Generic placeholder image

Current Pharmaceutical Biotechnology

Editor-in-Chief

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

Confocal Fluctuation Spectroscopy and Imaging

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

Volume 11, Issue 6, 2010

Page: [639 - 653] Pages: 15

DOI: 10.2174/138920110792246618

Abstract

Currently, work with subnanomolar concentrations is routine while femtomolar and even single-molecule studies are possible with some efforts getting high on single-molecule biophysics and biochemistry. Methodological breakthroughs, such as reducing the background light contribution in single-molecule studies, which has plagued many studies of molecular fluorescence in dilute solution, and particularly in live cells, have recently described by us. We first demonstrated how optimized time-gating of the fluorescence signal, together with time-correlated single-photon counting, can be used to substantially boost the experimental signal-to-noise ratio about 140-fold, making it possible to measure analyte concentrations that are as low as 15 pM. By detection of femtomolar bulk concentrations, confocal microsopy has the potential to address the observation of one and the same molecule in dilute solution without immobilization or hydrodynamic/ electrokinetic focusing at longer observation times than currently available. We present relevant physics. The equations are derived using Einsteins approach showing how it fits with Ficks law and the autocorrelation function. An improved technology is being developed at ISS for femtomolar microscopy. The general concepts and provided experimental examples should help to compare our approach to those used in conventional confocal microscopy.

Keywords: Fluctuation microscopy, fluctuation spectroscopy, fluctuation imaging, lifetime imaging, FLIM, fast FLIM, single-molecule detection in dilute solution, live cell, physical concepts, Brownian motion, optimized ISS fluorescence fluctuation system ALBA-JZ™, how-to-do approach


© 2024 Bentham Science Publishers | Privacy Policy