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Proceedings Paper

Focal volume characterization using multiphoton fluorescence correlation spectroscopy (MP-FCS)
Author(s): Vijay Iyer; Molly J Rossow; M. Neal Waxham
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Paper Abstract

Fluorescence correlation spectroscopy(FCS) normally employs measured fluctuations in molecular fluorescence from within a defined observation volume created by a laser focus to extract information about dynamic processes associated with those molecules. In order to extract quantitative information about these processes, the laser focal volume must be well characterized. We show in this work, similar to others before, that a description of the focal volume in terms of vector diffraction theory allows quantitative analysis of FCS sufficient to resolve the diffusion coefficients of small molecules-Rhod 6G and Alexa 546, here-in the diffraction-limited volume of a high numerical aperture objective lens. We subsequently demonstrate the utility of a “reverse” FCS experimental paradigm in which the focal volume itself is characterized for a known molecular sample. Using a combination of wide-field and localized detection, we characterize the focal volume location and relative size as the beam collimation and correction collar are varied. We find that Gaussian beam theory can predict the focal volume location over a wide range of collimation conditions. It can also predict the relative focal volume size for converging and collimated beams, provided that spherical aberration is corrected for each collimation condition using the correction collar.

Paper Details

Date Published: 21 June 2004
PDF: 14 pages
Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); doi: 10.1117/12.537000
Show Author Affiliations
Vijay Iyer, Univ. of Texas Health Science Ctr./Houston (United States)
Rice Univ. (United States)
Molly J Rossow, Univ. of Texas Health Science Ctr./Houston (United States)
M. Neal Waxham, Univ. of Texas Health Science Ctr./Houston (United States)

Published in SPIE Proceedings Vol. 5323:
Multiphoton Microscopy in the Biomedical Sciences IV
Ammasi Periasamy; Peter T. C. So, Editor(s)

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