Structured illumination microscopy with direct wavefront sensing

Abstract

With sub-diffraction resolution in three dimensions and good optical sectioning capability, three-dimensional superresolution structured illumination microscopy (3D-SRSIM) can provide eight-fold more information than conventional widefield microscopy. However, the application of SR-SIM is limited to single cells due to optical aberrations in thick tissues. The destructive impacts of aberrations include the decrease in spatial resolution and signal-to-noise ratio (SNR), the distortion of sample morphology, and, even worse, the failure of SIM reconstruction. There are several adaptive optics (AO) methods to correct the optical aberration, including direct wavefront sensing using a Shack Hartmann wavefront sensor (SHWFS). The SHWFS possesses good wavefront measurement accuracy and high-speed response but works best with an isolated guide-star. Therefore, combining SHWFS with widefield microscopy poses difficulties and remains challenging. To effectively apply the direct wavefront sensing method, we built a reconfigurable microscopy system that can switch to a confocal setup for measuring the wavefront where the fluorescence light emitted from the confocal illumination spot is used as the “guide-star” for wavefront measurements. We experimentally demonstrate that the confocal illumination based direct wavefront sensing AO method can precisely correct the sample induced optical aberrations and help to improve the image quality and fidelity of 3D-SIM imaging in thick samples, exhibiting enormous potential for in vivo biomedical research.