光谱学与光谱分析 |
|
|
|
|
|
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.
|
Received: 2007-05-16
Accepted: 2007-08-26
|
|
Corresponding Authors:
HUANG Yao-xiong
E-mail: tyxhuang@jnu.edu.cn
|
|
[1] Graselli J G, Bulkin B. Analytical Raman Spectroscopy. New York: Wiley and Sons, 1991. [2] Puppels G J, De Mul F F M, Otto C, et al. Nature, 1990, 347: 301. [3] Puppels G J, Garritsen H S P, Seger-Nolten G M J, et al. J. Biophys., 1991, 60(5): 1046. [4] Puppels G J, Garritsen H S P, Kummer J A, et al. Cytometry, 1993, 14(3): 251. [5] Puppels G J, Bakker Schut T C, Sijtsema N M, et al. J. Mol. Struct. 1995, 347: 477. [6] Kerstin Ramser, Katarina Logg, Mattias Goksor, et al. J. Biomed. Opt. 2004, 9(3): 593. [7] XU Yi-ming(许以明). Raman Spectrum and the Application in Structure Biology(拉曼光谱及其在结构生物学中的应用). Beijing: Chemical Industry Press(北京: 化学工业出版社),2005. [8] ZHAO Yuan-li, L Jing, GE Xiang-hong, et al(赵元黎,吕 晶,葛向红,等). Spectroscopy and Spectral Analysis(光谱学与光谱分析),2006,26(7):1267. [9] Wood B R, McNaughton D J. Raman Spectrosc.,2002, 33: 517. [10] Wood B R, Tait B, McNaughton D. Biochimica et Biophysica Acta-Molecular Cell Research-Moleculer Cell Research(BBA),2001, 1539: 58. [11] Wood B R, McNaughton D. Biospectroscopy,2002, 67: 259. [12] Wood B R, McNaughton D J. Raman Spec.,2002, 33: 517. [13] Wood B R, Hammer L, Davis L, et al. Biomed. Opt.,2005, 10(1): 014005. [14] Ramser K, Bjerneld E J, Fant C, et al. Proc. SPIE. 2002, 4614: 20. [15] Ramser K, Bjerneld E J, Fant C, et al. J. Biomed. Opt.,2003, 8(2): 173. [16] Puppels G J, Olminkhof J H, Segers-Nolten G M, et al. Experiment Cell Research, 1991, 195(2): 361. [17] Cary P R. Biochemical Application of Raman and Resonance Raman Spectroscopies, New York: Academic Press, 1982. [18] Songzhou H, Kevin M S, Thomas G S. J. Am. Chem. Soc., 1996, 118: 12638. |
[1] |
WANG Jin-shuang1, FU Ying-ying1, FU Min-rui1, GAO Bin1*, ZHENG Da-wei1, CHANG Yu2. Study on Erythrocyte Sublethal Damage Under Different Shear Stress Based on Raman Spectroscopy[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2021, 41(06): 1811-1815. |
[2] |
WANG Lei1, LIU Gui-dong2*, LIU Li1, LIU Yu-jie1, LOU Hong-jun1, QI Chao2, XIAO Hong-bin1*, CHI Wen-cheng1 . Difference of Nonlinear Degree between Healthy and Diabetic Rat Erythocyte Fluorescence Spectrum[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2016, 36(10): 3207-3210. |
[3] |
LUO Man1*, HUANG Yuan-yuan1, SU Bao-chang2, SHI Yun-feng3, ZHANG Hong1, YE Xun-da1 . Study on RBC Oxygen-Carrying Function with the Incubation Time [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2015, 35(12): 3350-3355. |
[4] |
WANG Lei1, LIU Gui-dong2*, MU Xin1*, XIAO Hong-bin1*, QI Chao2, ZHANG Si-qi3, NIU Wen-ying1, JIANG Guang-kun1, FENG Yue-nan1, BIAN Jing-qi1. Red Blood Cells Raman Spectroscopy Comparison of Type Two Diabetes Patients and Rats [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2015, 35(10): 2776-2780. |
[5] |
WU Zheng-jie1, WANG Cheng2, LIN Zheng-chun3, JIAO Qing-ze4 . Raman Spectra of Single Human Living Erythrocyte with the Effect of pH and Serum Albumin [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2014, 34(05): 1279-1283. |
[6] |
ZHANG Bao-ju1, LEI Qing1, LI Gang2, LIN Ling2, WANG Hui-quan2, Jean Gao1 . Noninvasive Measurement of the Human RBC Concentration Based on BP NN Model [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2012, 32(09): 2508-2511. |
[7] |
LI Gang1, ZHAO Jing1, LI Jia-xing1, 2, XIONG Hui1, 3, LIN Ling1, TONG Ying1, 4, ZHANG Bao-ju4* . Noninvasive Prediction of Red Blood Cell Counts by Normalized Reflection Spectroscopy for Tongue Inspection[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2011, 31(05): 1328-1331. |
[8] |
WANG Gui-wen1, PENG Li-xin1, YAO Hui-lu1, HUANG Shu-shi1, CHEN Ping2, LI Yong-qing3 . The Effect of Abnormal Cell Shape on the Spectral Distinguishing of Erythrocytes Using Laser Tweezers Raman Spectroscopy [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2009, 29(08): 2117-2121. |
[9] |
QIN Xi-yun1,LI Jun-hui2*,YANG Yu-hong1,CAI Gui-min2. Influence of Spectrometer Scanning Requirements in Homemade Grating Diffuse NIR Instrument on NIR Veracity[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2007, 27(02): 411-413. |
[10] |
FENG Shang-yuan1,2,CHEN Rong1,LI Yong-zeng1,YANG Wen-qin2,XIE Shu-sen1 . Fourier Transform Infrared and Near-Infrared Raman Spectra of Human Red Blood Cell [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2005, 25(05): 712-714. |
[11] |
YAO Cheng-can1, LI Xiao-kun1, HUANG Yao-xiong2* . Instant Effect of Temperature on the Oxygen Carrying Capacity of Single Living Intact Red Blood Cell [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2005, 25(04): 613-616. |
[12] |
YAN Xun-ling1, DONG Rui-xin1,2 , WANG Qiu-guo1 . Resonance Raman Spectra of Single Red-Cell from Human Blood [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2004, 24(05): 576-578. |
|
|
|
|