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

Raman microspectroscopy allows the classification of populations of human embryonic stem cells (hESCs) in different stages of differentiation based on the relative intensities of certain amino acid and nucleic acid bands. Here, we report the results of a comparative study of the Raman spectra of live cells versus cells killed and fixed by rapid desiccation, focusing on the ratio of intensities at 757 cm−1 (tryptophan) and 784 cm−1 (DNA and RNA). We observe that the same temporal pattern emerges over a 3‐week time course in both sample types. This suggests that prolonged observations of dry‐fixed cells can yield high signal‐to‐noise chemical images that cannot be obtained from colonies of living cells where the time scale of significant biological changes are comparable to the time scale of the measurement. This permits, for example, comparison of the spatial distributions of cells at different stages of differentiation within the same colonies. Copyright © 2010 John Wiley & Sons, Ltd. Raman microspectroscopy allows the classification of populations of human embryonic stem cells in different stages of differentiation based on the relative intensities of certain amino acid and nucleic acid bands. Prolonged observations of dry‐fixed cells can yield high signal‐to‐noise chemical images that reveal spatially resolved inhomo‐geneities in differentiating cells. Such images can not be obtained from colonies of living cells where the time scale of significant biological changes are comparable to the time‐scale of the measurement.