CN103512874A - Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method - Google Patents
Ultrasonic perforation-laser tweezer cell surface enhanced Raman spectroscopy method Download PDFInfo
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Abstract
The invention provides a method for identifying cells based on ultrasonic perforation effect in combination with laser tweezer cell surface enhanced Raman spectroscopy. The ultrasonic wave is used to act on cells to result in a perforation effect which instantly enhances the cell membrane permeability, so that metal nanoparticles are rapidly introduced into the cells to be used as an SERS enhancement substrate, and the laser tweezer technology is used to capture viable cells and perform surface enhanced Raman spectroscopy at the same time, as a result, the effect of pathological examination and screening of cancer cells is achieved by means of SERS. The method comprises the following steps of: preparing a silver gel solution; injecting the high-concentration silver gel into viable cells and performing surface enhanced Raman spectroscopy; and establishing a cell surface enhanced Raman spectroscopy diagnosis and identification mode for identifying whether the cells are healthy cells or cancer cells. The method provided by the invention has the characteristics of simple and repaid operation, strong reliability and high sensitivity, and can be widely applied to identify different types of cancerous cells.
Description
Technical field
The present invention relates to a kind ofly utilize ultrasonic perforating effect and in conjunction with the method for laser optical tweezer Surface enhanced raman spectroscopy (SERS) technical Analysis cell.
Background technology
Multiple biomedical technology has been applied to qualitative, quantitative going and has surveyed, screened normal or sick cell.Such as present radiology, pharmacology, histology, cytology and molecules technology can be applied in the analysis of cell.But these technology are all to have destructively, simultaneously their specificity is not ideal enough, and many times wants multiple technologies coupling just can effectively analyze, but also may be subject to the interference of cell biology means.We are badly in need of a kind of technological means now can overcome above-mentioned several cell analysis technical limitation, can in clinical diagnosis, easily realize accurate cancer cell analysis and screening.
Surface enhanced raman spectroscopy (SERS) technology is compared and can be made Raman signal enhancer reach 10 with conventional Raman spectrum
14, in some condition, even can realize Single Molecule Detection.Meanwhile, SERS technology can also suppress the interference of biological autofluorescence background to a great extent.Therefore, SERS technology has been widely used in the research of biomedical sector.There have been in the world a plurality of research groups to utilize SERS spectrum to carry out cell research, but want to detect cell SERS spectral signal, just must by certain specific process make metal nanoparticle transfered cell inner or make nano particle and cell can adsorbed close together with, the SERS spectral signal of cell so just can be detected.Make nano particle enter cell interior and can carry out SERS detection, more conventional way is metal nanoparticle and cell co-culture, hatches now, through cultivating for a long time (generally wanting about 24 hours), utilize the endocytosis effect of cell, nano particle just can " be gulped down " and be entered cell interior, thereby realize the analysis of cell SERS spectral detection, but this method expends time in very much; Second method is exactly to utilize the autonomous reduction mechanism of biological cell, solution with metallic ion and cell are hatched jointly, at cell interior spontaneous " growing " nano-metal particle, this method need to be carried out cell cultivation equally for a long time, and the metal nanoparticle out-of-shape that is reduced out at cell interior; Third method is to utilize electroporation technology fast metal nanoparticle to be imported to intracellular conveying, but this method needs special electroporation device, operation more complicated, efficiency low, and the distribution of nano particle in cell is very inhomogeneous.
Weak point in view of above the whole bag of tricks, patent of the present invention proposes to utilize the ultrasonic acoustic effect of holes by the quick transfered cell of metal nanoparticle, and laser optical tweezer Raman spectroscopy is combined with this supersonic induced technology can detect and obtain high reproducible cell SERS spectrum, we also further utilize cell SERS spectrum to carry out cancer cell and Normocellular screening study, obtain ideal differentiation effect.
Summary of the invention
The object of this invention is to provide a kind of ultrasonic acoustic effect of holes of utilizing and in conjunction with laser optical tweezer Surface enhanced raman spectroscopy technology, carry out the method for cell analysis.It is to utilize high-energy ultrasound effect moment generation acoustic horn effect, make pericellular micro-bubble violent contraction occur, expand and finally collapse and go out, meanwhile the bubble of these generation acute variation and cell generation heat effect and mechanical effect produce perforation effect on cell membrane, at surface of cell membrane, produce reversibility cavernous structure, permeability of cell membrane moment is strengthened greatly; Simultaneously metal nanoparticle forms single dispersion and in high-speed motion state under ul-trasonic irradiation, the aperture that so just can produce by surface of cell membrane brief moment strengthens substrate using the quick transfered cell of metal nanoparticle inside as SERS, and utilize laser optical tweezer technology to catch cell and carry out Surface enhanced raman spectroscopy detection simultaneously, finally the cell SERS spectroscopic data obtaining is carried out to multivariate statistical analysis (PCA-LDA) and process, by the diagnostic equation in scatter diagram, can differentiate cell is cancer or normal cell.We take nasopharyngeal carcinoma cell and normal nasopharyngeal cell as model is to cancer or Normocellularly carry out discriminatory analysis.In measuring process, utilize laser optical tweezer technology can maintain cell and under physiologically active state, carry out SERS spectrum test.The whole processing procedure time of the present invention only needs 10 minutes, simple to operate, quick, reliability is strong; Can make metal nanoparticle very efficiently in transfered cell, and metal nanoparticle distributes more evenly, can obtain the extraordinary cell SERS of reappearance spectrum.To the laser optical tweezer SERS spectrum of normal and cancer cell differentiate, analysis conclusion can be doctor to patient's diagnosis provide fast, objective appraisal standard.The method can be expanded, be applied to other kinds cancer cell and Normocellular discriminatory analysis.
For realizing the technical scheme of object employing of the present invention, be:
(1) get and in the deionized water that silver nitrate powder is dissolved in 200ml, be heated to boiling, then splashing into citric acid three sodium solution, to make the fulmargin that contains Nano silver grain standby;
(2) it is standby that competent cell trypsinization being got off is made single-cell suspension liquid with RPMI1640 cell culture fluid;
(3) by fulmargin with single-cell suspension liquid mixes and utilize ultrasonic perforating effect by the quick transfered cell of Nano silver grain;
(4) to unicellular, carry out the detection of laser optical tweezer Surface enhanced raman spectroscopy maintaining under physiological environment;
(5) set up cell surface and strengthen Raman spectrum data storehouse, utilize multivariate statistical analysis to obtain the scatter diagram corresponding to Surface enhanced raman spectroscopy of variety classes cell;
(6) utilize and to set up cell surface and strengthen Raman spectrum data storehouse, diagnosis cell is not differentiated.
The preparation process of described fulmargin is: get 36mg silver nitrate powder and be dissolved in the deionized water of 200ml, rapid stirring is also heated to boiling, splashing into 5ml concentration is 1% citric acid three sodium solution again, continue agitating heating 1 hour, prepare the fulmargin that contains Nano silver grain, and put at room temperature lucifuge and seal up for safekeeping standby;
The preparation process of described single-cell suspension liquid is: the competent cell that trypsinization is got off is made single-cell suspension liquid with RPMI1640 cell culture fluid, then by blood counting chamber, calculates the number of cell, and controlling cell density is 10
5～10
6individual/ml.
The described ultrasonic perforating effect of utilizing by the quick transfered cell internal procedure of Nano silver grain is: get pre-prepared good fulmargin 1 ml and carry out centrifugal, abandon the cell culture fluid that adds 0.5ml after supernatant, in the ratio of 1:1, put into centrifuge tube with the prefabricated active single-cell suspension liquid of getting ready, utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, they to be mixed, make active unicellular elargol mixed liquor; Centrifuge tube is put into ultrasonic pond, and by regulating hyperacoustic power, frequency, the temperature in ultrasonic pond, ultrasonic time parameter, by the ultrasonication of the unicellular elargol mixed liquor of the activity in centrifuge tube; The unicellular elargol mixed liquor of the activity of ultrasonication is mixed with PBS damping fluid and carry out low-speed centrifugal, clean three times, the silver nano-grain that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in RPMI1640 cell culture fluid.
Described hyperacoustic power is: 200W～400W.
Described hyperacoustic frequency is: 25HZ, 40HZ or 59HZ;
The temperature in described ultrasonic pond is: room temperature～60 ℃;
Described ultrasonic time is: 1min～30min.
Described is the laser optical tweezer system (model: OTKB) that is accompanied with Raman spectrum module that adopts U.S. Thorlabs company to produce maintaining under physiological environment to the unicellular laser optical tweezer Surface enhanced raman spectroscopy testing process of carrying out, utilize in this laser optical tweezer system acquisition RPMI1640 cell culture fluid active unicellularly, utilize the semiconductor laser that wavelength is 785nm to carry out cell SERS spectral detection as exciting light simultaneously.For obtaining high reappearance SERS spectrum, under the condition of cell, reduce excitation light power as far as possible and carry out cell SERS spectrum test guaranteeing to catch, can obtain high reappearance cell SERS spectrum.
The described cell surface of setting up strengthens Raman spectrum data storehouse model, and its process is: cellularity is differentiated and must be realized by a diagnostic model, and this model is set up by calibration process.For guaranteeing that data have statistical significance, need to gather the cell SERS spectrum of abundant confirmed cases, set up calibration spectra database.On this basis, use main element analysis method and linear discriminant analysis algorithm, set up the diagnostic equation being formed by cell SERS spectrum parameter.First we need cell SERS spectrum to detain fluorescence and intensity normalized, whole piece spectral line data input SPSS software is carried out to major component (PCA) analysis, then utilize independent variable T check to obtain having the PCA score of significant difference, and utilize the PCA score with significant difference to draw the scatter diagram with diagnostic equation, and further utilize linear discriminant analysis (LDA) to draw statistics.
The described built vertical cell surface that utilizes strengthens Raman spectrum data storehouse model, diagnosis cell is not differentiated, its process is: diagnosis cell is not made to the unicellular elargol mixed liquor of required activity according to the method described in technical scheme, and maintaining under physiological environment the unicellular laser optical tweezer Surface enhanced raman spectroscopy detection SERS spectrum that carries out, spectrum is imported to the cell surface having established and strengthen Raman spectrum data storehouse, utilize multivariate statistical analysis to obtain the not scatter diagram corresponding to Surface enhanced raman spectroscopy of diagnosis cell, according to the region that is distributed in diagnostic equation both sides in scatter diagram, differentiate cell.
Advantage of the present invention is to adopt ultrasonic perforation effect to make metal nanoparticle can enter fast cell, have that nano particle imports that efficiency is high, metal nanoparticle is in the cell interior more uniform feature that distributes, combine and can guarantee cytoactive with laser optical tweezer technology, can detect and obtain high reappearance cell SERS spectrum simultaneously; And with this cell SERS spectroscopic data, set up diagnostic model, can be used for the differentiation to nasopharyngeal carcinoma cell.Also the method can be expanded to the discriminatory analysis that is applied to the kinds cancer cells such as cancer of the esophagus, cutaneum carcinoma.
Accompanying drawing explanation
Fig. 1 be the present invention to 63 NP69 normal cells cell SERS spectrum averaging spectrum under ultrasonic perforation effect, shade and shadow partly represents the standard deviation of cell SERS spectrum.
Fig. 2 be the present invention to 67 C666 nasopharyngeal carcinoma cells cell SERS spectrum averaging spectrum under ultrasonic perforation effect, shade and shadow partly represents the standard deviation of cell SERS spectrum.
Fig. 3 is the present invention to 67 C666 cells and 63 NP69 cell SERS spectrum PCA score PC1 and PC2 two dimension scatter diagram.
Fig. 4 is the present invention to 67 C666 cells and 63 NP69 cell SERS spectrum PCA score PC1, PC2 and the three-dimensional scatter diagram of PC3.
Embodiment
Concrete ins and outs of the present invention are implemented to be described below for normal nasopharyngeal cell NP69, two types of cells of differentiated Nasopharyngeal Squamous Cell Carcinoma C666:
Embodiment 1
1, the solid nitric acid silver of getting 36mg is dissolved in the deionized water of 200ml, rapid stirring is also heated to boiling, then splashes into 1% trisodium citrate of 5ml, continues agitating heating 1 hour, obtain silver sol solution, and put at room temperature lucifuge sealing preservation in order to follow-up supersonic induced step use; Two kinds of competent cell NP69, C666 with pancreatin, just having digested are made to single-cell suspension liquid with commercially available RPMI1640 cell culture fluid respectively, then by blood counting chamber, calculate the number of cell, controlling cell density is 10
6individual/ml.
2, get the above-mentioned fulmargin 10ml preparing and carry out centrifugally, abandon after supernatant, be divided into two groups, every group adds respectively the cell culture fluid of 2.5ml to mix; Get respectively 0.5ml silver sol and NP69, C666 cell suspending liquid are put into respectively EP pipe separately in the ratio of 1:1 for every group; utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, silver sol suspending liquid and cell suspending liquid to be mixed, make unicellular elargol mixed liquor;
3, the centrifuge tube that silver sol and cell suspending liquid are housed is put into ultrasonic pond, by regulating the ultrasonic power in ultrasonic pond, be that 200W, ultrasonic frequency are 25HZ, 30 ℃ of ultrasonic pond temperature, ultrasonic time 1min, make the mixed liquor ultrasonication under best ultrasound condition in centrifuge tube.
4, the cell of ultrasonication mixed with PBS damping fluid and carry out low-speed centrifugal, cleaning three times, the metal nanoparticle that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in nutrient solution.
5, the sample cell that the cell suspending liquid cleaning is injected to laser optical tweezer Raman detection system carries out light tweezer SERS spectral detection, the semiconductor laser that excitation wavelength is 785nm in the environment of cell culture fluid.Test condition is 100 times of Nikon oil mirrors.
6, by definite normal nasopharyngeal cell and nasopharyngeal carcinoma cell SERS establishment of spectrum calibration spectra database, and all cells SERS spectral line is input to SPSS software carries out PCA analysis, is the concrete computation process that application PCA analyzes below:
(1) first utilize fitting of a polynomial to eliminate fluorescence background 63 normal NP69 nasopharyngeal cells and 67 C666 nasopharyngeal carcinoma cell SERS spectrum;
(2) for reducing experimental error impact, the normal and nasopharyngeal carcinoma cell SERS spectrum of eliminating fluorescence background is carried out to area normalization processing;
(3) utilize SPSS software that the normal and nasopharyngeal carcinoma cell SERS spectrum of processing through (1), (2) is carried out to PCA analysis;
(4) utilize on this basis the T method of inspection in SPSS, select to have most three PCA of significant difference LDA analysis of must assigning to further carry out;
Utilizing T check to obtain three has the PCA score of significant difference most and draws scatter plot distributions.
The present invention confirms that by independent variable T check PCA analyzes rear major component 1, major component 2 and major component 4 these three major components and has significant difference.We further draw major component 1 and the two-dimentional scatter diagram of major component 4 with diagnostic equation, as shown in Figure 3.Diagnostic equation is y=-2.73x-0.43, and this diagnostic equation line can distribute nasopharyngeal carcinoma cell point set to distribute and effectively separate to get up with normal cell point set.If utilize major component 1, major component 2 and the major component 4 in two class cell SERS spectrum PCA analyses with significant difference further to draw three-dimensional scatter diagram, as shown in Figure 4, the data point distribution that can find to represent normal nasopharyngeal cell and represent nasopharyngeal carcinoma cell is in two separate spaces, without any overlapping, distinguish effect very obvious each other.Further utilize follow-up LDA to analyze and draw statistics, the present invention reaches 100% to specificity and the sensitivity of the cancer cell identification in calibration spectra collection.Normal and nasopharyngeal carcinoma cell SERS spectrum PCA score scatter plot distributions is measurement result of the present invention, also can be diagnosis important reference is provided.
Embodiment 2
1, the solid nitric acid silver of getting 36mg is dissolved in the deionized water of 200ml, rapid stirring is also heated to boiling, then splashes into 1% trisodium citrate of 5ml, continues agitating heating 1 hour, obtain silver sol solution, and put at room temperature lucifuge sealing preservation in order to follow-up supersonic induced step use; Two kinds of competent cell NP69, C666 with pancreatin, just having digested are made to single-cell suspension liquid with commercially available RPMI1640 cell culture fluid respectively, then by blood counting chamber, calculate the number of cell, controlling cell density is 10
6individual/ml.
2, get the above-mentioned fulmargin 10ml preparing and carry out centrifugally, abandon after supernatant, be divided into two groups, every group adds respectively the cell culture fluid of 2.5ml to mix; Get respectively 0.5ml silver sol and NP69, C666 cell suspending liquid are put into respectively EP pipe separately in the ratio of 1:1 for every group; utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, silver sol suspending liquid and cell suspending liquid to be mixed, make unicellular elargol mixed liquor;
3, the centrifuge tube that silver sol and cell suspending liquid are housed is put into ultrasonic pond, by regulating the ultrasonic power in ultrasonic pond, be that 250W, ultrasonic frequency are 40HZ, 24 ℃ of ultrasonic pond temperature, ultrasonic time 20min, make the mixed liquor in centrifuge tube under best ultrasound condition, carry out ultrasonication.
4, the cell of ultrasonication mixed with PBS damping fluid and carry out low-speed centrifugal, cleaning three times, the metal nanoparticle that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in nutrient solution.
5, the sample cell that the cell suspending liquid cleaning is injected to laser optical tweezer Raman detection system carries out light tweezer SERS spectral detection, the semiconductor laser that excitation wavelength is 785nm in the environment of cell culture fluid.The object lens of test use are 100 times of Nikon oil mirrors.
6, by definite normal nasopharyngeal cell and nasopharyngeal carcinoma cell SERS establishment of spectrum calibration spectra database, and all cells SERS spectral line is input to SPSS software carries out PCA analysis, is the concrete computation process that application PCA analyzes below:
(1) 1) 1 first utilize fitting of a polynomial to eliminate fluorescence background 120 normal NP69 nasopharyngeal cells and 130 C666 nasopharyngeal carcinoma cell SERS spectrum;
(2) for reducing experimental error impact, the normal and nasopharyngeal carcinoma cell SERS spectrum of eliminating fluorescence background is carried out to area normalization processing;
(3) utilize SPSS software that the normal and nasopharyngeal carcinoma cell SERS spectrum of processing through (1), (2) is carried out to PCA analysis;
(4) utilize on this basis the T method of inspection in SPSS, select to have most three PCA of significant difference LDA analysis of must assigning to further carry out;
(5) utilizing T check to obtain three has the PCA score of significant difference most and draws scatter plot distributions.
The present invention confirms that by independent variable T check PCA analyzes rear major component 1, major component 2 and major component 4 these three major components and has significant difference.We further draw major component 1 and the two-dimentional scatter diagram of major component 4 with diagnostic equation.This diagnostic equation line can distribute nasopharyngeal carcinoma cell point set to distribute and effectively separate to get up with normal cell point set.If utilize major component 1, major component 2 and the major component 4 in two class cell SERS spectrum PCA analyses with significant difference further to draw three-dimensional scatter diagram, the data point distribution that can find to represent normal nasopharyngeal cell and represent nasopharyngeal carcinoma cell is in two separate spaces, without any overlapping, distinguish effect very obvious each other.Further utilize follow-up LDA to analyze and draw statistics, the present invention nearly all reaches 100% to specificity and the sensitivity of the cancer cell identification in calibration spectra collection.Normal and nasopharyngeal carcinoma cell SERS spectrum PCA score scatter plot distributions is measurement result of the present invention, also can be diagnosis important reference is provided.
Claims (9)
1. ultrasonic perforating-laser optical tweezer cell surface strengthens a Raman spectrum analysis method, it is characterized in that the method is comprised of following steps:
(1) get and in the deionized water that silver nitrate powder is dissolved in 200ml, be heated to boiling, then splashing into citric acid three sodium solution, to make the fulmargin that contains Nano silver grain standby;
(2) it is standby that competent cell trypsinization being got off is made single-cell suspension liquid with RPMI1640 cell culture fluid;
(3) by fulmargin with single-cell suspension liquid mixes and utilize ultrasonic perforating effect by the quick transfered cell of Nano silver grain;
(4) to unicellular, carry out the detection of laser optical tweezer Surface enhanced raman spectroscopy maintaining under physiological environment;
(5) set up cell surface and strengthen Raman spectrum data storehouse, utilize multivariate statistical analysis to obtain the scatter diagram corresponding to Surface enhanced raman spectroscopy of variety classes cell;
(6) utilize built vertical cell surface to strengthen Raman spectrum data storehouse, diagnosis cell is not differentiated.
2. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that the described ultrasonic perforating effect of utilizing by the quick transfered cell internal procedure of Nano silver grain is: get the prefabricated fulmargin of getting ready 1 ml and carry out centrifugal, abandon the cell culture fluid that adds 0.5ml after supernatant, in the ratio of 1:1, put into centrifuge tube with the prefabricated active single-cell suspension liquid of getting ready, utilize the liquid-transfering gun of 200ul by the mode of repeatedly aspirating, they to be mixed, make active unicellular elargol mixed liquor; Centrifuge tube is put into ultrasonic pond, and by regulating hyperacoustic power, frequency, the temperature in ultrasonic pond, ultrasonic time parameter, carry out ultrasonication by the unicellular elargol mixed liquor of the activity in centrifuge tube; The unicellular elargol mixed liquor of the activity of ultrasonication is mixed with PBS damping fluid and carry out low-speed centrifugal, clean three times, the silver nano-grain that does not enter cell interior is cleaned up, and the cell Eddy diffusion cleaning is standby in RPMI1640 cell culture fluid.
3. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that described ultrasound wave, and its power is 200W～400W.
4. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that described hyperacoustic frequency is: 25HZ, 40HZ or 59HZ.
5. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that described ultrasonic pond temperature is: room temperature～60 are ℃ continuous adjustable.
6. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that described ultrasonic time is that 1min～30min is adjustable continuously.
7. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that the described cell surface of setting up strengthens Raman spectrum data storehouse model, and its process is:
(1) by calibration process, set up diagnostic model;
(2) gather the cell SERS spectrum of confirmed cases, set up calibration spectra database;
(3) use main element analysis method and linear discriminant analysis algorithm, set up the diagnostic equation being formed by cell SERS spectrum parameter;
(4) utilize the PCA score with significant difference to draw the scatter diagram with diagnostic equation, and further utilize linear discriminant analysis to draw statistics.
8. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 7 strengthens Raman spectrum analysis method, it is characterized in that the described diagnostic equation of setting up is first to need cell SERS spectrum to detain fluorescence and intensity normalized, whole piece spectral line data input SPSS software is carried out to principal component analysis (PCA), then utilize independent variable T check to obtain having the PCA score of significant difference, and utilize the PCA score with significant difference to draw the scatter diagram with diagnostic equation.
9. a kind of ultrasonic perforating-laser optical tweezer cell surface as claimed in claim 1 strengthens Raman spectrum analysis method, it is characterized in that the described built vertical cell surface that utilizes strengthens Raman spectrum data storehouse model, diagnosis cell is not differentiated, its process is: diagnosis cell is not made to the unicellular elargol mixed liquor of required activity according to the method described in technical scheme, and to unicellular, carry out the detection of laser optical tweezer Surface enhanced raman spectroscopy maintaining under physiological environment, spectrum is imported to the cell surface having established and strengthen Raman spectrum data storehouse model, utilize multivariate statistical analysis to obtain the not scatter diagram corresponding to Surface enhanced raman spectroscopy of diagnosis cell, according to the region that is distributed in diagnostic equation both sides in scatter diagram, differentiate cell.
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| CN106645079A (en) * | 2016-09-30 | 2017-05-10 | 福建师范大学 | Human blood type identifying method based on erythrocyte laser tweezers Raman spectroscopy |
| CN114235773A (en) * | 2020-09-09 | 2022-03-25 | 南京大学 | Raman imaging method for dynamically monitoring cell membrane repairing process |
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