Research On Imaging Technologies Of Point Scanning Optical Microscope System

In recent years,the development of super-resolution microscopy has greatly promoted the development of fields such as cell biology and nanomaterials.Super-resolution microscopy is mainly divided into two categories.The first category is structured light illumination microscopy,which spatially modulates the fluorescence behavior of molecules in the diffraction limit range,so that the luminous point of the fluorescence is smaller than the diffraction limit.Typical example of such techniques is stimulated emission of depletion microscopy(STED).The second category is single molecule localization super-resolution microscopy(SMLM).Its typical representatives are stochastic light reconstruction microscopy(STORM),and photosensitive localization microscopy(PALM).The point scanning optical microscopy system is an important basic platform for constructing the first category of super-resolution microscopy techniques,such as stimulated emission of depletion microscopy.The point scanning optical microscopy system plays an important role in promoting the development of super-resolution optical microscopy.This paper aims to construct a point scanning optical microscopy system based on point illumination and point detection,and to study its key imaging techniques.Firstly,the principle and application of point scanning optical microscopy are outlined,and the relationship between diffraction limit and resolution is discussed.Taking the advantages of the advantages of time correlated single photon counting technology,a point scanning optical microscope was developed,where transverse scan and axial scan can be achieved.This system mainly includes the models of hardware,software,scanning control,and optical systems.Key issues such as data acquisition and processing,system synchronization control,illumination light generation,detection of fluorescent photons,system adjustment and optimization were solved.Finally,a test model was established and verified the performance of the imaging system.The innovation and contributions of this article are:1.A stead and simple setup of the point scanning microscope system was developed,which created conditions for the construction of stimulated emission of depletion microscopy.Using the constructed point scanning microscopy system,a 40 nm fluorescent bead was scanned and imaged to obtain a lateral resolution of 394 nm.2.A imaging software with complete functions and simple operation was designed and developed,by which data acquisition,image reconstruction and system synchronization control were realized.The software can be applied to many kinds of point scanning optical systems,such as confocal microscopy,stimulated emission of depletion microscopy,two-photon microscopy.3.A stable fluorescence detection system was designed and implemented,which can efficiently collect fluorescence photon signals,effectively suppress the interference of environmental noise,and improve the signal-to-noise ratio of the microscopy system.4.With the help of the wide field observation using CCD,we can quickly find the interesting area of the imaging focal plane,which save time and improve work efficiency.5.The preliminary exploration on the polarization modulation of fluorescent molecules is done.The fiber samples stained with ATTO590 under different polarization angels were imaged,which showed that the polarization dependence of the fluorescence.