Single molecule TIRF calibration determines number of molecules in cross section of individual fibrin fibers

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NIBIB	BIOPHY-9

Authors

• A Popescu-Hategan
• K Gersh
• D Safer
• J Weisel

Abstract

The bleaching behavior of fluorescently labeled fibrinogen molecules observed in total internal reflective fluorescence microscopy (TIRF) together with analysis of labeling probability were used to determine the number of active fluorophores attached nonspecifically to each fibrinogen molecule and characterize the uniformity of nonspecific labeling. The predominant labeling was shown to be one active dye molecule per fibrinogen, for bulk labeling ratios of up to 4 dyes/molecule. From the total intensity of the bleaching steps—as single molecule signature events—and the distribution of active labeling of fibrinogen molecules, was obtained a single molecule intensity calibration, which accounts for all, including the “not seen” molecules. Live observation of fibrin polymerization in TIRF was translated by this calibration in molecular information on the growth kinetics of fibers. The molecular analysis of a network formed by diffusive mixing showed that fibrin fibers thickened in time to thousands of molecules across, whereas some fibers reached early a stationary state.

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