No abstract

Complex biomolecules absorb in the mid-infrared ( ‫؍‬ 2-20 m), 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. Paraffinembedded slices of normal human gut were sectioned (10 m thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 m ؋ 15 m aperture) FTIR microspectroscopy in reflection mode, synchrotron (<10 m ؋ 10 m 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 ( 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 ␤-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. 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.

Infrared microspectroscopic imaging data of single human prostate cancer cells, on an artificial extracellular matrix (Matrigel) thin-film surface, are presented. The spectral intensity maps, obtained in reflection mode, appear to show that the protein intensity distribution observed at the location of a cell changes dramatically depending on the concentration and/or thickness of the underlying Matrigel layer. Specifically, cells adhered to a low protein concentration or thin surface exhibit a higher protein intensity signal than the surrounding layer whereas those on a high protein concentration or thick surface exhibit a lower protein intensity signal. These results are qualitatively explained by a simple model that takes into account the fact that radiation scattered from cells can enter the collection optics of the microscope without passing through the Matrigel layer. This leads to an apparent reduction in absorption at the cell.

Markers of gastrointestinal (GI) stem cells remain elusive. We employed synchrotron Fourier-transform infrared (FTIR) microspectroscopy to derive mid-infrared (IR) spectra along the length of human GI crypts. Tissue sections (10-μm thick) were floated onto BaF2 windows and image maps were acquired of small intestine and large bowel crypts in transmission mode with an aperture of ≤10 μm×10 μm. Counting upwards in a step-size (≤10 μm) fashion from the crypt base, IR spectra were extracted from the image maps and each spectrum corresponding to a particular location was identified. Spectra were analyzed using principal component analysis plus linear discriminant analysis. Compared to putative crypt base columnar/Paneth cells, those assigned as label-retaining cells were chemically more similar to putative large bowel stem cells and, the small intestine transit-amplifying cells were closest to large bowel transit-amplifying cells; interestingly, the base of small intestine crypts was the most chemically-distinct. This study suggests that in the complex cell lineage of human GI crypts, chemical similarities as revealed by FTIR microspectroscopy between regions putatively assigned as stem cell, transit-amplifying and terminally-differentiated facilitates identification of cell function.