Technique of Confocal Raman Microscopy on Erythrocytes
KANG Li-li1,2,HUANG Yao-xiong1*, LUO Man1
1. Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China 2. Department of Biomedical Engineering, Southern Medical University, Guangzhou 510515, China
Abstract：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.
康立丽1,2,黄耀熊1*,罗曼1. 红细胞显微激光共焦拉曼散射光谱扫描技术研究[J]. 光谱学与光谱分析, 2008, 28(10): 2343-2347.
KANG Li-li1,2,HUANG Yao-xiong1*, LUO Man1. Technique of Confocal Raman Microscopy on Erythrocytes. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2008, 28(10): 2343-2347.
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