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Label-free determination of the cell cycle phase in human embryonic stem cells by Raman microspectroscopy.

Authors
Type
Published Article
Journal
Analytical Chemistry
1520-6882
Publisher
American Chemical Society
Publication Date
Volume
85
Issue
19
Pages
8996–9002
Identifiers
DOI: 10.1021/ac400310b
PMID: 23968373
Source
Medline
License
Unknown

Abstract

The cell cycle is a series of integrated and coordinated physiological events that results in cell growth and replication. Besides observing the event of cell division it is not feasible to determine the cell cycle phase without fatal and/or perturbing invasive procedures such as cell staining, fixing, and/or dissociation. Raman microspectroscopy (RMS) is a chemical imaging technique that exploits molecular vibrations as a contrast mechanism; it can be applied to single living cells noninvasively to allow unperturbed analysis over time. We used RMS to determine the cell cycle phase based on integrating the composite 783 cm(-1) nucleic acid band intensities across individual cell nuclei. After correcting for RNA contributions using the RNA 811 cm(-1) band, the measured intensities essentially reflected DNA content. When quantifying Raman images from single cells in a population of methanol-fixed human embryonic stem cells, the histogram of corrected 783 cm(-1) band intensities exhibited a profile analogous to that obtained using flow-cytometry with nuclear stains. The two population peaks in the histogram occur at Raman intensities corresponding to a 1-fold and 2-fold diploid DNA complement per cell, consistent with a distribution of cells with a population peak due to cells at the end of G1 phase (1-fold) and a peak due to cells entering M phase (2-fold). When treated with EdU to label the replicating DNA and block cell division, cells with higher EdU-related fluorescence generally had higher integrated Raman intensities. This provides proof-of-principle of an analytical method for label-free RMS determination in situ of cell cycle phase in adherent monolayers or even single adherent cells.

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