FTIR Micro-spectroscopy Identifies Symmetric PO₂^- Modifications as a Marker of the Putative Stem Cell Region of Human Intestinal Crypts

¹ Biomedical Sciences Unit, Department of Biological Sciences, Bailrigg, Lancaster University, Lancaster LA1 4YQ, UK
² Centre for Diabetes and Metabolic Medicine, Queen Mary's School of Medicine and Dentistry, Institute of Cell and Molecular Science, 4 Newark Street, Whitechapel, London E1 2AT, UK
³ Department of Physics, Lancaster University, Bailrigg, Lancaster LA1 4YB, UK
⁴ Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan
⁵ Synchrotron Radiation Department, Daresbury laboratories, Science and Technologies Facilities Council, Warrington WA4 4AD, UK
⁶ European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble, France
⁷ Biomedical Sciences Unit, Department of Biological Sciences, Bailrigg, Lancaster University, Lancaster LA1 4YQ, UK; Sharoe Green Unit, Lancashire Teaching Hospitals, Preston, UK

^* To whom correspondence should be addressed. E-mail: f.martin{at}lancaster.ac.uk.

	Abstract

Complex biomolecules absorb in the mid-infrared ( = 2-20 µm) giving vibrational spectra associated with structure and function. We employed Fourier transform infrared (FTIR) micro-spectroscopy to "fingerprint" locations along the length of human small and large intestinal crypts. Paraffin-embedded slices of normal human gut were sectioned (10-µm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected employing globar (15 µm x 15 µm aperture) FTIR micro-spectroscopy in reflection mode, synchrotron (10 µm x 10 µm aperture) FTIR micro-spectroscopy in transmission mode, or near-field photothermal micro-spectroscopy (PTMS). 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 towards symmetric (_s)PO₂^- (1080 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 -catenin or CD133. This pattern was subsequently confirmed by image mapping and points to a novel approach of non-destructively identifying a tissue's stem cell location. _sPO₂^-, 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.

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