Pixel reassignment in image scanning microscopy with a doughnut beam: example of maximum likelihood restoration

Abstract

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images–of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.