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 (IR) absorbance of cellular biomolecules generates a vibrational spectrum, which can be exploited as a “biochemical fingerprint” of a particular cell type. Biomolecules absorb in the mid-IR (2–20 μm) and Fourier-transform infrared (FTIR) microspectroscopy applied to discriminate different cell types (exfoliative cervical cytology collected into buffered fixative solution) was evaluated. This consisted of cervical cytology free of atypia (i.e. normal; n = 60), specimens categorised as containing low-grade changes (i.e. CIN1 or LSIL; n = 60) and a further cohort designated as high-grade (CIN2/3 or HSIL; n = 60). IR spectral analysis was coupled with principal component analysis (PCA), with or without subsequent linear discriminant analysis (LDA), to determine if normal versus low-grade versus high-grade exfoliative cytology could be segregated. With increasing severity of atypia, decreases in absorbance intensity were observable throughout the 1,500 cm−1 to 1,100 cm−1 spectral region; this included proteins (1,460 cm−1), glycoproteins (1,380 cm−1), amide III (1,260 cm−1), asymmetric (νas) PO2− (1,225 cm−1) and carbohydrates (1,155 cm−1). In contrast, symmetric (νs) PO2− (1,080 cm−1) appeared to have an elevated intensity in high-grade cytology. Inter-category variance was associated with protein and DNA conformational changes whereas glycogen status strongly influenced intra-category. Multivariate data reduction of IR spectra using PCA with LDA maximises inter-category variance whilst reducing the influence of intra-class variation towards an objective approach to class cervical cytology based on a biochemical profile.
Stem cells have great potential in clinical medicine. Sensitive methods for stem cell identification are a requirement for the development of medical interventions involving these cells. To date, a definitive stem cell marker has not been discovered. We are exploring the use of photothermal microspectroscopy (PTMS) for the purpose of stem cell characterisation and identification in human corneal epithelium. PTMS measures heat fluctuations associated with infrared radiation absorption. The technique is advantageous over existing Fourier transform infrared (FTIR) spectroscopy methods in having a spatial resolution which is not diffraction limited, thus allowing examination at a subcellular scale. PTMS measurements are unaffected by IR opacity of the sample, giving the method a further edge in comparison to FTIR spectroscopy. We show that PTMS spectra can be used for the characterisation of stem cells and differentiated cells in the human corneal stem cell model. We demonstrate for the first time that PTMS spectra derived from these cell types segregate into separate data clusters after principal component analysis. The predominant wavenumbers responsible for this separation appear to be associated with nucleic acid structure and function. PTMS offers great promise as a technique for stem cell identification in tissue samples where spatial resolution at the cellular scale or better is required.
SummaryThe identification of stem cells in adult tissue is a challenging problem in biomedicine. Currently, stem cells are identified by individual epitopes, which are generally tissue specific. The discovery of a stem-cell marker common to other adult tissue types could open avenues in the development of therapeutic stem-cell strategies.We report the use of the novel technique of Fourier transform infrared near-field photothermal microspectroscopy (FTIR-PTMS) for the characterization of stem cells, transit amplifying (TA) cells and terminally differentiated (TD) cells in the corneal epithelium. Principal component analysis (PCA) data demonstrate excellent discrimination of cell type by spectra. PCA in combination with linear discriminant analysis (PCA-LDA) shows that FTIR-PTMS very effectively discriminates between the three cell populations. Statistically significant differences above the 99% confidence level between IR spectra from stem cells and TA cells suggest that nucleic acid conformational changes are an important component of the differences between spectral data from the two cell types.FTIR-PTMS is a new addition to existing spectroscopy methods based on the concept of interfacing a conventional FTIR spectrometer with an atomic force microscope equipped with a near-field thermal sensing probe. FTIR-PTMS spectroscopy currently has spatial resolution that is similar to that of diffraction-limited optical detection FTIR spectroscopy techniques, but as a near-field probing technique has considerable potential for further improvement. Our work also suggests that FTIR-PTMS is potentially more sensitive than synchrotron radiation FTIR spectroscopy for some applications.
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