Complex biomolecules absorb in the mid-infrared ( = 2-20 m) 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-m thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected employing globar (15 m 15 m aperture) FTIR micro-spectroscopy in reflection mode, synchrotron (10 m 10 m 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 between cells in situ 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 of crypts. This was subsequently confirmed by image mapping and points to a novel approach of deriving an integrated biochemical fingerprint of a tissue’s stem cell niche and identifying its spatial location in a non-destructive fashion. These results suggest that DNA conformational alterations associated with cells residing in the putative stem cell region of crypts can be used as a means of identification, which may have utility in other tissues where the location of the niche is unclear.