激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开，在光谱谱面处设置两个可移动缝片形成出射狭缝，两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽，其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较，结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验，获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像，实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像，扩大了共聚焦显微镜的适用范围。

Spectral imaging system on laser scanning confocal microscopy

A spectral imaging module was added into a laser scanning confocal microscopic system to discriminate different fluorescence components in biological tissures. A prism was used as a beam split part, and two movable slit edges were used to form a slit whose width and position could both be adjusted on the spectral image plane. The two slit edges were installed on a stepping motor. By adjusting two slit edges, the laser scanning confocal spectral microscopy could work at wavelengths of 400-700 nm and its minimum spectral wavelength for confocal imaging was less than 5 nm. The actual slit positions corresponding to spectral lines of 488 nm laser and low pressure mercury lamp were tested and compared with the theory positions. The results show that the differences of the actual slit positions and theory values are all less than 0.1 mm. A laser scanning confocal imaging experiment with a full spectrum confocal imaging and 2.5 nm spectrum bandwidth (50 μm slit width) confocal imaging was carried out using a mouse kidney tissue slide. The images of nuclei labeled by DAPI and glomeruli labeled by Alexa Fluor^® 488 were obtained and the different components in biological tissues were distinguished. It concludes that the system can complete the confocal spectrum imaging, by which the application area of laser scanning confocal microscopy has been extended.