Raman Spectroscopy of Retina


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schematic saggital section human eye

The retina is the photosensitive tissue that lies at the rear of the eye, supported by the vitreous in front and the RPE/ Bruch's membrane behind (Figure 1). This tissue is comprised of three layers of highly organized cells, and each part of the cells are arranged in a highly structured arrangement, which results in a distinctive pattern of layers within the tissue, as illustrated in Figure 2. The retina is subdivided in several ways, the crudest being a simple division into two halves, the outer layers being those at the rear of the eye and the inner layers towards the front. The retina is three cells deep, with the rearmost being responsible for detecting the light falling on the retina. The intermediate and foremost layers are responsible for signal amplification, crude image processing and as a conduit for the nerve impulses to the optic nerve. The cells themselves are very highly structured with nuclei forming uniform bands in the two outmost layers and cell synapses forming a uniform band where each layer of cells meet. The photoreceptors take this to the extreme, even segmenting the energy production into a distinct band from the photosensitive disks.

schematic cross section human retina
Raman spectra retina DHA heme kyneurinine cytochrome c DNA

The Raman data obtained from the retina are complex and contributed to by 50 separate biochemicals, some of which are shown in Figure 3. The biochemicals detected included heme (haemoglobin), dodecahexenoic acid (DHA) cytochrome c, DNA, kyneurinine and four different proteins and a monounsaturated fatty acid. The Raman maps in Figure 4 show the distribution of some of the selected signals. Fatty acids were found to be localised most strongly in the photoreceptor outer segments, with little present in the remaining outer layers then a diffuse distribution in the inner layers. The composition of the fatty acid was radically different between the photoreceptor outer segments and the inner layers, with the former exhibiting a much larger degree of unsaturation consistent with the relatively high proportion of DHA in the photo-sensitive disks. The DNA formed two distinct bands called the outer and inner nuclear layers while the third layer of cells did not have its nuclei localised to a distinct band. The DNA is present in a much lower concentration in the inner nuclear layer as the cell density is much lower. The heme signal was highly localised to the capillaries that extend upwards through the ganglion cell layer. Cytochrome c was localised to the inner segments of the photoreceptor layer as these are the engine rooms that provide the metabolic energy that drives the detection of light.

Raman spectroscopy distribution map retina heme cytochrome c dna protein fatty acid dha

This foundational work is important for progressing to pathologies where the underlying biochemistry of the eye is disrupted. We are currently working on assessing retinas from retinitis pigmentosa using the Raman. Retinitis pigmentosa is a degenerative genetic group of diseases that, though a variety of mechanisms, cause photoreceptor cell death in the periphery of the retina. This leads to loss of vision around the central field creating tunnel vision and night blindness (the more sensitive rod cells are thos found in the periphery). Figure 5 presents some preliminary results from this study, namely a principal component score plot comparing normal and RPRaman spectroscopy retina retinitis pigmentosa principal component analysis score plot photoreceptor layers. Principal component analysis separates out data based on how different they are, with the consequence that the closer two samples are the more alike, and conversely the further apart the more different. It is clear from the PC score plot that the RP retina is completely different from the normal retina as there is a clear separation between the two with no overlap. Furthermore, the normal retina shows a clear trend with depth into the retina, as would be expected in a layered sample. In contrast the RP retina does not show consistent variation with depth, suggesting a much more disordered structure. It is anticipated that when completed the study will provide insight into the biochemical malfunctions involved in RP.

1.            Beattie, J. R., Brockbank, S., McGarvey, J. J., and Curry, W. J. (2007) Raman microscopy of porcine inner retinal layers from the area centralis. Molecular Vision 13, 1106-1113

2.            Beattie, J. R., Brockbank, S., McGarvey, J. J., and Curry, W. J. (2005) Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis. Molecular Vision 11, 825-832

 

British Retinitis Pigmentosa Society


   

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