Synchrotron FTIR maps, focal plane array and linear array images recorded of 4 μm cervical biopsy sections from the surface epithelium and glandular endometrium are compared in terms of spatial resolution and applicability to the clinical environment. Synchrotron FTIR maps using a 10 μm aperture appear to provide a better spatial resolution capable of discerning single nuclei in the tissue matrix. Unsupervised hierarchical cluster analysis performed on the synchrotron, focal plane array and linear array data in the 1700-1400 cm-1 region show very similar clusters and mean-extracted spectra, demonstrating the robustness of FTIR microscopy and UHCA in the analysis of tissue sections. Maps recorded with the focal plane array using a conventional globar source take one-fortieth of the time but the spatial resolution precludes true single cell analysis in the tissue matrix. The high spatial resolution achieved with the synchrotron shows potential as a gold standard for FTIR diagnosis of cervical samples.
With the advent of Raman spectrometers based on CCD array detectors, instruments have been coupled to optical microscopes leading to all the advantages of bright field microscopy with the added advantage of a direct chemical probe. The primary biological solvent, water, is a weak Raman scatterer and so these instruments can now be used to investigate the chemistry of living systems at spatial resolutions of 1 μm and below. We have developed techniques that allow us to study functional red blood cells and monitor the exchange of ligands and the development and chemistry of disease processes. These techniques take advantage of Aggregated Enhanced Raman Spectroscopy, which enables us to use the haem group of the haemoglobins and related haem pigments, such as the malarial pigment haemozoin, as a sensitive probe for changes in oxidation state, spin state and electronic structure. We have used the Raman microprobe to investigate the effect of drugs such as quinoline on the food vacuole of the malarial parasite in vivo. Sickle cell disease affects 1 out of 600 African American births and is caused by a mutant form (β6 glu→val) of haemoglobin (HbS). HbS polymerizes and forms higher order aggregates under hypoxic conditions, leading to distortion and rigidity of the erythrocyte. These rigid cells can block the microvasculature resulting in tissue ischaemia, organ damage, and ultimately death. The sensitivity of the Raman technique to haem aggregation provides a tool with which we can analyse the changes that occur between normal and sickle cells.
The oxygenation process of a human erythrocyte is monitored using a Raman microimaging technique. Raman images of the 1638 cm–1 band are recorded in the oxygenated and deoxygenated state using only 120 s of laser exposure and ~1 mW of defocused laser power. The images show hemoglobin oxygenating and deoxygenating within the cell. Prolonged laser imaging exposure (<180 s) at low temperatures results in photoinduced and/or thermal degradation. The effect of thermal degradation is investigated by recording spectra of erythrocytes as a function of temperature between 4 and 52°C. Five bands at 1396, 1365, 1248, 972, and 662 cm–1 are identified as markers for heme aggregation. Raman images recorded of cells after prolonged laser exposure appear to show heme aggregation commencing in the middle and moving toward the periphery of the cell. UV-visible spectra of erythrocytes show the Soret band to be broader and red shifted (~3 nm) at temperatures between 45 and 55° indicative of excitonic interactions. It is postulated that the enhancement of the aggregation marker bands observed at 632.8-nm excitation results primarily from excitonic interactions between the aggregated hemes in response to protein denaturation. The results have important medical implications in detecting and monitoring heme aggregation associated with hemopathies such as sickle cell disease.