Quick-freeze/deep-etch electron microscopy visualization of the mouse posterior pole

Abstract

The mouse is one of the most commonly used mammalian systems to study human diseases. In particular it has been an invaluable tool to model a multitude of ocular pathologies affecting the posterior pole. The aim of this study was to create a comprehensive map of the ultrastructure of the mouse posterior pole using the quick-freeze/deep-etch method (QFDE). QFDE can produce detailed three-dimensional images of tissue structure and macromolecular moieties, without many of the artifacts introduced by structure-altering post-processing methods necessary to perform conventional transmission electron microscopy (cTEM). A total of 18 eyes from aged C57BL6/J mice were enucleated and the posterior poles were processed, either intact or with the retinal pigment epithelium (RPE) cell layer removed, for imaging by either QFDE or cTEM. QFDE images were correlated with cTEM cross-sections and en face images through the outer retina. Nicely preserved outer retinal architecture was observed with both methods, however, QFDE provided excellent high magnification imaging, with greater detail, of the apical, central, and basal planes of the RPE. Furthermore, key landmarks within Bruch's membrane, choriocapillaris, choroid and sclera were characterized and identified. In this study we developed methods for preparing the outer retina of the mouse for evaluation with QFDE and provide a map of the ultrastructure and cellular composition of the outer posterior pole. This technique should be applicable for morphological evaluation of mouse models, in which detailed visualization of subtle ocular structural changes is needed or in cases where post-processing methods introduce unacceptable artifacts.

Keywords: Bruch's membrane; Choriocapillaris; Mouse eye; Quick-freeze/deep-etch; Retinal pigment epithelium; Transmission electron microscopy.

Fig 1. cTEM of RPE-BrM-Choroid complex with attached sclera

Cross-section of the complex architecture of the posterior pole of a wild type mouse eye including the RPE, Bruch’s membrane, choroid and subjacent sclera. Regions of pigment infiltration into the OCL and expanded regions of the OCL into the CC are marked by black arrows. The interface and boundaries between the CC and Bruch’s membrane are not linear. Scale bar: 5 μm.

Fig 2. Correlative imaging of apical RPE with cTEM and QFDE

A) An en face cTEM section of apical RPE. Remnants of microvilli can be seen on the left side of the image (MV). RMels of varying shapes and sizes appear discretely or as clusters. RPE nuclei are readily identified (N). B) QFDE of a non-fractured, etched surface of the apical RPE with the retina removed. The flat regions marked by * (top and left sides) are either on the surface of the RPE cell under the retina where the microvilli were completely removed or the open space within the cytoplasm of the RPE cells. As is typical at the apical RPE there are clusters of both vertical (white arrows, inset a) and horizontal (black arrows, inset b) RMels. The areas marked with “B” are areas of torn replica. C) QFDE of the non-fractured, etched surface of the apical RPE with the retina removed. Black arrows point to microvilli that are collapsed, lying flat (normally upright when intact and in contact with the retina). The white arrow on the right points to a lightly outlined microvilli process. D) & (E) compare the typical shapes of RPE melanosomes (RMel, D), and choroidal melanosomes (CMel, E). Black arrows point to collagen fibrils in (E). Scale bar: (A) 5 μm; (B) 10 μm (inset a = 2 μm; inset b = 2 μm; (C) 15 μm; (D) 400 nm; (E) 400 nm.

Fig 3. Correlative imaging of central RPE with cTEM and QFDE

A) QFDE image of an intact nucleus (N, inset (b) shows filaments on the surface) surrounded by organelles (ER is labeled *). The background material in the image surrounding the nucleus and the organelles is the cytoskeleton of the cell, most likely composed of intermediate filaments (insert (a) shows higher mag). B) cTEM thin-section of the central area of the RPE showing two adjacent cells. RMels are visible in abundance in this plane through the RPE as are numerous other small dense structures (*). Scale bar: 6 μm; inset a = 1 μm; inset b = 2 μm.

Fig 4. Correlative imaging of basal infolding membrane and RPE basal lamina with cTEM and QFDE

A) cTEM cross-section of BrM, CC, Choroid and Sclera with the RPE removed, leaving the RPE-BL as the most apical structure along with occasional remnants of BIM (not shown here). B) cTEM en face view of the BIM, all layers of BrM [RPE-BL, ICL (not visible), EL, OCL, and CC-BL], and the CC with its fenestrations (bottom left inset highlights the OCL-CC interface). The section depth of this image moves from the top right (RPE BIM) to the bottom left (choroid) at a relatively shallow slope. The RPE-BL appears as a grey diffuse layer. The ICL should follow, but due to its thinness in the mouse eye is difficult to appreciate in oblique and cross sections. The prominent collagen layer that occupies the majority of the figure is OCL. The EL is the dark, semi-fibrous layer comprising randomly oriented short, straight, fibers located between the OCL and the RPE-BL. The capillaries of the CC are visible with their characteristic fenestrations (arrows). C) & D) Show RPE-BL using two methods of pre-freezing QFDE preparations. In (C) the RPE was left intact prior to freezing then freeze fractured to expose this layer. In (D) the RPE was gently removed (same as A) prior to freezing and no fracturing was used. E) QFDE of non-fractured basal RPE structures following RPE removal prior to freezing. The image shows significant amounts of BIM with a periodic appearance, attached to a dense field of RPE-BL. Scale bar: (A) 6 μm; (B) 9 μm (inset: 500 nm); (C) 2 μm; (D) 2 μm; (E) 2 μm.

Fig 5. en face side-by-side comparison of views into BrM from the RPE BIM

A) QFDE: In the center of the image the fracture plane descends briefly into BrM revealing the RPE-BL and numerous fibrous structures (EL and OCL). The ICL is difficult to observe in the mouse, but is likely indicated by short curly fibril ends protruding from the RPE-BL (white arrow). “Below” the OCL there is a flat surface, which is likely the unetched ice front. The EL is shown in the inset, arrows pointing to elastin fibers. The mesh like structure surrounding the view into BrM is etched BIM. B) cTEM: The RPE-BL is traversed (black arrows) over a short distance exposing the dark, fibrous structure of the EL (white arrows) and the collagen fibrils of the OCL (black arrow heads). The ICL cannot be readily discerned in this image, but may be indicated by the short fibrils protruding from the basement membrane of the RPE (white arrowheads). (C) & (D) show two aspects of the EL. C) The elastin, in enface section, appears as knobby fibers that are densely packed but still distinct. This is observed when the fracture plane remains in the EL. D) EL is a flat, continuous sheet (arrows) with occasional “bumps” or protrusions. This is observed when the fracture plane descends rapidly and obliquely through the EL. Scale bar: (A) 2 μm (inset: 500 nm); (B) 2 μm; (C) 2 μm; (D) 1 μm.

Fig 6. QFDE of OCL and Choriocapillaris complex

A) Fracture plane traverses the BIM of the RPE and proceeds obliquely to the capillaries of the choriocapillaris complex. The replica reveals the principal structures that separate the outer retina from its primary blood supply. Among the many structures revealed in this image, there are two features that stand out: 1) thickly populated OCL and 2) clear arrays of capillary fenestrations (Fen). (B), (C), & (D) are magnifications of the boxed regions in panel A. B) A high magnification of the CC basal lamina, fenestrations of the endothelium, as well as the inner layers of BrM. The fracture plane descends rapidly from the BIM (top, right) through the RPE-BL/ICL/EL (arrow) and the OCL, which is mostly fractured away except to the far right of the image (OCL). The asterisk indicates an area of the CC-BL with both the lamina rara and densa. The fenestrated endothelium of the choriocapillaris can be readily identified by the clustering of small “holes” adjacent to an open space (capillary lumen). C) High magnification of the fenestrations found in panel B. Two structures indicating the presence of fenestrations were found. The classic wagon wheel was present in what we assume are intact fenestra (black arrows) and ‘holes’ where, presumably, the internal structure of the “wagon wheel” has been pulled away (white arrows). D) Dense collagen networks of the OCL decorated with proteoglycans, fine filaments and remnants of the EL (arrows). E) A fractured CC vessel is surrounded by CMels. white arrows point to small groups of loosely organized fenestrations which we believe are on the posterior side of the capillary. The mesh material in the center is likely to be etched, fixed, plasma proteins within the exposed capillary lumen. Scale bar: (A) 4 μm; (B) 1 μm; (C) 500nm; (D) 1 μm; (E) 2 μm.

Fig 7. Correlative imaging of the choroid with QFDE and cTEM

A) QFDE and B) cTEM. In the post-CC area, discrete groups of CMels are observed (unlike the infrachoroid where larger areas include CMels) separated by melanocytes (Mcytes) and semi-organized collagen fibrils. This QFDE image is part of a larger mosaic (SI Fig. 5), in which collagen structures appear as both disorganized and highly-aligned bundles. Scale bars: 2 μm.

Fig 8. QFDE of the infra, central, and outer choroid

A) Shows the typical structure the infrachoroid, posterior to the choriocapillaris, comprising large clusters of CMels with few collagenous structures apparent. Mcyte membranous surfaces are visible in this area and a displaced red blood cell (RBC) can be seen (possibly an artifact of perfusion during tissue processing), indicating proximity to the CC. B) The central choroid is composed of small collagen strands in abundant ECM. In some areas, CMels have been dislodged exposing their encapsulating extra-cellular matrix. The right half of the image comprises mostly of dislodged melanosomes while in the left side the CMels are intact. The left inset is a higher magnification of a melanosome secure in its ECM while the right inset shows empty supporting matrix (arrows surround the object in each). C) As the choroid approaches the sclera we begin to observe larger and more frequent aligned collagen bundles. In this central-outer layer, the collagen appears in fiber like bundles while still fully surrounded by CMels. The insert shows a cTEM image of a cross-section of a region similar to, but orthogonal in orientation, to the corresponding QFDE. Scale bar: (A) 10 μm (inset: 1 μm); (B) 5 μm (insets: 500 nm); (C) 2 μm.

Fig 9. Correlative imaging of scleral lamellar collagen layers with QFDE and cTEM

A) Cross section through the sclera, illustrating the lamellar arrangement of collagen fibrils with cTEM. B) Deeper fracture with vitrified ice, allowing further imaging of the sclera with QFDE. In this image, we can appreciate layers of differentially oriented collagen lamellae, in which some fibrils have been fractured and recoiled into curved shapes (a common, mild artifact of fracturing collagen). On the right side of the image (*) is an area of partially etched, vitrified ice. Scale bar: (A) 3 μm; (B) 2 μm.