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The photon counting histogram (PCH) analysis of the fluorescence fluctuations provides the molecular brightness (epsilon) and the average number of fluorophores (N) in an open observation volume. PCH, which is based on the analysis of the whole of the photon counting histogram, has been recently improved by taking into account the detector dead time effect, which is relevant at high fluorescence rates. We investigate here the possibility of quantitatively applying the PCH analysis in the simplified form of photon moment analysis, in which only the first two moments of the photon counting histogram are computed. We have applied this analysis to low fluorescence signals from living cells in the presence of cell micro-movements and molecular photo-bleaching and describe a simple algorithm for its routine application. The algorithm has been tested on Saccharomyces Cerevisiae (yeast) cells labeled with Dimethyl-pepep and Rhodamine 6G, and Chinese Hamster Ovary (CHO) cells stably expressing the regulatory subunit (RII) of protein kinase A fused to the cyan-emitting variant of GFP (CFP). Our statistical analysis allows us to estimate the local concentrations and the brightness of the fluorophores in different cellular compartments (nucleus, membrane, and cytoplasm) despite the occurrence of microscopic cell movements and significant photo-bleaching.

Original publication

DOI

10.1366/0003702053084981

Type

Journal article

Journal

Appl Spectrosc

Publication Date

02/2005

Volume

59

Pages

227 - 236

Keywords

Algorithms, Animals, CHO Cells, Cricetinae, Cricetulus, Cyclic AMP-Dependent Protein Kinases, Fluorescence Recovery After Photobleaching, Fluorescent Dyes, Image Interpretation, Computer-Assisted, Microscopy, Fluorescence, Multiphoton, Photobleaching, Photons, Saccharomyces cerevisiae Proteins