3D Super-resolution Optical Fluctuation Imaging with Temporal Focusing two-photon excitation

Abstract

3D super-resolution fluorescence microscopy typically requires sophisticated setups, sample preparation, or long measurements. A notable exception, SOFI, only requires recording a sequence of frames and no hardware modifications whatsoever but being a wide-field method, it faces problems in thick, dense samples. We combine SOFI with temporal focusing two-photon excitation -- the wide-field method that is capable of excitation of a thin slice in 3D volume. Both methods are easy to implement in a standard microscope, and by merging them, we obtain super-resolved 3D images of neurons stained with quantum dots. Our approach offers reduced bleaching and an improved signal-to-background ratio that can be used when robust resolution improvement is required in thick, dense samples.