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A volume‐exclusion normalization procedure for quantitative Raman confocal microspectroscopy of immersed samples applied to human embryonic stem cells
Author(s) -
Schulze H. Georg,
Konorov Stanislav O.,
Okuda Kadek,
Piret James M.,
Blades Michael W.,
Turner Robin F. B.
Publication year - 2012
Publication title -
journal of raman spectroscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.748
H-Index - 110
eISSN - 1097-4555
pISSN - 0377-0486
DOI - 10.1002/jrs.3045
Subject(s) - raman spectroscopy , raman microspectroscopy , embryonic stem cell , normalization (sociology) , chemistry , stem cell , raman scattering , confocal , analytical chemistry (journal) , optics , biology , chromatography , microbiology and biotechnology , biochemistry , physics , sociology , anthropology , gene
Raman microspectroscopy is a quantitative instrumental method with considerable promise for the nondestructive analysis of living biological samples. Amongst samples of particular interest are human embryonic stem cells because of their therapeutic potential and because examination using Raman microspectroscopy does not appear to adversely affect this potential. However, it can be difficult to compare different spectra obtained with this technique and to quantify the native cellular constituents of such samples because their characteristic dimensions are difficult to establish or may vary from point to point. We present here a method to normalize spectra and estimate sample thicknesses based on a reference component present in the basal cell culture medium when we perform spectroscopy on colonies of living cells. Because more basal medium is displaced from the sampling volume as the cell layer increases in thickness, and because this component is present in the medium but excluded from cells, a concomitant decline therefore occurs in the intensity of the Raman scattering from the reference component. This permits comparisons between samples because their spectra can be scaled in inverse relation to their excluded volumes. Furthermore, estimations of sample thicknesses can also be obtained based on the same concept. Thus, the absolute quantification of cellular components becomes possible because cell sample volumes can be determined. Although applied to human embryonic stem cells, the approach is sufficiently general to be adapted for use with other samples. Copyright © 2011 John Wiley & Sons, Ltd.