Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems.