Fixatives
While Raman spectroscopic imaging can be performed on living cells, most Raman cell imaging studies are performed on fixed cells or tissues in order to preserve cellular structure over the long imaging times required. As such, a suitable choice of fixative is important in terms of preserving structural and biochemical (and hence spectral) information content.
In the context of Raman spectroscopy, the main fixatives that have been investigated are formalin (37% paraformaldehyde solution in H2O), methanol, ethanol, acetone, and Carnoy's fixative. Each of these fixatives act in different ways, and with different affinity for certain classes of molecules (e.g. formalin forms cross-links, while organic solvents tend to dehydrate cells and cause lipid leaching). In addition to selecting a suitable fixative, other parameters to consider include the length and temperature of fixation, and the addition of further processing steps. Fortunately, several groups have investigated different fixatives and fixation protocols and the effects they have on Raman spectral information relative to Raman spectra of living cells [1-5].
These studies have shown that all of the fixation methods investigated have an effect on the Raman spectra of the cells, though to varying extents. Formalin fixation has repeatedly shown to display Raman spectra that most closely align with those of living cells, with a recent study suggesting the formalin fixation at room temperature is preferable [5]. However, depending on the particular cell type or analysis technique, alternative fixatives may be selected. Regardless of the choice of fixative (or none), it is imperative that comparisons are only made between samples treated equivalently.
Below we detail potential fixative choices and evaluate them in terms of their Raman spectral preservation, extent of use in cell biology, and relative cost.
References:
These studies have shown that all of the fixation methods investigated have an effect on the Raman spectra of the cells, though to varying extents. Formalin fixation has repeatedly shown to display Raman spectra that most closely align with those of living cells, with a recent study suggesting the formalin fixation at room temperature is preferable [5]. However, depending on the particular cell type or analysis technique, alternative fixatives may be selected. Regardless of the choice of fixative (or none), it is imperative that comparisons are only made between samples treated equivalently.
Below we detail potential fixative choices and evaluate them in terms of their Raman spectral preservation, extent of use in cell biology, and relative cost.
References:
- Chan, J. W., Taylor, D. S., Thompson, D. L., Biopolymers, 91, 2, 2009.
- Mariani, M. M., Lampen, P., Popp, J., Wood, B. R., Deckert, V., Analyst, 134, 2009.
- Meade, A. D., Clarke, C., Draux, F., Sockalingum, G. D., Manfait, M., Lyng, F. M., Byrne, H. J., Analytical and Bioanalytical Chemistry, 396, 2010.
- Draux, F., Gobinet, C., Sule-Susso, J., Trussardi, A., Manfait, M., Jeannesson, P., Sockalingum, G. D., Analytical and Bioanalytical Chemistry, 397, 2010.
- Hobro, A. J., Smith, N. I., Vibrational Spectroscopy, 91, 2017.