红细胞显微激光共焦拉曼散射光谱扫描技术研究

采用显微激光共焦拉曼散射光谱扫描系统对活态红细胞进行拉曼光谱(点测定、线扫描、二维扫描)测定及成像的技术与方法进行了研究，并对514 nm激光对红细胞在拉曼扫描中的影响进行了评价。通过扫描前后细胞的拉曼光谱变化和亮场图像变化确定了在不同扫描模式下既可获得较好的拉曼散射信号，又不会影响细胞生命活动与功能的合适扫描参数的设置。对于点扫描模式，样品激光功率是重点调节的参数，一般应小于1.5 mW。对于线扫描模式，照射激光功率和扫描间隔(步进)是要重点关注的参数。小扫描间隔意味着激光能量相对聚集，易对细胞造成损伤；大扫描间隔可以较好地降低激光对细胞的损伤，但是空间分辨率会因此而下降。对于线扫描，建议扫描间隔大于0.5 μm、照射激光功率小于0.7 mW。对于二维扫描，除照射激光功率、扫描间隔需要调节外，其他扫描参数也要作相应调节以降低激光对红细胞的影响，可适当降低样品温度和增加共焦孔径尺寸降低二维扫描过程中激光对红细胞的影响。1.0 μm扫描间隔、0.7 mW的照射激光功率和500 μm共焦孔径，以及样品温度适当调低可得到较好的二维红细胞拉曼图像。对于所有扫描模式，如果得到的红细胞的拉曼信号足够强，也可适当降低曝光积分时间以降低激光对红细胞的影响。实验前进行实验过程的优化对活态细胞的拉曼测试也非常重要。

Technique of Confocal Raman Microscopy on Erythrocytes

The technique of confocal Raman scanning microscopy (point scanning, line scanning and 2D scanning) and bright field imaging of living erythrocytes was investigated as a function of different scanning conditions at the excitation wavelength of 514 nm. The biological effect of the 514 nm laser radiation on the erythrocytes was also evaluated, so that a set of proper scanning parameters for different scan modes can be determined to obtain strong enough Raman signal while without damage on the living cells by evaluating the change of Raman spectra and lighted field images of the cells. For the point scanning mode, the laser power at sample is the most important parameter to be adjusted, which normally should be less than 1.5 mW. For the line scanning mode, the laser power at sample and scanning step should be considered at first. Small scanning step means the energy of laser accumulated at a small region, which can easily damage to erythrocytes. Large scanning step can reduce the damage; however the spatial resolution decreases also. It is recommended that scanning step should be more than 0.5 μm and laser power at sample should be less than 0.7 mW. For the 2D scanning mode, besides the laser power at sample, scan step needs to be adjusted, and other scan parameters need to be adjusted properly for reducing the effect of laser on erythrocytes. Large pinhole and relative low temperature of sample are the remedies, which can reduce the effect of laser on erythrocytes. 1.0 μm scanning step, 0.7 mW laser power at sample, 500 μm pinhole and proper low temperature can get better 2D Raman image of erythrocytes. For all scanning modes, if the Raman signal is strong enough, the exposure time can be shortened properly, thus reducing the effect of laser on erythrocytes. The optimization of experiment process is also important for Raman test on living cells.