Optimizing Photophysical Conditions in Confocal Fluorescence Correlation Spectroscopy (FCS)

Abstract

FCS is a powerful method to measure diffusional and rotational properties of molecules as well as intramolecular transitions (e.g. triplet transition rates and isomerization) and intermolecular interactions (e.g. protein-protein, DNA-protein, etc.). Since the introduction of the confocal measuring principle by Rigler et al. and single photon counting avalanche photodiodes, FCS techniques attained a tremendous revival due to an increased sensitivity enabling even the study of single fluorescent molecules traversing the open, optically confined volume element. Despite these improvements it still remains desirable to further enhance the most important parameter in FCS measurements, i.e. the count rate per molecule (CRpM), in order to further improve the overall performance of FCS measurements (e.g. to enhance signal-to-noise and signal-to-background ratios, and, simultaneously, to minimize acquisition times).To this end we present here investigations showing an increase of the CRpM of > 200 kHz for Rhodamine Green (excit. laser power > 0.4 mW, 488 nm) along with an effective confocal volume in the subfemtoliter range (0.76 fl). However, photophysical processes like photobleaching, saturation, Rayleigh and Raman scattering as well as triplet transitions and/or isomerization have to be taken into account to perform optimized FCS measurements. Under these conditions we succeeded to yield signal-to-background ratios (i.e. CRpM/BGCR with BGCR background count rate due to scatter) of > 400 at relatively low laser power (0.1-0.3 mW). Further increasing the laser power results in increased triplet transition rates, bleaching and linearly increasing BGCR whereas CRpM levels out into a plateau. Thus, higher excitation energies (above 0.5mW) are in case of Rhodamine Green of counterproductivity. Finally, thanks to the improved single molecule detection sensitivity, we demonstrate that pico- and even femtomolar concentrations of fluorophores can be detected by confocal FCS under optimized photophysical conditions.