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Background Suppression in Frequency-Domain Fluorometry

✍ Scribed by Joseph R. Lakowicz; Ignacy Gryczynski; Zygmunt Gryczynski; Michael L. Johnson

Publisher: Elsevier Science
Year: 2000
Tongue: English
Weight: 155 KB
Volume: 277
Category: Article
ISSN: 0003-2697
DOI: 10.1006/abio.1999.4346

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✦ Synopsis

Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry. The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and instrumental conditions, the data can be analyzed as usual for frequency-domain data without further processing. We show by simulations that this method can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence.

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