Fast Structured Illumination Super-resolution Microscopy Based On Laser Interference And Its Applications

Optical microscopy performs an increasingly important role in clinical diagnosis and basic scientific research.With the development of novel fluorescence probes and detectors,super-resolution optical microscopy breaks through the diffraction limit and becomes a powerful tool for modern biomedicine.For structured illumination microscope(SIM),this is achieved by using spatially coded structured illumination which down modulates spatial frequencies beyond the cutoff into the passband of the microscope.SIM shows lower phototoxic,higher imaging speed,and no special requirements for fluorescent probes,which has significant potential in application to biomedical research.This thesis focuses on the theory and experimental study of laser-interference fast super-resolution SIM,and its applications in biology.The research details are as follows:1.I proposed a zero-order vortex half-wave retarder(VHR)based polarization control method and develop a compact SIM system.I also designed a synchronous control timing sequence,and developed multi-thread automatic control software.The commercial SIM system has drawbacks like slow imaging speed,and complex equipment.Thus we proposed the VHR based polarization control method,which is very efficient in terms of simple system configuration,ease of use,and high light energy utilization efficiency.I propose a synchronous method based on the s CMOS camera,which simplify the system without the external chip.I also developed the multi-thread control software based on C++ language and QT environments,realizing automatic control of the system.The physical dimensions of the compact SIM system are 600 mm × 600 mm × 200 mm.The system has four different illumination light wavelengths.The maximum super-resolution imaging frame rate is 162 fps@512pixel × 100 pixel.2.A super-resolution reconstruction algorithm based on image recombination transformation is proposed,which is suitable for arbitrary phase shifts.The image recombination transformation(IRT)algorithm can obtain the precise initial phase.It is conducive for SIM to obtain high quality reconstruction images under weak fluorescent signals.However,the existing IRT algorithm only applies to the DMD-based SIM system,which has two stripe directions and fixed ?/2 or 2?/3 phase shifts.This cannot sufficient for the laser-interference SIM with three grating directions and a variety of phase shifts.In this thesis,I proposed a super-resolution reconstruction algorithm based on image recombination transform.The IRT algorithm can obtain the precise initial phase and is suitable for arbitrary phase shifts.This extends the application of the IRT algorithm,especially for live cell imaging under weak light illumination.3.The laser-interference fast super-resolution SIM system is applied to the live cell imaging under a low light dose and observes a variety of cell biological processes for the first time.The current super-resolution methods increase the resolution at the expense of increasing excitation laser power,leading to a short duration time for live-cell imaging and a relatively low signal-to-noise ratio.In this thesis,I image the motion of live cells for a long time under a low laser power and successfully record several dynamic biological processes,including the changes of SSB localization in E.coli during the response to DNA damage,the dynamic process of the mitochondrial DNA in the mitochondria,and the speed of the vesicles along different microtubes.These results provide direct observation evidence for the corresponding biological process,which proves the advantages of laser-interference fast super-resolution SIM systems in cellular biology.