Optical fluctuation microscopy based on calculating local entropy values

We demonstrate a novel and easy-to-use method to dramatically reduce noise and background contributions in advanced fluorescence microscopy experiments. The underlying idea is that the entropy value increases for systems with a large number of accessible energy states. Intensity fluctuations originating from photophysical or photochemical effects lead to an increased information content. Calculating the pixel-wise entropy value results in an enhancement of the signal-to-noise ratio by a factor of 90–100. Comparing ECI (entropy-based contrast-enhanced imaging) to superresolution methods such as STORM and SOFI, we find that this technique also bears substantial potential for enhancing fluctuation-based superresolution microscopies.