FluorescenceCorrelation Spectroscopy at MicromolarConcentrations without Optical Nanoconfinement.

Fluorescence correlation spectroscopy(FCS) is an important techniquefor studying biochemical interactions dynamically that may be used in vitroand in cell-based studies. It is generally claimedthat FCS may only be used at nM concentrations. We show that thisgeneral consensus is incorrect and that the limitation to nM concentrationsis not fundamental but due to detector limits as well as laser fluctuations.With a high count rate detector system and applying laser fluctuationcorrections, we demonstrate FCS measurements up to 38 μM withthe same signal-to-noise as at lower concentrations. Optical nanoconfinementapproaches previously used to increase the concentration range ofFCS are not necessary, and further increases above 38 μM maybe expected using detectors and detector arrays with higher saturationrates and better laser fluctuation corrections. This approach greatlywidens the possibilities of dynamic measurements of biochemical interactionsusing FCS at physiological concentrations. [ABSTRACT FROM AUTHOR]