Research On Multi-color Confocal Laser Scanning Fluorescence Microendoscopic System

Confocal laser scanning microscope endoscope combines confocal laser scanning microscope(CLSM)with traditional fiber optic endoscope to achieve microscopic imaging of living tissues and cells,which not only takes advantage of the flexible insertion of optical fibers into living tissue,but also retains the high resolution and high contrast of the confocal microscope.Confocal laser scanning microscope endoscope is currently the only endoscopic instrument capable of imaging cells labeled with fluorescence.Confocal laser scanning microscope endoscope can be used for in vivo microscopic imaging at the cellular level,suitable for detection of cell structure or morphology under normal and pathological conditions,detection of protein expression levels and detection of marker molecules,etc.This paper describes the development and verification of multi-color confocal fluorescence microscopy system based on fiber bundles.This article provides a general description of the optical structure,important components,designed and assembled probes,system software and data processing of the multi-color confocal laser scanning fluorescence microendoscopic system.Multi-color confocal laser scanning fluorescence microendoscopic system uses two semiconductor lasers with wavelengths of 488 nm and 650 nm respectively as light sources.The x and y directions of the image in the system are scanned by a two-dimensional scanning galvanometer.The Z axis scanning is determined by the Z axis movement of the three axis displacement table of the sample table.The three-dimensional confocal scanning can be completed by combining the galvanometer scanning and the Z axis scanning.The resolution of the system is calibrated by using fluorescent beads with a size of500 nm,and the full width at half maximum of the fluorescent beads is 510 nm,the imaging field of view of the system is 151 ?m,and the working distance is about 80 ?m.The fluorescence dyes FITC and Di A are excited by 488 nm laser.The 650 nm laser excite fluorescent dye R3.The imaging ability of the system was tested using mirror-stained paper stained with fluorescent dyes,and the system was used to image three fluorescent dye-labeled cells and mouse tissue sections in vitro,indicating that the system can achieve three channels for three-stained cells and tissues.It also verifies the system's ability to analyze and discern the structure information of tissue samples.Finally,a fluorescent dye is injected into the tail vein of living mice,and the system performs in vivo imaging of multiple organs and tissues(capillaries,liver,small intestine,muscles)of mice.The imaging effect verifies that the endoscopic system can be used to monitor the dynamic process of the physiological activities of various fluorescent labeling substances in small animal living cells in real time.