Optimization Of Structured Illumination Light Field And Characterization Of System Point Spread Function In Structured Illumination Microscopy(SIM)

Fluorescent microscope with the particular function of real time dynamic imaging living cells with special marker is widely used in cell biological study. However, due to the existence of the diffraction limit, resolution of traditional fluorescent microscope is limited. Hence, it cannot be used in sub-cellular level research. In order to break the diffraction limit and improve the resolution, a series of super-resolution fluorescent microscope have been proposed. Even so, long time research of living cells is still difficult.Structured Illumination Microscopy(SIM), based on Moiré effects, works by the follow steps. Firstly, it excites fluorescence with geometric light beam. Secondly, it modulates the invisible high-frequency signal of experimental sample to low-frequency band of fluorescence. Thirdly, it changes the orientations and phases of the geometrics and records multiple sets of data. Finally, it extracts information carrying the high-frequency signals from the data recorded. and rebuilds the super-resolution image. Theoretically, the lateral resolution of SIM can achieve twice of traditional fluorescent microscope. What?s more, SIM has traits of fast imaging and no special requirements for fluorescent marker. It has widely application prospects in sub-cellular level research such as stoichiology and cyto-dynamics with these traits.In this paper, a structured illumination super-resolution fluorescent microscope with LED illuminant and programmable digital micro-mirror device(DMD) has been designed and set up, via combining the designed microscope with an Olympus X73 microscope. The design of the super-resolution rebuilt algorithm is very important. We have carried on the theoretical analysis about using LED, DMD and objective to generate cosine structure illumination, and put forward the new algorithm of measuring system resolution using fluorescent microspheres. The results of imaging of cell action filament, which excitation wavelength is 650 nm and emission wavelength is 668 nm, with magnification 40 and numerical aperture 0.75, shows the system resolution is 307 nm. It is 1.78 times of the traditional fluorescent microscope resolution which was measured 545 nm. The measurement of fluorescent beads, with 200 nm in diameter, with magnification 100 and numerical aperture 1.4, shows resolutions are 328 nm and 203 nm respectively. The system resolutions respectively are 312 nm and 170 nm. It is 1.84 times of traditional fluorescent microscope resolution.Experiments show that the structured illumination fluorescence microscopy system that based on LED and DMD has been break through the diffraction limit, and has realized the super resolution imaging.