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J.R. Beattie, S. Brockbank, W.J. Curry, J.J. McGarvey; Preliminary Investigation on the Use of Raman Microscopy to Characterise Porcine Retina Sections . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5505.
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Raman microscopy is a powerful spectroscopic method for analysing variation in multiple biochemicals on a micron scale of spatial resolution. Extensive use of resonance enhanced Raman (RRS) has been employed in the study of carotenoid content in the macula but there is no significant body of work on the application of non resonant Raman spectroscopic methods to retinal tissues. The aim of this work is to highlight the extent of the information that can be extracted from the Raman microscopic analysis of retinal sections.
Porcine retina was fixed in paraformaldehyde and stored in 5% sucrose/PBS prior to sectioning (20 µm). The Raman data were recorded using a Jobin–Yvon LabRam HR equipped with a 0.1 µm step xy stage and using 633 nm excitation.
Principal Component Analysis (PCA) was employed to identify the main biochemical components within each layer within the retina sections. Restricting the results to the most important variable within each layer yielded a total of 11 biochemical constituents, some of which are shown in the figure below. It was found that the outer layers each had distinct spectral signals, with lipid signals found in the POS, cytochrome c in the PIS and DNA in the ONL. Distinct protein signals were also observed for each of these layers. The signal from the INL was found to show lower intensity for DNA and the presence of another lipid which was more saturated than the lipid found in the POS. A common protein signal was found to extend from the OPL through to the GCL, which would match the distribution of Muller cells. The lipid signal found in the INL was also found within the other inner layers. Strong signals due to haemoglobin were found distributed in a vertical line through the GCL and IPL, suggesting that the sectioning process had cut through a vertical capillary.
The study demonstrated that the Raman method is capable of simultaneously analysing the concentration and properties of multiple biochemical components of the retina. The authors would like to thank the BBSRC, R&D office (NI), British Retinitis Pigmentosa Society and Frazer Foundation
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