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Current Pharmaceutical Biotechnology

Editor-in-Chief

ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

Author(s): Zeno Foldes-Papp, Shih-Chu Jeff Liao, Tiefeng You and Beniamino Barbieri

Volume 10 , Issue 5 , 2009

Page: [532 - 542] Pages: 11

DOI: 10.2174/138920109788922137

Price: $65

Abstract

We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

Keywords: Single-molecule fluorescence fluctuation spectroscopy and imaging, solution, fluorescence correlation, FCS, scattered background, autofluorescence, pulsed excitation, modulated excitation, detector gating, time-correlated single-photon counting


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