|
US researchers have used laser tweezers Raman spectroscopy (LTRS) to help them characterize the effects of different chemical fixation procedures on the spectra of healthy cells and leukaemia cells and to preclude the misinterpretation of data. James Chan of the Applied Physics and Biophysics Division, at Lawrence Livermore National Laboratory, Livermore, and the NSF Center for Biophotonics Science and Technology, University of California-Davis, Sacramento, in California, together with UC Davis' Douglas Taylor and Deanna Thompson, hoped to find a way to reduce errors in Raman spectra. The team investigated individual samples of normal and diseased, transformed lymphocytes, that were either unfixed or fixed with paraformaldehyde or methanol. Three different cell lines were analysed with the LTRS technique. "When compared to the spectra of unfixed cells, the fixed cell spectra show clear, reproducible changes in the intensity of specific Raman markers commonly assigned to DNA, RNA, protein, and lipid vibrations in mammalian cells," the researchers explain. The markers were found at 785, 1230, 1305, 1660 cm-1, they add, these are all of biomedical importance as they are commonly used in research to discriminate between normal and cancerous lymphocytes. The team carried out a statistical analysis of the Raman data and used principal component analysis and linear discriminant analysis to classify the results. They demonstrated that methanol fixation is the most worrying method. "[It] induces a greater change in the Raman spectra than paraformaldehyde," the researchers say. Perhaps more worryingly, the study also revealed that the changes in the spectra caused by the cell-fixation process also have an adverse effect on the accurate discrimination using Raman of normal and cancerous cells using Raman. "The spectral artefacts created by the use of [common] fixatives indicate that the method of cell preparation is an important parameter to consider when applying Raman spectroscopy to characterize, image, or differentiate between different fixed cell samples," the team says. Raman spectroscopy has become a widely used and powerful technique in biomedical research. It has uses in the detection of cancer in whole tissues, it can be used to characterize neurodegenerative diseases, and identify stem cells, among other applications. Raman can also be used to obtain a "molecular fingerprint" from a sample for biochemical analysis. By combining the technique with laser tweezers it has also been demonstrated to be a powerful technique in studying single cells in suspension. Optical trapping simplifies the process even further and reduces the number of pre-preparation steps. The approach even has potential in lab-on-a-chip, or microelectromechanical (MEMS), devices, such as a Raman-based cell sorter. The team points out that the analysis of chemically fixed cells is almost unavoidable despite these advances. Their latest study, revealing the impact fixation can have on the spectra, serves not only as a note of caution to other researchers, but may also point the way forward in developing alternative approaches that preclude the issues. Further studies are now required to help the researchers understand why these differences in Raman spectra arise depending on the chosen chemical fixation method. "This information is critical for future planned LTRS studies aimed at correlating the Raman data to clinical fluorescence flow cytometry results of fixed specimens to avoid misinterpretation of spectral artefacts arising from sample preparation," the researchers conclude, "In addition, it remains to be seen if these observations can be extended to other cellular systems." "It is probable that this may be a problem for other cell types and fixation methods," Chan told SpectroscopNOW, "I think it has to be evaluated on a case-by-case basis for each specific cell type, system, and situation." He suggests that for each cell type, one would need to determine what spectral changes occur as a result of the fixation process, and whether those changes directly involve/affect the Raman markers used to identify/discriminate between the cell types. "I don't think old results are necessarily wrong," he emphasises, "Our old results discriminating normal and leukaemia cells were all performed on unfixed cells, so we are confident that those results are valid. This was our first endeavour to determine the effect of fixation. We have simply shown that this is an important thing to consider for the case of leukaemia cells." Related links:
|
|
