Fluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra.

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.