Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

<div><p>Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM.</p></div

Correlative light electron and microscopy of cryostat sections can be used to identify rhodopsin enriched phagosomes in the retinal pigment epithelium (RPE) cell layer.

<p>The same region of RPE viewed by (A) fluorescence microscopy (FM) and (B) electron microscopy (EM), with an overlay of the two in (C). The boxed regions in (A-C) are shown at higher magnification in (D) highlighting regions that include FM rhodopsin staining (green) overlapping with phagosomes seen by EM. (E) Higher magnification of phagosomes (Ph) boxed in (D), surrounded by melanosomes (Me) and mitochondria (M). Scale bar = (A-C)– 10um (D)– 1um (E)– 250nm.</p

Correlative light and electron microscopy of cryostat sections of control iPSC optic cup using dual fluorescence microscopy (FM) and nano-gold rhodopsin labelling.

<p>(A) Diagram outlining the differentiation of inducible pluripotent stem cells (iPSCs) into optic cups with photoreceptors. (B) Confocal FM overlay on top of differential interference contrast (DIC) image (C) FM overlay on top of electron microscopy (EM) image. (D) EM with (E) a high magnification region showing concentrated rhodopsin localisation in the photoreceptor OS region of the optic cups. The connecting cilia in (D) is indicated by CC. Scale bar = (B & C) 100um, (D) 2um and (E) 200nm.</p

Immuno-electron microscopy (iEM) labelling of cryostat sections allows the identification of peroxisomes in the RPE, illustrating the close contact with phagosomes and melanosomes.

<p>(A-B) FM images showing the localisation of peroxisomes (red) around rhodopsin enriched phagosomes (green) in the RPE cell layer. (C) Low magnification electron microscopy image of RPE cell layer with gold labelled peroxisomes that are localised towards the basal cell surface close to the basal infolding (Bi) and Bruch’s membrane (Br) and away from the apical processes (Ap). The box highlights a region with a phagosome (Ph) that is shown at higher magnification in (D) with a gold labelled peroxisome in close proximity and other organelles nearby, including melanolipofuscin granules (MeLi) and mitochondria (M). (E) Gold labelled peroxisomes in contact with melanosomes (Me) with a high magnification image (F). Contacts between the peroxisomes and melanosomes (black arrows) and in the zoomed insert tethers between the organelles can be seen (white arrows) Scale = (A)– 10um (B)– 5um (C)– 1um (D-E)– 250nm (F)– 100nm.</p

Mouse retinal tissue prepared conventionally and from OCT embedded sections for electron microscopy.

<p>(A & B) Conventional fixed tissue and (C & D) OCT sections of tissue prepared for EM show little difference in the preservation quality. Organelles including mitochondria (M), melanosomes (Me) and phagosomes (P), in addition to membranes such as basal infoldings (BI) and photoreceptor outer segment (OS) discs and structural features such as collagen in Bruchs membrane (Co) and fenestrae of choroidal endothelial cells (F) are well preserved. (A & C) the connecting cilium is indicated by CC and the photoreceptor inner segment IS. Scale bar = 500nm.</p

Outline method for correlative light and electron microscopy of cryostat sections.

<p>Outline method for correlative light and electron microscopy of cryostat sections.</p

Impaired melanosome distribution in <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice.

<p>Electron micrographs of the RPE of 7-month old <i>Chm^Flox</i> (A), <i>Chm^Flox, Tyr-Cre⁺</i> (B) and <i>ashen</i> (C) mice. Scale bars: 5 µm. The percentage of melanosomes in the apical processes in <i>Chm^Flox</i> and <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice was determined (D). No melanosomes were ever found in the apical processes of <i>ashen</i> mice. Results are mean+/−SEM of 5 to 6 observations. *P<0.05.</p

Irregularity of basal infoldings and basal laminar deposits in <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice.

<p>Electron micrographs of the RPE of 5-month old <i>Chm^Flox</i> (A), littermate <i>Chm^Flox, Tyr-Cre⁺</i> (B and C) and 1-year old <i>Chm^Flox</i>, <i>Tyr-Cre⁺</i> (D–E) mice. BIs are very regular in control mice (parenthesis in A). They disappear in some areas or expand in the cytoplasm of <i>Chm^Flox</i>, <i>Tyr-Cre⁺</i> mice (B). The box in B is enlarged in panel C and shows early BLamDs underneath BIs. Membrane debris and membrane bound vesicles accumulate in late BLamDs (D and E). Panel E is a magnification of the rectangular box in D. Note thickening of BrM in D. Small arrowheads indicate fibrillar materials in BLamDs, asterisks highlight striations, big arrowheads indicate membrane debris, double arrows show BrM thickness. Scale bars: 10 µm (A, B), 0.5 µm (C–E).</p

Thickening and abnormalities of Bruch’s Membrane in <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice.

<p>Electron micrographs of 5-month old <i>Chm^Flox</i> (A), littermate <i>Chm^Flox</i>, <i>Tyr-Cre+</i> (B–C) and 1-year old <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice (D). An enlargement of the box in B is shown in C. In <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice BrM becomes thicker with time. Double arrows show BrM thickness, small arrowheads indicate endothelial cell protrusions into BrM. Scale bars: 0.5 µm (A and C), 2 µm (B), 1 µm (D). (E) BrM thickness was measured in four 7-month old <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice (black square) and their littermate controls (grey dots). In each mouse 10 areas of retina were analysed. The two means are significantly different. ***P = 0.009. (F) Example of the variation of the measurements of BrM thickness along the retina for one <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mouse (black square) and its littermate control (grey dots).</p

Increased number of phagosomes in <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice.

<p>Frozen sections of eyes from 6-month old <i>Chm^Flox</i>, <i>Tyr-Cre+</i> mice and littermates <i>Chm^Flox</i> mice harvested at the indicated times after light onset were immunostained with antibody (RetP1) for rhodopsin and analysed by confocal microscopy. Phase images were used to identify the RPE within the tissue. (A) Overview of the choroid, RPE, and part of the retina in <i>Chm^Flox</i>, <i>Tyr-Cre+</i> sample. Scale bar: 50 µm. The boxed region is magnified in the panel beneath and shows an area of the RPE with overlaying POS, scale bar: 5 µm. (B) Projections of 9 confocal sections. Scale bar: 10 µm. (C) Phagosomes were counted and results were normalised to the control and are presented as mean+/−SEM of 3–5 observations. *P<0.05. (ONL) Outer nuclear layer, (IS) Inner segment, (OS) Outer segment.</p