Hybrid Illumination Optical Sectioning Fluorescence Microscopy Based On Optical Fiber

Fluorescence microscope is an important tool in life science research. In common wide-field fluorescence microscopy, the out-of-focus signal can be also detected by the detector, which severely degrades the image quality. Those microscopes that can reject outof-focus background such as confocal microscopy, two-photon microscopy, are usually of slow imaging speed, expensive and complex. The optical sectioning method based on speckle and uniform illumination has low cost and its axial resolution is close to confocal microscopy and promising on the miniaturized device application. Traditional optical sectioning with hybrid speckle and uniform illumination uses diffuser in free space scattering light to generate speckle, which occupies too much room and is bad to integration with commercial microscope.With guiding light with optical fiber and vibrating multimode fiber to eliminate speckle, this thesis improved the traditional optical sectioning with hybrid speckle and uniform illumination, and demonstrates a method to obtain optical sectioning with hybrid illumination fluorescence microscopy based on optical fiber. It has good optical sectioning apprching confocal microscopy, and advantages such as less exposed optical access, flexible alignment, simple building and low cost(about 20000 yuan). It's very generalizable to a variety of commercial microscope. The main work is as below.Firstly, based on commercial microscope, optical sectioning system with hybrid speckle and uniform-illumination fluorescence microscopy based on optical fiber was designed. Then theory of how to obtain optical sectioning with hybrid speckle and uniform illumination was deduced and the algorithm was provided. Moreover, the theory of speckle reduction by optical fiber vibration was analyzed.Secondly, the factors on speckle reduction by optical fiber vibration was quantitatively studied with experiment. These studies shows that better speckle reduction can be got with higher driven voltage, and the higher driven voltage is, the lower the increasing efficiency is. Large model number of multimode fiber can help speckle reduction. And twisting the fiber is slightly bad for speckle reduction. Moreover, we designed the speckle reduction module and its engineering package, which produces a speckle reduction module. It's plug and play, easy to adjust, low cost, and suitable for various optical system.At last, we built the imaging system, and programmed the Matlab code. Pollen grains and 100 ?m-thick mice brain slice(neurons and nerve fibers) were imaged, which showed remarkable optical sectioning.