule on silica (trace A) and a bare silica substrate (background) < 10 nm under an NSOM probe (trace B). Center Panel Decays obtained for a higher coverage surface with the tip at a distance of <10 nm (trace C) and 1.1 micrometers (trace D) above the surface. Bottom Panel Traces obtained at lateral positions of maximum fluorescence intensity ( + lOnm) over different single Rhodamine-6G molecules. The extracted lifetime values are 4.6 (trace E), 3.4 (trace F), 2.1 (trace G) and 1.3 ns (trace H). The rates are normalized to 10 per second at zero time for slope comparisons. Background traces were subtracted in the center and bottom panels. Farfield powers (Top) 33 nW, (Center) 1.8 nW, and (Bottom) 20 nW. The bin width was 30.8 ps. Adapted from Ref. 21.

A related experimental method is Time-Correlated Single Photon Counting (TCSPC), which generates a histogram representing the fluorescence intensity over time. This is an efficient method because it counts photons and records their arrival time, which directly represents the fluorescence decay. 

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