Technique Of Confocal Raman Micro-spectroscopy On Single Living Cell Measurements And Its Applications

The detection of molecular information of a living cell level has become a major research hot point with the development of life science.The molecular information provided by Raman spectrum can be used for the study of physiological of living creatures,the mechanism and process of diseases.So Raman scatter technique is a powerful tool for biological sample,especially for in situs,noninvasive,and real time or near real time measurement on living single cell.The confocal Raman micro-spectroscopy technique has many advantages over other techniques in doing so.For example,it can provide the information about the molecular component and structure of different samples without special preparation and any labeling,and it can detect the micro-region in living cells with 3D scanning.In this thesis,an inverted confocal Raman scatter spectroscopy system produced by Horbia JY(equipped with 514.5nm laser) was used to study living cells.The work we have done includes:(1) Fast test and calibration of performance parameters for the confocal Raman spectroscopy system in measuring living cells.(2) The techniques for living cells measurement including point scan, line mapping and 2D mapping and the technique of measuring the variation of the molecular conformation with time;(3) The optimization of scan parameters for living cells with different scan modes.The analysis and processing technique of Raman spectrum.(4) Confocal Raman micro-spectroscopy on erythrocytes and lymphocytes based on the work mentioned above.The measurements include:(a) The Raman spectra of erythrocytes with different cell age and states of intracellular Hb.(b) The variation of the Raman spectra of lymphocytes under the radiation of 900MHz electromagnetic filed and 254nm UV exposure respectively.According to the study and the experiment results,we can come to the following conclusions:(1) The fast test and calibration of performance parameters are vital to obtain high quality,reliability and comparable ability of Raman spectra.The wave riuinber precision,the stability:of system,the position of laser spot and the laser power density etc.should be calibrated.For confocal scanning,the confocal performance testing is also needed.(2) In order to meet the requirement of performing in situs,noninvasive measurement,the scanning parameters should be optimized for different samples. The optimized parameters mainly included the;laser power density at sample, exposure time,scan step,pinhole etc.The Raman spectrum of cells in vivo can be achieved with optimization of scan parameters.(3) In order to obtain high quality fingerprint spectrum,a series of process should be taken,such as the deletion of cosmic ray,classification,average and smooth of Raman spectra,etc.The classification of Raman spectra is significant in enhancing the bands,which are sensitive to the structure of molecule to highlight the fingerprint spectrum.(4) The diversity of Raman spectra of erythrocytes had been achieved by our developed method.The Raman spectra of erythrocytes can be divided into T,M1, M2,M3,M4 and R states according to the oxidation band and O₂ level marker band.The different states of Hb's Raman spectra can show the changing of its structure.Moreover,we qualitatively and semi quantitatively analyzed the allosteric effect of Hb using the v4 band(514.5nm excitation line) in living erythrocytes.(5) Differences in the signal intensity,molecule distribution,globin structure and the ability of carrying O₂ are shown among the young,old and old with added salic acid(old+SA) erythrocytes.(a) For old erythrocytes,its Raman spectrum is very strong and smooth; however,the Raman spectrum of young erythrocytes is relatively weak and coarse. And the result of "old+SA" is between that of young and old erythrocytes.(b) The distribution of Hb in old erythrocytes is not uniform,and exhibits high signal at the membrane of cells.The distributions of Hb in young and "old+SA" erythrocytes are more uniform.The results suggest that crosslink and aggregation of Hb around the cell membrane appeared in old erythrocytes.(c) The Raman spectrum of old erythrocytes shows obvious information of globin.The exposure of tyrosine(Tyr),the increase of unordered coil structure and decrease of ordered structure happen easily in old erythrocytes.However,in "old+SA" erythrocytes,the Tyr becomes embedded,the ordered structure increases and the unordered structure decreases.The signal of globin in young erythrocytes is very weak.(d) The transition speed of the Hb from T(tense) state to R(relaxed) state in old erythrocytes is slower than that of young ones,and the results of "old+SA" is intervenient.The T to R transition results shows that young erythrocytes have better ability of carrying oxygen than that of old ones.SA improves the ability of carrying oxygen in old erythrocytes.(e) SA not only decreases the intensity of Hb's Raman spectrum in old erythrocytes,but also improves the uniformity of the Hb distribution and the T to R transition speed in old ones,which indicates the SA has effect on the function of Hb in aging erythrocytes.(6) The intensity and background of the Raman signal of erythrocytes under the normal condition(PH=7.4) are weaker than that of erythrocytes under the acidic (PH＜7.0) or alkaline(PH＞7.6) condition.Greater the deviation from normal condition,higher the intensity and background counts.The intensity of the band at 755cm^-1 increases with the decrease of PH value in acidic situation.This suggests the structure and function of Hb will change if the PH value is deviant(for example,sickness).(7) The Raman spectra of distorted erythrocytes exhibits dramatical enhance at the distortion which suggests the aggregation and denaturation of Hb.(8) The results of lymphocytes under 900MHz electromagnetic field shows there are no obvious change in their Raman spectra,only appeared slight change in protein when the exposure time is short(less than 20min).However,if the exposure time were up to 40min,the characteristic bands of Raman spectra in protein and DNA would change.For example,the hydrophobic amino acid appeared exposed,the base damage etc.When the exposure time is up to 60min, there would be more changes in the Raman spectra of protein and DNA,such as the decrease of ordered structure,the increase of unordered structure and damage in single chain.(9) When the time of 254nm UV(power density was 10W/cm²) exposure to lymphocytes is equal to or less than 5min,there are slight increase in the unordered structure and slight decrease in the ordered structure in protein;When the exposure time is longer than 10min,not only the situation mentioned above would developed further,but also the hydrophobic amino acid would be exposed and the intensity of some bands increase for the reason of "hyperchromic effect".The results indicated the base-base effect was damaged and the CPD may be appeared as the photoproduct of UV.When the exposure time is up to 15min,the changes mentioned above become more obvious.(10) More divers Raman spectra for the lymphocytes under UV exposure than those under RF radiation based on analysis of the bands at 1156cm^-1 and 1520cm^-1. The two bands were assigned to the carotenoids,when the bands of carotenoids appeared,the cells is slightly damaged,indicating that the lymphocytes have released carotenoids at Gall bodies to protect themselves and eliminated the free radical as the antioxygen agent.With the UV exposure time increasing,the amount of carotenoids in cell decreases and the damage become obvious.Our innovations in this work are as follow:(1) Designing a fast method and sequence of the test and calibration of Raman confocal micro-spectroscopy.(2) Optimization of the scanning parameters and data process for point scan,line scan and 2D scan modes.And apply the technique on erythrocytes and lymphocytes measurements.(3) Have developed the classification and average method to incarnate the abundant fingerprint spectrum.(4) Classify the Raman spectrum of Hb into 6 states:T,M1,M2,M3,M4 and R based on the classification and average method and parameters' optimization.(5) Have performed qualitative and semi-quantity analysis of alloestric effect of Hb in living erythrocytes based on the change of v₄ band in Raman spectrum of erythrocytes.(6) Report the Raman spectrum of young,old and old+SA erythrocytes first time. Design an easy and practical method to do the T to R state transition experiment and realize dynamic test on the variation of the molecular structure in living cells.(7) Report the Raman spectra of erythrocytes at different PH value and the distortion erythrocytes first time.(8) Study the effects of 900MHz electromagnetic field and 254nm UV on the lymphocytes with the confocal Raman micro-spectroscopy,and report the diversity of Raman spectrum of lymphocytes and the carotenoids changes after the radiations.In summary,the confocal Raman micro-spectroscopy we developed for single living cell measurements,especially the line scan,2D scan methods,classification and average method can provide helpful tool in living cell studies,and they are expected to have a variety of application on the measurements of different single living cells.