3D super-resolution optical fluctuation imaging with temporal focusing two-photon excitation.

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 exciting a thin slice in 3D volume. Temporal focusing is simple to implement whenever the excitation path of the microscope can be accessed. The implementation of SOFI is straightforward. By merging these two methods, we obtain super-resolved 3D images of neurons stained with quantum dots. Our approach offers reduced bleaching of out-of-focus fluorescent probes and an improved signal-to-background ratio that can be used when robust resolution improvement is required in thick, dense samples.