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 mu m 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 mu m scanning step, 0.7 mW laser power at sample, 500 mu m 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.