Optical Design And Data Processing In Stochastic Optical Reconstruction Microscopy (STORM)

Stochastic Optical Reconstruction Microscopy (STORM) is a key technique forsuper-resolution imaging. To achieve super-resolution by STORM, the dataprocessing method was discussed and the reconstruction algorithm was designed. Atthe same time, an optical system and the related controlling system were set up bywhich the super-resolution imaging was achieved.Firstly, we discussed the single molecule localization based super-resolutionmicroscopy algorithm used in STORM. Several kinds of frequently used algorithmswere compared and a single PSF(Point Spread Function) algorithm and multiple PSFsalgorithm fitting with Gaussian function were designed. We simulated the imagingprocess by software of MATLAB and then we achieved the single moleculelocalization and image reconstruction using the designed algorithm.Secondly, the optical system of STORM was set up based on the Olympus’sinverted fluorescence microscope, including laser selection subsystem and totalinternal reflection subsystem. The image acquisition and optical controlling systemwere designed. We first analyzed the controlling time sequence, based on which asingle-chip microcomputer circuit was designed including interface analysis, chipselection and the drawing of electrical schematic diagram and printed circuit boarddiagram.Thirdly, as the imaging density in single molecule based super resolutionmicroscopy is very important, we tried to optimize the imaging density. To obtain thebest imaging density quantitatively, we simulated the super-resolution imagingprocess and localized marker positions with single or multiple PSFs. The twoalgorithms are compared with regard to localization error, recall ratio and number of valid molecules then the optimized imaging densities corresponding to differentmolecules and algorithms are obtained.Finally, we imaged fluorescent microspheres, organic fluorescent molecules andfluorescent actin filaments by the STORM setup we constructed. Meanwhile, theimages obtained by STORM and conventional fluorescence microscope werecompared.