Study Of A Three-Dimensional Super Resolution Microscope Based On Bessel Light Sheet Illumination And Its Application

Super resolution microscopy has now been an important tool in investigating the subcellular structures and activities.However,most super resolution microscopes are still limited to single-cell imaging.Three-dimensional super resolution imaging in live thick tissues remains a challenge.Here,we present a 3D super resolution method combining Bessel light sheet illumination with super resolution algorithms to extend the application of super resolution microscope to three-dimensional imaging in live tissue.The main content of this work is as follows:We improved the Bayesian analysis of blinking and bleaching(3B)algorithm by first extending it to the third dimension and then combining super resolution radial fluctuation(SRRF)algorithm with 3B method,which we termed super resolution radial fluctuation guided three-dimensional Bayesian(SRTB)microscopy to accelerate the convergence of 3B's iteration for about 100 folds.We built the Bessel light sheet illumination microscope for thick tissue imaging.By generating a light sheet whose thickness is about 1?m and can cover a region of about 650?m to illuminate the sample,we can reduce the photobleaching and photodamage as well as provide raw images with high signal-to-noise ratio.The illumination of our method is discontinuous,while present super resolution methods are adapting continuous illumination.Thus,we first tested the fluorophore blinking under the discontinuous illumination of our system.We find there did exist blinking events under the scanning illumination if the power density isn't lower than 1kw/cm~2.We demonstrate our method can achieve 70nm lateral resolution and 170nm axial resolution and has good validity on attached cell.After the validation,we applied our method to investigate the subcellular structures inside live drosophila brain.