Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

The interaction between the retinal neuronal and vascular elements in healthy and disease states is traditionally explored by immunofluorescent studies on physical sections of paraffin- or cryo-fixed retina tissue or on retina flat preparations¹. However, tissue sectioning disrupts retina neuronal and vascular continuity, and although three-dimensional reconstruction of the adjacent retina sections is suggested as a possible solution, it is still subject to errors and artifacts. Retina flat mount preparations also markedly disturb the integrity of retinal vascular and neuronal elements and the geographic connection between adjacent retinal areas². Alternatively, intact whole retina imaging has recently been introduced to visualize the three-dimensional projections of retinal neuronal and vascular components in their natural anatomic position^2,3,4,5.

In intact whole retina imaging, fluorescent signals from the vascular and neuronal elements of adjacent retina areas (tiles) of an intact whole retina are captured using a light sheet microscope; these tiles are then “stitched” together to reconstruct a three dimensional view of the entire whole retina^2,3,4,5,6. Intact whole retina imaging provides an unprecedented view of the retina for studying the pathogenesis of retinal vascular, degenerative, and inflammatory diseases^2,3,4,5,6. For example, Prahst et al. revealed a previously “un-appreciated” knotted morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics in an oxygen-induced retinopathy (OIR) model using live imaging of an intact whole retina². Similarly, Henning et al., Singh et al., and Chang et al. demonstrated the complex three-dimensional retinal vascular network in intact whole retinas^3,4,6. Vigouroux et al. used an intact whole eye imaging method to show the organization of the retina and visual projections in perinatal period⁵. In order to be able to create such unparalleled three-dimensional views of the retina, intact whole retina imaging protocols have overcome two major limitations: 1) the presence of opaque and pigmented coatings of the eyeball (sclera, choroid, and RPE) and 2) the limited penetration of the light through full retina thickness caused by the light scattering properties of the retinal nuclear and plexiform layers. Henning et al. and Vigouroux et al. applied H₂O₂ bleaching of choroid/RPE pigments so as to be able to image an intact retina^3,5. However, bleaching is not suitable for animal strains with endogenous fluorophores such as green fluorescent protein (GFP) or after in-vivo immunofluorescent stainings^3,5,7. In addition, Henning et al.’s method of H₂O₂ treatment was carried out in aqueous conditions which may generate microbubbles that result in retinal detachment. Moreover, the H₂O₂ treatment was performed at 55 ˚C, a condition that further deteriorates tissue antibody affinity. Furthermore, bleaching may introduce heavy autofluorescence originating from oxidized melanin⁸. Other depigmentation protocols for eye sections using potassium permanganate and oxalic acid were able to remove RPE pigments in embryonic sections but this depigmentation method also has been shown to reduce the efficacy of immunolabeling^9,10. As an alternative to bleaching, Prahst et al., Singh et al., and Chang et al. removed sclera and choroid and cornea to render a whole retina reachable to microscope light^2,4,6. However, removing cornea, lens, and peripheral retina may distort and disrupt peripheral retina and hyaloid vessels making these methods unsuitable for studying peripheral retina and hyaloid vasculature.

All currently available intact whole eye imaging protocols include the use of a tissue optical clearing step to overcome the light scattering properties of retinal layers^2,3,4,5. Tissue optical clearing renders retina transparent to microscope light by equalizing the refractive index of a given tissue, here retina, across all of its cellular and intercellular elements to minimize light scattering and absorption¹¹. Choroid and RPE should be removed or bleached before tissue optical clearing is applied to the retina as the pigmented coatings of the eyeball (choroid and RPE) cannot be sufficiently cleared^{6,12,13,14,15,16,17,18}.

The participation and contributions of vitreous and hyaloid vascular system in pathologic conditions such as retinopathy of prematurity (ROP), persistent fetal vasculature (PFV), Norrie Disease, and Stickler Disease is best studied when retina and hyaloid vessels are not disrupted in tissue preparation^{19,20,21,22,23}. Existing methods for intact whole retina imaging either removes the anterior segment of the eye, which naturally disrupts the vitreous and its vasculature, or apply bleaching agents, which may remove endogenous fluorophores. Published methods for visualizing the vitreous body and vasculature in their intact, untouched condition are lacking. We describe here a whole retina and vitreous imaging method that consists of surgical dissection of pigmented and opaque coatings of the eyeball, a modified tissue optical clearing optimized for retina, and light sheet fluorescent microscopy. Sample preparation, tissue optical clearing, light sheet microscopy, and image processing steps are detailed below.