Single-cell raman spectroscopy for the non-destructive, non-invasive analysis of cells and cellular components
Raman spectra of cells, such as normal human T- and B-cells from peripheral blood or human tonsil and the corresponding transformed cells are obtained by optically trapping the cells and obtaining their Raman spectra. The trapped cells can be subjected to one, two, or more different excitation wavelengths, and each wavelength of the corresponding Raman spectra can be stored in a separate channel. In preferred embodiments, two spectra are subtracted from each other to give a difference spectrum and each channel is analyzed independently to characterize the trapped cell. Alternatively, the Raman spectrum can be subjected to Principal Component Analysis (PCA) in order to characterize the trapped cell. The trapped cell thus classified can be sorted, or further manipulated.
1. A method for non-destructively characterizing a cell, the method comprising:
providing a sample comprising at least one intact cell in a solution, wherein the at least one intact cell is a white blood cell;
optically trapping a single intact cell from the sample using a laser using an excitation wavelength selected from the group of about 800-1800 nm, 780-950 nm, and 780-800 nm, wherein the excitation wavelength is selected to minimize the deleterious effects of heating the single intact cell;
illuminating the single intact trapped cell with the laser to generate a Raman spectrum, wherein the laser is not deleterious to the single intact trapped cell;
analyzing the Raman spectrum to characterize the single intact trapped cell;
identifying the single intact trapped cell as normal or abnormal based on the analyzed Raman spectrum; and
if the single intact trapped cell is identified as abnormal, removing the trapped intact abnormal cell from the sample using the laser.
2. The method of claim 1 , wherein the white blood cell is a T-lymphocyte.
3. The method of claim 1 , wherein the white blood cell is a B-lymphocyte.
4. The method of claim 1 , wherein the white blood cell is a normal cell or an abnormal cell.
5. The method of claim 4 , wherein the abnormal cell is a lymphoma cell.
6. The method of claim 4 , wherein the abnormal cell is a leukemia cell.
7. The method of claim 4 , wherein the abnormal cell is a myeloma cell.
8. The method of claim 1 , wherein the Raman spectrum is stored in a plurality of channels wherein each channel stores a single wavelength.
9. The method of claim 8 , wherein one or more of the plurality of channels is subjected to a statistical analysis.
10. The method of claim 9 , wherein the statistical analysis comprises obtaining a 95% to 99% confidence limit.
11. The method of claim 9 , wherein the statistical analysis comprises obtaining the variability index.
12. The method of claim 9 , wherein the statistical analysis comprises performing a t-test.
13. The method of claim 9 , wherein said statistical analysis identifies a significant channel, the method further comprising comparing the significant channel to one or more channels of a normal cell Raman spectrum, or to one or more channels of an abnormal cell Raman spectrum, or to one or more channels of both.
14. The method of claim 13 , wherein the one or more channels comprise between 1 and 100 channels.
15. The method of claim 14 , wherein the one or more channels comprise between 1 and 15 channels.
16. The method of claim 8 , wherein the Raman spectrum is subjected to Principal Component Analysis (PCA).
17. The method of claim 16 , wherein principal components are compared to principal components of a set of preprocessed spectra obtained from cells of known pathology.
18. The method of claim 16 , wherein PCA includes between 2 and 6 principal components.
19. The method of claim 18 , wherein PCA includes 2 or 3 principal components.
20. The method of claim 1 , wherein the analyzing step comprises comparing the produced Raman spectrum to a Raman spectrum of a normal cell, or to a Raman spectrum of an abnormal cell, or to both.
21. The method of claim 1 , wherein removing the abnormal cell further comprises destroying the abnormal cell.
22. The method of claim 1 , wherein removing the abnormal cell comprises sorting the abnormal cell away from the sample.