Focal volume characterization using multiphoton fluorescence correlation spectroscopy (MP-FCS)

Vijay Iyer; Molly J Rossow; M. Neal Waxham

doi:10.1117/12.537000

21 June 2004 Focal volume characterization using multiphoton fluorescence correlation spectroscopy (MP-FCS)

Vijay Iyer, Molly J Rossow, M. Neal Waxham

Proceedings Volume 5323, Multiphoton Microscopy in the Biomedical Sciences IV; (2004) https://doi.org/10.1117/12.537000
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

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.

Citation Download Citation

Vijay Iyer, Molly J Rossow, and M. Neal Waxham "Focal volume characterization using multiphoton fluorescence correlation spectroscopy (MP-FCS)", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); https://doi.org/10.1117/12.537000

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