Fourier transform infrared microspectroscopy identifies symmetric PO(2)(-) modifications as a marker of the putative stem cell region of human intestinal crypts

Abstract

Complex biomolecules absorb in the mid-infrared (lambda = 2-20 microm), giving vibrational spectra associated with structure and function. We used Fourier transform infrared (FTIR) microspectroscopy to "fingerprint" locations along the length of human small and large intestinal crypts. Paraffin-embedded slices of normal human gut were sectioned (10 microm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 microm x 15 microm aperture) FTIR microspectroscopy in reflection mode, synchrotron (<or=10 microm x 10 microm aperture) FTIR microspectroscopy in transmission mode or near-field photothermal microspectroscopy. Dependent on the location of crypt interrogation, clear differences in spectral characteristics were noted. Epithelial-cell IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in "hyperspace" might on the basis of multivariate distance reveal biophysical differences along the length of gut crypts. Following spectroscopic analysis, plotted clusters and their loadings plots pointed toward symmetric (nu(s))PO(2)(-) (1,080 cm(-1)) vibrations as a discriminating factor for the putative stem cell region; this proved to be a more robust marker than other phenotypic markers, such as beta-catenin or CD133. This pattern was subsequently confirmed by image mapping and points to a novel approach of nondestructively identifying a tissue's stem cell location. nu(s)PO(2)(-), probably associated with DNA conformational alterations, might facilitate a means of identifying stem cells, which may have utility in other tissues where the location of stem cells is unclear.