Research On Structured Illumination Fluorescence Microscopy Imaging System

Optical microscope, as an important tool, greatly promotes the development oflife science research with its improvement. Especially the fluorescence microscopy iswidely used because of its advantages: undamaging, non-invasion, molecule-specificlabeling and allowing the observation of structures inside a live sample in real time.While as a far-field optical microscope, conventional fluorescence microscope suffersthe diffraction limit and owns a limited spatial resolution, in the lateral direction is200-300nm and500-700nm in the axial direction. The limited spatial resolutionlimits the application of conventional fluorescence microscope in observingsub-cellular structures. A series of novel super-resolution imaging techniques forfluorescence microscopy have been invented to break the diffraction limit.This thesis makes a study on two-dimensional structured illuminationfluorescence microscope on the base of the investigation of several currently usedsuper-resolution techniques for fluorescence microscopy and summarizes thetwo-dimensional structured illumination microscope with its principle, imagereconstruction algorithm and apparatus. The next three sections will list the things wehave done.(1)We design and set up the two-dimensional structured illuminationfluorescence microscopy imaging system on Olympus IX83inverted fluorescencemicroscope using digital micro-mirror device (DMD) for spatial light modulation andLED for light source. From the fitting result, we find that the biggest deviation of phase interval’s fitting result with its preset value is just only6.8%and0.2%forspatial frequency of fringes. With simulation, these deviations have no obvious effecton reconstructed images.(2)With the deep analysis of the algorithm for the imagereconstruction of two-dimensional structured illumination fluorescence microscope,we optimize the separation process of high frequency information and accomplish theimage reconstruction in real domain using recorded sample image illuminated bythree illumination fringes with different initial phase. Then the frequency domainbased three-phase image reconstruction algorithm, the real domain based three-phaseimage reconstruction algorithm and the real domain based four-phase imagereconstruction algorithm are translated to MATLAB programs. Then simulating thethree programs with noise interference and non-noise interference, from the result wefind that whether there is noise containing in recorded image, the reconstructionimages have a high resolution compared with the images using normal illuminationmode. Furthermore, the resolution improved ability agrees well with the calculatedresult based on the period of illumination fringes in theory. Comparing thereconstruction speed in the same conditions, we find that the real domain basedthree-phase image reconstruction algorithm is faster than the other two imagereconstruction algorithm.(3)We use100nm fluorescence microsphere and actinfilaments to test our system. Result shows that the resolution of reconstructionmicrosphere image is442nm with illumination fringes period is nearly1100nmwhile the conventional image just have584nm resolution. As for the constructionimage of actin filaments the resolution is obvious improved compared withconventional image.