James M. Nicholson

Fourier Transform Infrared Microspectroscopy Identifies Symmetric PO2− Modifications as a Marker of the Putative Stem Cell Region of Human Intestinal Crypts

Complex biomolecules absorb in the mid‐infrared (λ = 2–20 μm), 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 μm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 μm × 15 μm aperture) FTIR microspectroscopy in reflection mode, synchrotron (≤10 μm × 10 μm 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 (νs)PO2− (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 β‐catenin or CD133. This pattern was subsequently confirmed by image mapping and points to a novel approach of nondestructively identifying a tissues stem cell location. νsPO2−, 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.

FTIR micro-spectroscopy identifies symmetric PO2- modifications as a marker of the putative stem cell region of human intestinal crypts.

Complex biomolecules absorb in the mid‐infrared (λ = 2–20 μm), 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 μm thick) and mounted to facilitate infrared (IR) spectral analyses. IR spectra were collected using globar (15 μm × 15 μm aperture) FTIR microspectroscopy in reflection mode, synchrotron (≤10 μm × 10 μm 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 (νs)PO2− (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 β‐catenin or CD133. This pattern was subsequently confirmed by image mapping and points to a novel approach of nondestructively identifying a tissues stem cell location. νsPO2−, 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.

Tracking the cell hierarchy in the human intestine using biochemical signatures derived by mid-infrared microspectroscopy

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.

Identification of reaction compounds in micrometric layers from gothic paintings using combined SR-XRD and SR-FTIR

Synchrotron radiation X-ray diffraction (micro-SR-XRD) and Fourier transform infrared spectroscopy (micro-SR-FTIR) are used in the non-destructive identification of reaction and aging compounds from micrometric ancient painting layers. The combination of the micrometer size and non-destructive nature of the techniques together with the high resolution and brilliance of the synchrotron radiation has proved to be a procedure most advantageous for the study of reaction, aging and degradation processes. Copper, lead and calcium carboxylates and oxalates are determined in the chromatic, preparation and alteration layers from 15th century egg tempera and oil paintings. Their nature and crystallinity have been assessed. Some hypothesis about the mechanisms of development of both carboxylates and oxalates are presented.

Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: An FTIR microspectroscopy study

Batch cultures of Chlamydomonas reinhardtii were used to study carbon allocation in relation to growth phase and phosphorus availability. Cultures were grown at initial phosphorus (PO4-P) concentrations of 500 µg l−1 (high-P) and 50 µg l−1 (low-P). Cellular carbon allocation was monitored using Fourier transform infrared (FTIR) microspectroscopy with the ratio of the band intensities at 1736 cm−1 (lipid) and the 1180–950 cm−1 region (carbohydrate) to 1652 cm−1 (amide I) used as an index of changing carbon balance. Cellular phosphorus concentrations (P quota) were measured by energy dispersive X-ray microanalysis (EDXRMA). Both treatments entered stationary phase on day 18. Increased cell counts in the high-P treatment (max. 3.0 × 106 cells ml−1 at stationary phase) led to a rapid decrease in external P availability to <20 µg l−1 during early log phase, with a subsequent decrease in P quota from 0.5% to <0.1% DW. The fall of P quota to <0.1% led to an increase in the lipid/protein ratio (0.13 to 0.23) and carbohydrate/protein ratio (0.37 to 1.57), with ratios increasing further (lipid:protein 1.85; carbohydrate:protein 2.77) in late stationary phase. In the low-P treatment external P concentrations (<20 µg l−1 from day 1) restricted population growth (max. 0.75 × 106 cells ml−1 at stationary phase). P quotas fell to <0.1% in early log phase, with the carbohydrate/protein ratio increasing from 0.15 to 3.7 and remaining high into stationary phase while the lipid/protein ratio increased from 0.2 to 1.2. In both treatments increasing synthesis of lipid and carbohydrate storage products resulted in an increased cell volume. Transfer of P-deficient cells (late stationary) to fresh media led to a rapid stimulation of growth, a rapid reduction in lipid/protein and carbohydrate/protein ratios, and decreased cell volumes.

An investigation of the RWPE prostate derived family of cell lines using FTIR spectroscopy.

Interest in developing robust, quicker and easier diagnostic tests for cancer has lead to an increased use of Fourier transform infrared (FTIR) spectroscopy to meet that need. In this study we present the use of different experimental modes of infrared spectroscopy to investigate the RWPE human prostate epithelial cell line family which are derived from the same source but differ in their mode of transformation and their mode of invasive phenotype. Importantly, analysis of the infrared spectra obtained using different experimental modes of infrared spectroscopy produces similar results. The RWPE family of cell lines can be separated into groups based upon the method of cell transformation rather than the resulting invasiveness/aggressiveness of the cell line. The study also demonstrates the possibility of using a genetic algorithm as a possible standardised pre-processing step and raises the important question of the usefulness of cell lines to create a biochemical model of prostate cancer progression.

Derivation of a subtype-specific biochemical signature of endometrial carcinoma using synchrotron-based Fourier-transform infrared microspectroscopy

Endometrial carcinoma consists of endometrioid (type I) and serous papillary (SP; type II) subtypes; a rarer form is malignant mixed müllerian tumours (MMMT; type II/mixed). We set out to determine whether one might be able to biochemically signature these subtypes using Fourier-transform infrared (FTIR) microspectroscopy and distinguish non-tamoxifen associated from tamoxifen-associated cases. Paraffin-embedded blocks were obtained from non-tamoxifen associated cases reported as endometrioid (n=7), SP (n=4) or MMMT (n=4). From tamoxifen-associated cases, endometrioid (n=1), SP (n=3) and MMMT (n=4) blocks were retrieved; benign tissues (n=3) were also analysed. Exploiting synchrotron-based radiation, sections (10-microm thick) on BaF(2) windows were interrogated through a 10 microm x 10 microm aperture. Point spectra were derived from >or=10 locations in each of six glandular elements per tissue; a further 20 stromal spectra were obtained. Following normalisation to Amide I, average spectra (1800-900 cm(-1)) per gland or stroma were analysed for variance using principal component analysis (PCA) and linear discriminant analysis (LDA). In scores plots, segregation of spectra from different subtypes or benign tissues was noted and it proved possible to distinguish tamoxifen-associated cases. In the PCA-LDA loadings plots, the wavenumbers that highlighted variance for benign or endometrioid carcinoma tissues were in the protein region (1800-1480 cm(-1)) whereas those contributing most to SP or MMMT segregation were primarily in the DNA/RNA region (1425-900 cm(-1)) of the vibrational spectrum. Our results suggest that the application of FTIR microspectroscopy is a powerful new approach in disease diagnosis and characterisation.

Rapid characterisation of archaeological midden components using FT-IR spectroscopy, SEM-EDX and micro-XRD

Samples taken from middens at the Neolithic site of Catalhöyük in Turkey have been analysed using IR spectroscopy backed up by powder XRD and SEM-EDX. Microcomponents studied include fossil hackberries (providing evidence of ancient diet and seasonality), mineral nodules (providing evidence of post-depositional change) and phytoliths (mineralised plant cells, providing evidence of usage of plant species). Finely laminated ashy deposits have also been investigated allowing chemical and mineralogical variations to be explored. It is found that many layers which appear visually to be quite distinctive have, in fact, very similar mineralogy.

Molecular response of Anabaena flos-aquae to differing concentrations of phosphorus: A combined Fourier transform infrared and X-ray microanalytical study

The response of Anabaena flos‐aquae to varying levels of P availability was determined using the combined techniques of Fourier transform infrared (FTIR) microspectroscopy and energy‐dispersive X‐ray microanalysis (EDXRMA). Batch cultures of Anabaena were grown at initial P concentrations of 50 μg (low‐P culture), 500 μg (intermediate‐P) and 5000 μg (high‐P) (PO4‐P L−1) and were monitored for total biovolume, chlorophyll a, media nutrients (PO4‐P and NO3‐N), cellular P quota (EDXRMA) and carbon allocation (FTIR spectroscopy). The high‐P culture showed a sixfold increase in total biovolume over the sampling period. Phosphorus in the media remained at more than ∼4000 μg L−1 and intracellular P concentration (P quota) as determined by EDXRMA showed no decline, remaining at more than 0.20% dry weight (DW). The intermediate‐P culture showed a similar increase in total biovolume, but P concentrations in the media fell to ∼20 μg L−1, and this was reflected in a decline in the cellular P quota from 0.24% to 0.06% DW. Although the high‐ and intermediate‐P treatments differed markedly in both internal and external P, analysis of the FTIR spectra from the two treatments, using both hierarchical cluster analysis (HCA) and principal component analysis (PCA), indicated no difference in carbon allocation. Cells from the low‐P treatment showed strong evidence for P deficiency. P concentrations in the media were undetectable (<5 μg L−1), total biovolume was much reduced, and P quotas were low, falling from 0.08% to 0.01% DW. HCA and PCA clearly separated FTIR spectra from low‐P cells from those of intermediate‐ and high‐P cultures, with low‐P cells having increased absorbance in the region of the spectra associated with carbohydrate. Both FTIR spectroscopy and EDXRMA have the resolution to study the elemental and macromolecular composition of single species within mixed phytoplankton populations and the combined use of these techniques has considerable potential for the study of cyanobacterial responses to environmental stress.

Changing patterns of carbon allocation in lake phytoplankton: an FTIR analysis

Fourier transform infra-red (FTIR) spectroscopy was used to assess carbon allocation in selected phytoplankton (Pediastrum duplex, Ceratium hirundinella, Anabaena flos-aquae) in a eutrophic lake. In contrast to bulk-analysis techniques, FTIR spectroscopy can provide information on carbon allocation at the species level within natural mixed populations. Carbon allocation was determined using lipid/protein (L/P) and carbohydrate/protein (C/P) ratios and multivariate analysis of complete spectra and showed considerable intraspecific heterogeneity within samples dates, and clear seasonal changes in the spectra of Pediastrum and Anabaena, with both alga showing increased L/P and C/P ratios on selected sampling dates. Although increased ratios can indicate nutrient deficiency, the alga were not nutrient deficient during these periods, as indicated by continued population growth and high (>0.1%) internal phosphorus concentrations. FTIR spectra from Ceratium did not show any clear seasonal change. In all three algae, the transition from population growth to decline was not accompanied by any significant change in ratios, nor were low epilimnetic nutrients correlated with increased ratios, marking a significant contrast to laboratory batch cultures (previous studies) where low nutrients and the transition to stationary phase led to marked spectral changes.