High resolution MALDI imaging mass spectrometry of retinal tissue lipids

Abstract

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism's surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4(-/-) knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.

Figure 1

H&E stain of C57 mouse retinal tissue showing the multi layered structure of mouse retinal tissue.

Figure 2

Positive ion mode mass spectra from distinct cell layers observed in positive ion MALDI-TOF images (Figure 3 a-i). (a) Mass spectrum from nerve fiber/ganglion cell layer. (b) Mass spectrum from inner plexiform layer. (c) Mass spectrum from outer plexiform layer. (d) Mass spectrum from outer nuclear layer (e) mass spectrum from photoreceptor layer. (f) Mass spectrum from the choroid layer. (g) mass spectrum from the sclera. (h) mass spectrum from retinal pigment epithelium.

Figure 3

Positive MALDI-TOF images of mouse Abca4^−/− retina tissue displaying eight out of nine distinct layers distinguished by unique lipid signals in an overlaid image (center) along with individual images (a) m/z 782.6, (b) m/z 784.6, (c) 810.6, (d) m/z 756.6, (e) m/z 900.6, (f) m/z 780.6, and (g) m/z 725.6 which form the overlaid image.

Figure 4

MALDI-TOF images of m/z 592.5 and 746.5 in Abca4-/- (a,b) and Sv129 (c,d) retinal tissue along with zoomed in regions (inserts) showing signals originating from the retinal pigment epithelium (RPE) region of the Abca4-/- retinal tissue.

Figure 5

Negative ion mode mass spectra from layers observed in negative ion MALDI-TOF image (Figure 6 a-e). (a) Mass spectrum from nerve fiber/ganglion cell layer. (b) Mass spectrum from inner plexiform layer. (c) Mass spectrum from outer nuclear layer. (d) Mass spectrum from photoreceptor layer (e) Mass spectrum from retinal pigment epithelium.

Figure 6

Negative MALDI-TOF images of mouse Abca4^−/− retina tissue displaying seven out of nine layers distinguished by unique lipid signals in an overlaid image (center) along with individual images. (a) m/z 885.5, (b) m/z 774.5, (c) m/z 718.6, (d) m/z 834.5, and (e) 865.5 which form the overlaid image.