The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the effect of different chemical fixation procedures on the Raman spectra of normal and leukemia cells. Individual unfixed, paraformaldehyde-fixed, and methanol-fixed normal and transformed lymphocytes from three different cell lines were analyzed with LTRS. 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 (e.g. 785, 1230, 1305, 1660 cm(-1)) in mammalian cells, many of which are important markers that have been used to discriminate between normal and cancer lymphocytes. Statistical analyses of the Raman data and classification using principal component analysis and linear discriminant analysis indicate that methanol fixation induces a greater change in the Raman spectra than paraformaldehyde. In addition, we demonstrate that the spectral changes as a result of the fixation process have an adverse effect on the accurate Raman discrimination of the normal and cancer cells. The spectral artifacts created by the use of 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 to avoid potential misinterpretation of the data.