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Proceedings Paper

Using POPOP's viscosity-dependent lifetime as a picosecond resolution standard in near-UV fluorescence lifetime imaging microscopy
Author(s): Mary-Ann Mycek; Paul Urayama; Katherine Heyman; Megan Bussey
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Paper Abstract

Fluorescence lifetime imaging microscopy (FLIM) using near-UV excitation is being developed to probe endogenous fluorophores in cells and tissues. Here, we describe such a UV-FLIM system and present a useful method for measuring the fluorescence lifetime discrimination of the system using the viscosity dependent lifetime of 1,4-Bis(5-phenyloxazol-2-yl)benzene (POPOP). The time-domain UV-FLIM system employed a nitrogen laser (337.1 nm) fiber-optic coupled to an inverted microscope. Wide-field fluorescence images were obtained at controlled time delays with a 200 ps gated, intensified-CCD camera. Lifetimes were calculated from the intensity decay on a pixel-by-pixel basis. The system was capable of imaging endogenous fluorescence in living cells using UV excitation. POPOP is a nanosecond lifetime standard suitable for UV excitation (325-375 nm). Its single-exponential lifetime (1.4 ns in ethanol) is comparable to endogenous lifetime values measured in living cells. Increasing solvent viscosity via the incremental addition of glycerol produced a series of POPOP lifetime standards having a range of 1 ns. The FLIM system’s ability to discriminate lifetime differences of 50 ps was demonstrated using the POPOP series. Thus, POPOP’s viscosity dependent lifetime represents a useful and convenient resolution standard for UV-FLIM calibration.

Paper Details

Date Published: 19 June 2003
PDF: 8 pages
Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003);
Show Author Affiliations
Mary-Ann Mycek, Univ. of Michigan (United States)
Paul Urayama, Univ. of Michigan (United States)
Katherine Heyman, Dartmouth College (United States)
Megan Bussey, Dartmouth College (United States)

Published in SPIE Proceedings Vol. 4962:
Manipulation and Analysis of Biomolecules, Cells, and Tissues
Dan V. Nicolau; Joerg Enderlein; Robert C. Leif; Daniel L. Farkas, Editor(s)

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