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

Abstract

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.

Evidence of marked glycogen variations in the characteristic Raman signatures of human embryonic stem cells

“…the upper-left arrows point to low glycogen, but high cell density, and the lower-left arrows point to high glycogen, but low cell density). Chemical fixing can significantly distort the Raman spectra of cells, [31] while dry-fixing better preserves important Raman features. [21] We therefore used a dry-fixed sample to accommodate wileyonlinelibrary.com/journal/jrs the long acquisition times needed for Raman mapping of the 480 cm −1 glycogen marker band after PAS and DAPI staining.…”

Raman microspectroscopic evidence that dry‐fixing preserves the temporal pattern of non‐specific differentiation in live human embryonic stem cells

“…Chemical fixatives, however, contribute their own spectral signatures which may interfere with spectral features of interest from the cells. [16,17] Dry fixing by rapid desiccation would be an attractive alternative [16] provided changes in the spectra due to dehydration effects are sufficiently consistent across experimental variables that trends in spectral variations due to biological variations are preserved. Fixing in such a manner that preserves the information content of the sample has the further benefit of facilitating archiving.…”

Application of silver nanoparticle‐based SERS spectroscopy for DNA analysis in radiated nasopharyngeal carcinoma cells

“…The main band positions and the corresponding assignments are shown in Table 2. [14][15][16][17][18][19][20][21] It can be seen that none of the component type has changed along with the dose from 6 to 20 Gy. Hence, whether the mean SERS spectra of DNA extracted out of radiated CNE2 cell line have really changed is still unascertainable.…”