Molecular Vision 2005; 11:825-832 <http://www.molvis.org/molvis/v11/a98/> Received 18 January 2005 | Accepted 24 July 2005 | Published 30 September 2005

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Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis

J. Renwick Beattie,^1,2 Simon Brockbank,³ John J. McGarvey,^1,2 William J. Curry³
 
 

¹School of Chemistry and ²Center for Clinical Raman Microscopy, Faculties of Medicine and Science and Agriculture, Queen's University Belfast, Belfast, Northern Ireland; ³Ophthalmic Research Center, Institute of Clinical Science, Queen's University Belfast, Belfast, Northern Ireland

Correspondence to: Professor John J. McGarvey, School of Chemistry, Queen's University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK; Phone: +442890975450; FAX: +442890974890; e-mail: j.mcgarvey@qub.ac.uk

Abstract

Purpose: Raman microscopy, based upon the inelastic scattering (Raman) of light by molecular species, has been applied as a specific structural probe in a wide range of biomedical samples. The purpose of the present investigation was to assess the potential of the technique for spectral characterization of the porcine outer retina derived from the area centralis, which contains the highest proportion of cone:rod cell ratio in the pig retina.

Methods: Retinal cross-sections, immersion-fixed in 4% (w/v) PFA and cryoprotected, were placed on salinized slides and air-dried prior to direct Raman microscopic analysis at three excitation wavelengths, 785 nm, 633 nm, and 514 nm.

Results: Raman spectra of each of the photoreceptor inner and outer segments (PIS, POS) and of the outer nuclear layer (ONL) of the retina acquired at 785 nm were dominated by vibrational features characteristic of proteins and lipids. There was a clear difference between the inner and outer domains in the spectroscopic regions, amide I and III, known to be sensitive to protein conformation. The spectra recorded with 633 nm excitation mirrored those observed at 785 nm excitation for the amide I region, but with an additional pattern of bands in the spectra of the PIS region, attributed to cytochrome c. The same features were even more enhanced in spectra recorded with 514 nm excitation. A significant nucleotide contribution was observed in the spectra recorded for the ONL at all three excitation wavelengths. A Raman map was constructed of the major spectral components found in the retinal outer segments, as predicted by principal component analysis of the data acquired using 633 nm excitation. Comparison of the Raman map with its histological counterpart revealed a strong correlation between the two images.

Conclusions: It has been demonstrated that Raman spectroscopy offers a unique insight into the biochemical composition of the light-sensing cells of the retina following the application of standard histological protocols. The present study points to the considerable promise of Raman microscopy as a component-specific probe of retinal tissue.

Introduction

The Raman effect, which is the basis of Raman spectroscopy and microscopy, arises from the scattering of light by matter. When a beam of monochromatic light is directed onto a sample, most of the light is scattered elastically. However, a tiny fraction of the scattered light (0.1% or less) will differ in color from that of the incident beam, hence giving rise to inelastic scattering. The differences in color between the incident and the inelastically scattered beams, more conveniently expressed as frequency differences, correspond to the frequencies at which chemical bonds of the molecules in the sample typically vibrate. The important implication of this is that each molecular species possesses its own group of bond vibrations, and the collection of vibrational frequencies for a particular species constitutes a unique spectrum, which is in effect a "fingerprint" of that species.

Studies on a wide range of biological and biomedical samples have demonstrated that Raman spectroscopy has the capacity to determine the secondary structure and composition of proteins [1] and the chemical and physical characteristics of lipids [2], DNA [3] and saccharides [4]. Conventional ("ground state") Raman spectroscopy generally does not enhance minor components above the bulk media, consequently it is useful in studying the dominant (in terms of percent by weight) proteins, lipids, saccharides, and nucleotides encountered in a wide range of tissues. However, when an excitation wavelength matching a UV/Visible absorption band is used, the Raman signal for the vibrational modes of the chemical groups giving rise to the absorption band is greatly enhanced, increasing the sensitivity of Raman spectroscopy. This enhanced Raman technique, known as Resonance Raman Spectroscopy (RRS), allows the detection of certain minor components even in the presence of a dominant matrix.

The coupling of Raman spectroscopy with optical microscopy (Raman microscopy) allows the investigation of the spatial variation on a micrometer scale of the vibrational spectrum (and thereby the study of the chemical and physical variation) within a sample. This coupling of techniques is both a useful addition to the tools at a biochemist's disposal, and also a powerful analytical tool with highly reproducible sensitivity and selectivity. Raman microscopy has been applied to a wide range of biomedical samples and has been used in pathology, histology and cytochemistry, among other fields [5]. Pathological analysis of the eye, in particular the externalized cornea and interior ocular fluids and tissues, is a viable target for in situ Raman spectroscopy analysis [6], while RRS has been extensively used to study carotenoids in the retina [7]. Although there has been limited application of ground state Raman spectroscopy to the overall analysis of retinal tissue, there has been a considerable range of other tissues studied using Raman microscopy. For instance, it has been demonstrated that Raman microscopy is an effective tool in biochemical mapping of tissues, allowing the spatial resolution of the constituents of samples such as breast tissue [8,9]. A unique aspect of Raman microscopic mapping allows the simultaneous measurement of a diverse range of biomolecules (e.g., proteins, lipids, DNA, carbohydrates, and vitamins) in situ within normal and pathological specimens. A primary objective of the present study was to exploit these unique features of Raman microscopy to initiate Raman spectral characterization of normal porcine retina and in particular the area centralis, which exhibits a rod:cone ratio comparable to that of the peripheral retina in human [10].

Methods

Sample preparation

Porcine eyes were enucleated at the abattoir within 30 min of death and transported to the laboratory in ice and immersion fixed in buffered 4% (w/v) paraformaldehyde (24 h, 4 °C), prior to initial cryoprotection in 5% (w/v) sucrose/phosphate buffered saline (PBS, 0.05 M Na phosphate, 0.14 M NaCl, pH 7.4) with storage in 30% (w/v) sucrose/PBS containing 0.01% (w/v) sodium azide. Cryostat-derived retinal cross-sections (20 μm) were prepared from the area centralis, which contains the highest proportion of cone cells in the pig retina [11]. The Raman measurements were repeated on two independent samples.

Cytochrome c

Since the basal regions of the photoreceptor inner segments possess a high density of mitochondria, the Raman spectrum of cytochrome c was evaluated using horse heart cytochrome c (gel filtration chromatography grade, Sigma Chemicals, St. Louis, MO). Crystals of the material were simply presented to the Raman microscope on a glass microscope slide, straight from the vial.

Rhodopsin extraction

Porcine eyes were enucleated at the abattoir within 30 min of death and transported to the laboratory in ice. The retinas (n=40) were dissected on an ice stage under dim red light and any retinal pigmented epithelium/choroid contamination removed by washing in chilled 0.1 M PBS containing 0.5 mM EDTA. The retinas were placed in 0.73 M sucrose in 0.1 M sodium phosphate buffer and mixed by shaking prior to filtration using 85 μm nylon mesh (Lockertex, Warrington, England) and rod outer segments (ROS) were isolated by sucrose (Fisher Chemicals, Loughborough, Leicestershire, UK) density step-gradient centrifugation (adapted from Kennedy [12]). Briefly, sucrose density steps consisted of 0.8 M, 1.0 M, and 1.2 M sucrose in 0.1 M phosphate buffer. The retinal suspension was layered on top of the gradient and centrifuged (L8-55 M Ultracentrifuge; Beckman, High Wycombe, Buckinghamshire, UK) at 60,000x g for 1 h at 4 °C. The band that contained the rod outer segments was collected from the interface between the 0.8 M and 1.0 M sucrose steps. The isolated rod outer segments were diluted in phosphate buffer (0.1 M sodium phosphate, pH 7.4) and pelleted by centrifugation at 27,000x g for 20 min at 4 °C. The pellet was resuspended in phosphate buffer and pelleted by centrifugation at 12,000x g for 20 min. This was repeated two more times to generate a crude rhodopsin preparation.

Purification of rhodopsin

Prior to high performance liquid chromatography (HPLC) the crude rhodopsin preparation was solubilized under dim red light in 0.06 M sodium phosphate pH 6.5 with 0.15 M sodium chloride and 30 mM octylglucoside (Sigma-Aldrich, Bookham, Surrey, UK). A Waters Breeze HPLC system (Model 1525) incorporating a Waters dual wavelength absorbance detector (Model 2487) and inline degasser was employed for the purification, which was carried out under dim red light at 20 °C, following the procedure described by Delucas and Moccio [13]. Crude rhodopsin (25 μl) was injected onto a TosoHass TSK-Gel G 4000 SW column (30 cmx21.5 mm inner diameter, 13 μm particle size; Supelco, Poole, Dorset, UK) at a flow rate of 0.5 ml/min with a pressure of 250 psi with an elution buffer consisting of 0.06 M sodium phosphate (pH 6.5) with 0.15 M sodium chloride and 30 mM octylglucoside. Absorbances (A) of eluents were simultaneously measured at wavelengths of 278 nm and 498 nm. The rhodopsin purity was determined from the absorbance ratio, A₂₇₈/A₄₉₈. The measured ratio was 1.61, indicating high rhodopsin content. The high purity of rhodopsin resulting from the Delucas and Moccio procedure [13] obviated the need for SDS-PAGE analysis to establish protein purity.

Raman microscopy

The Raman spectra were recorded using a Jobin-Yvon LabRam HR800 Raman microspectrometer. The spectra were generated from sections of cone rich retina (area centralis) using 514 (10 mW), 633 (20 mW), or 785 nm (100 mW) lasers as excitation sources, focused on the sample with a 50x objective in a nonconfocal arrangement (confocal hole diameter 800 μm), with the laser focused to about a 3 μm diameter spot. The microspectrometer was fitted with a 1 μm step xyz stage. A 300 groove mm^-1 diffraction grating was used with 785 and 633 nm excitation, while a 600 groove mm^-1 diffraction grating was used in conjunction with 514 nm excitation. Spectral resolutions were 10, 12, and 8 cm^-1 with 514, 633, and 785 nm excitation, respectively. All optical images and spectral maps were recorded and processed using the Labspec software (Jobin-Yvon, Villeneuve d'Ascq, France). Statistical analysis was performed using Simca P8.0 (Umetrics, Umea, Sweden). Raman spectra were acquired at 3 μm spacing intervals for a depth of 80 μm across the retinal layers and for a length of 30 μm parallel to the layers. The maps from each wavelength were recorded sequentially at the same location, with the brightfield image checked between each map, and after the final map, to confirm that the stage had returned to precisely the same position (within 1 μm of the original position). Following Raman data acquisition the tissue section was stained using a standard hemotoxylin and eosin protocol to enable histological assignment of the retinal layers.

The intensity of a Raman spectrum needs to be standardized in some way, often by using a band in the spectrum as an internal standard. In general, the method used to normalize the spectra is dependent on the comparisons being assessed. The phenylalanine band at 1003 cm^-1 (assignments of bands are given in Table 1) is a popular internal standard as it is insensitive to environmental factors, but it is affected by the protein composition and so is most useful when comparing the same protein in different situations. The CH₂ "scissor" band at 1440-1460 cm^-1 (Table 1) is also a much used intensity standard, but is influenced by environment and has contributions from any molecule containing CH_x groups. If there is a large change in the relative proportions of, for example, DNA to protein, then standardising about the scissor band will not reflect the change as simply as a band that is exclusively DNA- or protein-linked in origin. In this study the amide I region was also used as a standard when comparing situations where the DNA/protein ratio did indeed significantly change. The spectra were normalized about the total area of the amide I region, which should approximate to normalizing on the protein content, leaving the main variation as the contribution from DNA.

The total intensity of the spectrum is affected by every factor that affects the Raman spectrum, and so normalizing using the average intensity of the entire spectrum is useful for simultaneously comparing all the changes occurring in the spectrum.

Multivariate analysis

The raw data accumulated in the course of a Raman experiment contain contributions from other optical phenomena that occur at the same wavelength of light. The Raman effect is extremely weak (only about 10^-6 of the incident radiation will be scattered with a Raman shift), so even weak optical phenomena can easily swamp the Raman signal. Since this background is not closely related to the actual Raman effect, the spectral maps were baselined to remove the non-Raman background. The spectra were normalized about the total area of the spectrum. The spectra were assigned to the different retinal layers, based on the histological analysis of the stained sections, and mean-centered. Principal Component Analysis [8,9] was carried out for each layer using Simca P8.0 (Umetrics, Umeâ, Sweden). The scores for the first component of each layer were used to determine the main component spectra. High score and low score spectra were averaged and these average spectra were used to construct a Raman map of component distribution by linear combination in the Labspec software.

Results

Rod and cone photoreceptors are elongated cells, which are anatomically divided into distinct functional domains. The regions of these cells adjacent to the retinal pigmented epithelium are known as the photoreceptor outer segments (POS). This region contains the photoreceptor discs, which array the proteins responsible for the transduction of light into electrical impulses. Electron microscopic analysis of the adjoining region, the photoreceptor inner segments (PIS), reveals a high density of mitochondria, therefore indicating that this region is dedicated to cellular energy production. The nuclei of these cells reside in another anatomically defined layer, the Outer nuclear layer (ONL), which gives rise to axonal projections that form synapses with cells from the inner nuclear layer; this defined layer is called the outer plexiform layer (OPL). The primary spectral data recorded at each of the three wavelengths used for excitation of the Raman scattering from the different regions just described will now be considered.

785 nm excitation

Average unbaselined Raman spectra of each of the ONL, PIS and POS layers of the retina acquired at 785 nm are presented in Figure 1. The spectra are dominated by the vibrational features (hereafter termed "modes") characteristic of proteins and lipids, and only a very minor background was observed in the spectra recorded for the ONL region.

Analysis of the photoreceptor segments revealed a clear difference between the inner and outer domains in the spectroscopic regions sensitive to protein conformation near 1640-1680 and 1200-1300 cm^-1 (known as the amide I and III regions, respectively, see Table 1 for assignments [14]). The amide I region is sharper and centered around 1660 cm^-1 in the POS, with some of the intensity spreading to 1640 and 1680 cm^-1 in the PIS. The region from 1400-1500 cm^-1, which is a region sensitive to CHx groups in the protein backbone, amino acid residues, lipids and carbohydrates, also changes shape, with greater intensity around 1440 cm^-1 in the POS, while the 1470 cm^-1 region is more intense in the PIS. Another noteworthy change occurs in the sharp band around 1003 cm^-1, a band attributed to phenylalanine amino acid residues. The bands at 1660, 1400-1500, and 1270 are regions where fatty acid based lipids are also known to contribute to the observed Raman scattering.

The PIS layer contains only mitochondrial DNA and nuclear derived mRNA, while the ONL region contains the nuclei of all the cell types found in the outer segments. Thus, it is expected that the proportion of DNA in the ONL would be significantly higher than in the PIS, and for this reason the spectra were normalized about the total area of the amide I region to allow approximate normalization on the basis of protein content. Indeed, the average Raman spectrum of the ONL in Figure 1 showed a characteristic series of bands at frequencies comparable to those of the vibrational modes of the nucleotides and the phosphate backbone of DNA and RNA. Bands at 1558, 1490, 1375, 1345, 1095, 787, and 735 cm^-1 clearly increase in the ONL spectra, compared to those of PIS (see labeled peaks in the ONL-PIS difference spectrum in Figure 1). The amide I region displayed minimal change at 1640-50 cm^-1, but a sharp intensity decrease was evident at 1660 cm^-1 and this was balanced by a broad intensity increase in the region centered around 1680 cm^-1 (Figure 1). The amide III region was masked by changes in the DNA/RNA modes.

633 nm excitation

The average 633 nm excited Raman spectra for each of the three layers evident in the histological tissue (Figure 2, below) are presented in Figure 3. The spectra mirror those observed at 785 nm excitation for the amide I region.

A pattern of three main bands can also be seen in the PIS spectra in the region from 1500-1630 cm^-1 and a similar pattern in the 1330-1400 cm^-1 region, which were not observable using 785 nm excitation. These extra bands are apparently the result of resonance enhancement at 633 nm, possibly due to absorption by cytochrome c at this wavelength, which would be expected to give a pattern of three bands in this region. This segment contains a high density of mitochondria, and consequently of the enzyme cytochrome c that is an essential part of the catabolism to produce the energy required by the photoreceptors.

514 nm excitation

The average 514 nm excited Raman spectra for the three layers are arrayed in Figure 4. In general, the ONL and POS spectra closely resemble those recorded at 785 and 633 nm, again suggesting an absence of resonance enhancement in the Raman scattering originating from these layers. However, there was a significant difference in the PIS layer, with the cytochrome c features in the 1500-1630 cm^-1 and 1330-1400 cm^-1 ranges showing larger signal enhancement than at 633 nm, though with a slightly different pattern of band intensities.

The high resolution Raman spectrum of commercial cytochrome c was recorded at each of the three wavelengths (Figure 5). These cytochrome C spectra can be compared with the band patterns observed at each wavelength for the PIS region of the retinal sections (Figure 1, Figure 3, Figure 4), a similar sequence of enhancement being evident as an increase in intensity in proceeding from 785 nm, to 633 nm to 514 nm excitation.

Raman mapping of retinal outer segments

As stated previously, Raman microscopy has been extensively used in the study of tissue biochemistry. Figure 2 shows the distribution (Raman map) of the 6 major spectral components (Figure 6) found in the retinal outer segments, as predicted by principal component analysis (PCA) of the data acquired using 633 nm excitation. The layered structure of the retina is very clear in its spectral distribution, and the banded pattern reflects closely that observed in the histological image, also shown in Figure 2. The distinct spectra associated with each layer are to be expected as each layer has specific biochemical compositions related to its specialist functions. The spectrum of isolated rhodopsin, in Figure 7, shows a distinctly different signal to that of the POS layer (Figure 1, Figure 3, Figure 4).

Discussion

One of the striking observations to emerge from this study is the relative lack of background fluorescence, which is often a problem in Raman spectroscopy, especially of biological specimens. However, a low fluorescence background should not be surprising in the layers of the retina section studied, the function of which would be considerably compromised if their components fluoresced greatly. This would have a significant and detrimental impact on the wavelengths of light interacting with the photoreceptor discs.

At all three wavelengths the three layers studied produce Raman signals dominated by protein vibrational modes, though fatty acid spectra contribute in the POS. The relationship between the Raman shift in the amide regions and the secondary and tertiary structure of proteins is well established [14], with α-helices associated with intensity in the regions around 1640-1655 cm^-1 and 1265 cm^-1 (globular tertiary structure) or 1305 cm^-1 (fibrous tertiary structure) while β-sheet structure imparts intensity around 1665-80 and 1225-35 cm^-1, with less systematically ordered (random) protein structure contributing intensity at frequencies lying between the α-helix and β-sheet regions. Thus, it is possible to assign spectral changes in these regions to variation in the secondary or tertiary structure of proteins.

These observed differences in the Raman spectra for the photoreceptor segments at 785 nm excitation would suggest that the PIS region contains a higher concentration of proteins with β-sheet structure (1670 cm^-1) and fibrous α-helices (1640 cm^-1), while the POS proteins contain greater concentrations of globular α-helices or random structure (1660 cm^-1). A large increase in the band at 1270 cm^-1 in going from the PIS to the POS spectra further confirms the increasing degree of α-helical or random structure. The bands in the POS occur at a position that is at the high end of the range of wavenumbers expected from α helices and the low end of the range of wavenumbers typical of random orientations, making assignment difficult. However, the main proteins expected in the outer segments are opsins that adopt an atypical helical structure with kinks in the α-helical sections as well as sections of 310 and α helices [15], which may account for the band position observed in the Raman spectrum. There are a considerable number of bands that can be attributed to individual amino acid residues, which are sensitive to composition, conformation, and environmental effects. For example the overall spectral profile near 1490 cm^-1, which gives greater intensity in the PIS may arise from a number of sources such as Trp, His, Phe, or DNA. DNA seems unlikely on account of the absence of other features, expected if DNA were present. However, further interpretation of these intensity changes would require much more extensive investigation beyond the scope of this article. The spectrum of the POS appears to be a composite of a protein signal and a fatty acid based lipid (bands at 1660 cm^-1, 1270 cm^-1, and 1440 cm^-1 [2]), which is a characteristic of photoreceptors containing a high proportion of rhodopsin and the highly unsaturated fatty acid dodecahexaneoic acid (DHA).

In the ONL there is no change in the proportion of α-helical content in the nuclear proteins, compared with the proteins in the PIS. However, there was a decrease in the less systematic (random) structure and an increase in β-sheet structure. The subtraction spectrum ONL-PIS (Figure 1) contains mainly bands which occur in regions characteristic of the individual nucleotide bases or phosphate stretching vibrations of the DNA/RNA backbone. The main spectral change in migrating from the PIS to the ONL demonstrates an increased signal due to DNA/RNA, indicative of this nuclear layer.

The detection of cytochrome c, the principal protein of the mitochondrial metabolic pathway, in the PIS spectra (Figure 3, Figure 4) is consistent with the high density of mitochondria. The difference in the pattern of spectral enhancement between 514 and 633 nm excitation is accounted for by different groups of bonds (chromophores) within the porphyrin ring absorbing the incident wavelengths, thereby enhancing the vibrational modes associated with those chromophores.

The POS, the region of phototransduction, is composed of a diverse array of proteins, the primary contributor being opsins such as rhodopsin. Rhodopsin adopts a highly ordered transmembrane conformation in the discs of the POS, which will be disrupted following its isolation from the plasma membrane using detergent based solubilization and chromatographic purification. A further complication is the presence of DHA within the photoreceptors, which has many bands that overlap those in the Raman spectrum of the rhodopsin preparation (Figure 7). For example, the helical protein mode of the rhodopsin coincides with the vibration of the unsaturated bonds in DHA at 1660 and 1270 cm^-1. Thus, extraction of the rhodopsin from the lipids of the POS is required to obtain a spectrum from the rhodopsin alone, but this has the consequence that the membrane lipids which play a major part in determining the physical organization of the protein are removed, as suggested above. In order to maintain consistency in terms of oxidation state and hydration the retinal tissue sections and the rhodopsin preparation were both air dried and exposed to light before measurement.

This study adopted published purification methods which enabled the separation of rhodopsin from bleached opsin and other POS proteins [12,13]. Purity was assessed by observing two maxima in the UV-Vis region: a protein band at approximately 280 nm and the chromophore-associated peak at 498 nm. The absorbance ratio A₂₈₀/A₄₉₈ of pure rhodopsin devoid of opsin is 1.6 [16]; in this study the ratio for the purified rhodopsin was assessed to be 1.61. Sequence analysis of bovine blue, green, and red cone opsins have demonstrated identities of 41%, 38%, and 37%, respectively, with rhodopsin [15]. Consequently, it is probable that unbleached porcine opsins were isolated during this purification process. Nonetheless, in this study the observed ratio (1.61) would suggest that rhodopsin is the primary protein from the HPLC purification.

PCA analysis of the individual layers identified a number of important spectral components which accounted for the variation observed within each layer. Detailed discussion of all these components is beyond the scope of this paper, so only the two major components will be examined. The spectra obtained from PCA are shown (Figure 6), where the high and low spectra correspond to the extremes in the variation of the biochemical composition within each of the layers. The high score spectra represent regions where one biomolecule has its highest contribution, while the other has its lowest. The low score spectrum corresponds to the opposite combination of the two biomolecules. As all the biochemicals contributing to the Raman spectrum are not completely resolved from each other on the 3 μm spatial resolution scale used for spectral acquisition, the spectra identified by PCA do not correspond to pure biochemicals, but rather to mixtures.

The high score spectrum in the POS has a very high intensity at 1660 cm^-1 (Figure 6), which is typical of a highly unsaturated fatty acid such as DHA, while the low score spectrum has a profile more typical of a protein. Thus, the purple regions of the Raman map (Figure 2) can be assigned to DHA, while the red regions can be assigned to a population of photoreceptor proteins. The low score spectrum from the PIS (green in the map) contains a high proportion of a porphyrin, while the high score spectrum (dark blue) contains a higher proportion of another protein population which has a higher β-sheet content than the POS proteins. Thus, the green and dark blue areas can be assigned to cytochrome c and PIS proteins, respectively. The ONL reveals a spectrum containing DNA modes (low score, cyan) and a spectrum containing no DNA modes (high score, yellow). Thus, the cyan areas represent DNA and the yellow areas, a third protein population which is distinct from those in the POS and PIS.

Direct comparison of the Raman map with its histological counterpart (Figure 2) revealed that the respective profiles within POS, PIS, and ONL regions appear to show a strong correlation between the two images. For example the spectra containing high contributions characteristic of DNA (cyan) were localized to the ONL, whereas those characteristic of DHA/photoreceptor proteins were localized to the POS and those attributed to cytochrome c (green) were primarily located in the PIS region. As expected, the DNA decreases beyond the ONL and is replaced by protein signals (yellow) which are distinct from the POS and PIS layers. The profile is possibly attributable to the Müller cell outer limiting membrane, which is located at the interface of the photoreceptor inner segment and photoreceptor outer segment junction.

Conclusion

This study has demonstrated the potential of Raman microscopy to investigate the distribution of proteins and DNA in the POS, PIS, and ONL. The spectral data indicate that 785 nm is an optimum wavelength for investigating the proteins in these retinal layers, while cytochrome c, which is indicative of the PIS, was most enhanced by 514 nm excitation. In addition, 633 nm excitation gave rise to excellent spectra for proteins and at the same time resulted in moderately enhanced cytochrome c spectra, thus enabling the simultaneous study of all proteins, including cytochrome c. Collectively, all three wavelengths employed in this study have been shown to be suitable for investigating the ONL, which contains mainly DNA and nuclear proteins. Finally, the construction of a Raman map provides an informative overview of the complexity of the biomolecules in the outer retina and demonstrates the potential of Raman spectroscopy as a component-specific retinal probe.

Acknowledgements

This research was supported by funds from R&D Office Northern Ireland (Grant No. SPI/2384/03), The British Retinitis Pigmentosa Society and Fraser Foundation, Ophthalmology, QUB. Purchase of the Raman microscope was assisted by funding from BBSRC (JREI grant number 18471). Special thanks are extended toward Mr. John Kennedy and Mr. Terence Archibald of Stevenson & Co., Cullybackey, County Antrim, Northern Ireland, for the collection of porcine ocular tissues.

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