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Danielle J. Harper, Martin Glösmann, Marion Gröger, Bernhard Baumann; Spectral reflectivity of the ex vivo mouse retina revealed by hyperspectral confocal microscopy. Invest. Ophthalmol. Vis. Sci. 2019;60(11):017.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the reflectivity of the ex vivo C57BL/6 mouse retinal layers as a function of wavelength.
The cornea and lens of a C57BL/6 mouse were removed from the eye and radial cuts were made to flatten the eyecup. The neural retina was lifted off from the retinal pigment epithelium (RPE), which was still attached to the underlying choroid and sclera, and both tissues were whole mounted in 70% glycerol and coverslipped. Both neural retina samples and RPE/choroid samples were imaged using a white light laser confocal microscope (Leica TCS SP8) in reflection mode with a 20x multi immersion objective. The samples were illuminated consecutively with light from 470 – 670 nm in 10 nm intervals throughout the whole sample at 5 μm intervals in depth. The hyperspectral spectra were then analyzed pixel by pixel in order to be able to characterize the wavelength dependent spectra in each retinal layer. Each image was color-corrected to account for the scattering/absorption which took place in the previous layers, using the dataset above as a reference. For visualization purposes (Fig. 1), the data were combined into an RGB image, with R = 610 – 670 nm, G = 540 - 600 nm and B = 470 - 530 nm.
The retinal nerve fiber layer/ganglion cell layer (RNFL/GCL) shows a heightened reflectivity in the shorter wavelengths. From the inner plexiform layer to the outer nuclear layer, the neural mouse retina displays a relatively constant reflectivity profile across the investigated wavelength range. In the RPE, the hexagonal-shaped cell walls also do not show a wavelength dependent reflectivity. The RPE melanin, however, has a very low reflectivity in the shorter wavelengths, resulting in a brownish appearance in the color representation (Fig. 1G).
The RNFL and the melanin in the RPE are the two regions in the ex vivo mouse retina which show largely wavelength dependent reflectivity. Since in vivo imaging methods are often later correlated to histology, this could give an insight into the accuracy of some in vivo spectroscopic imaging techniques (e.g. spectroscopic optical coherence tomography), and allow for corrections to be made in order to reconstruct the true in vivo reflectivity profiles.
This abstract was presented at the 2019 ARVO Imaging in the Eye Conference, held in Vancouver, Canada, April 26-27, 2019.
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