Active Structured Illumination Super-resolution Microscopy

Super-resolution microscopy is a powerful tool for revealing the structure,function,and mechanism of cells in real-time at the subcellular level,and obtaining a series of original scientific discoveries.Among these super-resolution microscopies,SIM has the advantage of rapid imaging speed,low photodamage,as well as compatibility with most fluorescent labeling protocols.The resolution of about 100 nm can meets the imaging requirements most organelles in live cells,so it is known as the most suitable super-resolution imaging technology for dynamic long-term live cells.This dissertation starts from the requirements of optical instruments for cell biology,mainly focusing on the problems of low dynamic range,non-uniform excitation,and invalid illumination in traditional SIM,developed three active structured illumination methods:high dynamic range SIM,flat-field SIM,and adaptive illumination region SIM.A modular,low-cost,and multi-imaging mode active structured illumination super-resolution imaging system compatible with fluorescent illuminator was built,named Active-SIM.The main contents of this dissertation are as follows:1.Existing SIM cannot reconstruct high-fidelity images for a high range of intracellular fluorescence signal intensities.To solve this problem,this dissertation proposed a high dynamic range SIM(HDR-SIM)method based on multi-exposure fusion,which collects a set of low dynamic range raw data under different illumination intensity and then fuses a high dynamic super-resolution image.The dynamic range of the traditional SIM imaging system is increased by 40 dB without loss of spatial resolution.With HDR-SIM,individual and clustered microspheres with 420 times intensity different can be visualized in the same scene at 100 nm resolution.Using HDR-SIM to observe the structure of cell vesicles at different scales,both single small and large aggregated vesicles can be clearly resolved.2.The illumination of existing SIM is non-uniform Gaussian beam,which limits its effective imaging field size and hinders potential application in histopathology.Aiming at the requirement of homogeneous and large field of view super-resolution imaging,this dissertation developed a flat-field SIM approach(Flat-SIM)based on spatio-temporal intensity joint modulation.By designing a set of holographic bit planes with different time weights,the incident Gaussian light can be actively controlled without destroying the plane wave front.The effective field of view of SIM is expanded by 2 times without reducing the temporal and spatial resolution.Furthermore,through the method of multi-field stitching,super-resolution of 1.32 mm×0.95 mm large field of view was carried out.Using large field Flat-SIM technology,the mitochondrial morphology in hundreds of osteosarcoma cells was quantitatively analyzed,and found that the mitochondrial morphology changed significantly after being treated with tumor inhibitors.The observation and statistical analysis of fluorescence in situ hybridization pathological sections found that it has higher positioning and counting accuracy than fluorescence microscopes,showing its potential advantages in pathological diagnosis.3.Existing SIM is a globally indiscriminate wide-field illumination method,which inevitably causes unnecessary light doses in non-interesting imaging regions.To address the above issues,this dissertation established an adaptive illumination regions SIM imaging technique(Adaptive-SIM).According to the spatial distribution characteristics of biological samples,the location and dose of illumination can be customized to reduce the impact of photobleaching and phototoxicity on live cells.Using Adaptive-SIM,this dissertation carried out twocolor imaging of mitochondria and nuclei.The 405 nm and 488 nm lasers were used to excite the nucleus and cytoplasmic area respectively,which effectively avoided the photobleaching and phototoxicity caused by the 405 nm laser to the mitochondria around the nucleus.4.Aiming at the problems of complex structure,high cost,and difficult use of the existing commercial SIMs,this dissertation designed and built a modularized,miniaturized,multi-imaging mode,and fluorescent illuminator-compatible ActiveSIM imaging system.The Active-SIM instrument has been fully optimized from the perspective of optical simulation design,optical-mechanical structure design,electronic control system,and software.A complete optical-mechanical adjustment scheme has been formulated,and performance test results are given.