Dual-color time-integrated fluorescence cumulant analysis.

Abstract:

:We introduce dual-color time-integrated fluorescence cumulant analysis (TIFCA) to analyze fluorescence fluctuation spectroscopy data. Dual-color TIFCA utilizes the bivariate cumulants of the integrated fluorescent intensity from two detection channels to extract the brightness in each channel, the occupation number, and the diffusion time of fluorophores simultaneously. Detecting the fluorescence in two detector channels introduces the possibility of differentiating fluorophores based on their fluorescence spectrum. We derive an analytical expression for the bivariate factorial cumulants of photon counts for arbitrary sampling times. The statistical accuracy of each cumulant is described by its variance, which we calculate by the moments-of-moments technique. A method that takes nonideal detector effects such as dead-time and afterpulsing into account is developed and experimentally verified. We perform dual-color TIFCA analysis on simple dye solutions and a mixture of dyes to characterize the performance and accuracy of our theory. We demonstrate the robustness of dual-color TIFCA by measuring fluorescent proteins over a wide concentration range inside cells. Finally we demonstrate the sensitivity of dual-color TIFCA by resolving EGFP/EYFP binary mixtures in living cells with a single measurement.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Wu B,Chen Y,Müller JD

doi

10.1529/biophysj.106.086181

subject

Has Abstract

pub_date

2006-10-01 00:00:00

pages

2687-98

issue

7

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(06)71981-6

journal_volume

91

pub_type

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