Open AccessFluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra
Author(s) -
Aleš Benda,
P. Kapusta,
Martin Hof,
Katharina Gaus
Publication year - 2014
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.22.002973
Subject(s) - fluorescence correlation spectroscopy , laurdan , fluorescence cross correlation spectroscopy , spectroscopy , optics , hyperspectral imaging , fluorescence spectroscopy , fluorescence , emission spectrum , cross correlation , spectral resolution , spectral line , spectral imaging , physics , materials science , chemistry , lipid bilayer , membrane , mathematical analysis , biochemistry , remote sensing , mathematics , quantum mechanics , astronomy , geology
We present a fluorescence correlation spectroscopy (FCS) approach to obtain spectral cross-talk free auto- and cross-correlation functions for probes with highly overlapping emission spectra. Confocal microscopes with either a hyperspectral EM-CCD or six-channel PMT array spectral detection were used, followed by a photon filtering correlation approach that results in spectral unmixing. The method is highly sensitive and can distinguish between Atto488 and Oregon Green 488 signals so that auto-correlation curves can be fitted without the need for cross-talk correction. We also applied the approach to the membrane dye Laurdan whose emission is dependent on the lipid order within the bilayer. With fluorescence spectral correlation spectroscopy (FSCS), we could obtain spectral cross-talk free auto- and cross-correlation functions corresponding to Laurdan located in liquid ordered and liquid disordered phases.