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

Summary

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Abstract

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Introduction

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.

Protocol

All experiments were approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee (IACUC). Animal use and care were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for use of animals in ophthalmic and vision research. All the materials required to carry out this procedure are listed in the Table of Materials. Wear powder-free gloves while performing each step. For steps 6 and 7, also refer to the official microscope operating manual.

1. Preparation of the animals

1. Euthanize the experimental mice in accordance with applicable Institutional Animal Care and Use Committee-approved protocol (anesthesia with a combination of Ketamine 60 mg/kg and Dexmedetomidine 0.5 mg/kg followed by cervical dislocation was used here). Immediately proceed to stabilizing the animal on a platform for dissection and heart perfusion.
   NOTE: Experimental animals are chosen based on the design of individual study.
2. Dissect the abdomen and thorax to expose the heart. Perform cardiac perfusion by transfusing the heart via a 27 G needle placed in the left ventricle and create a small (~1 mm) incision in the right atrium to allow egress of blood²⁴.
   1. First, transfuse 30–50 mL of ice-cold phosphate balanced saline solution (PBS) and then, 30–50 mL of freshly prepared 4% paraformaldehyde (PFA).
   2. To check for a successful PFA transfusion, check for visible muscle twitches throughout the body and tail. Proceed to the enucleation step.

2. Eyeball enucleation and fixation

1. Use a curved jeweler’s forceps to gently push over the upper or lower eyelid to force the eyeball out of its socket. Use another set of jeweler’s or similar forceps to puncture the conjunctiva from the side and hold the globe from the optic nerve side. Slowly lift the globe from its socket until it is severed from the optic nerve.
2. Transfer the globe to a tube containing freshly prepared ice-cold 4% PFA. Label the tube accordingly. Allow the globe to remain in 4% PFA in a 4 °C fridge for 12 h (overnight).
   NOTE: Use a plastic transfer pipette with a cut tip to transfer the globe. Widen the opening of the cut tip with a second pipette tip to avoid damaging the sample with sharp edges.

3. Dissection of the sample (Figure 1 and Figure 2)

1. Under a stereomicroscope, locate the cornea-sclera junction (Figure 1A) and, use the sharp cutting tip of a 30 G needle to make a very superficial cut at the sclera approximately 0.5–1 mm behind the cornea-sclera junction (Figure 1B).
2. Advance one of the blades of a sharp tip dissecting scissors through the incision that was just made into the potential space between the sclera/choroid/RPE and the retina (Figure 1C). Advance the scissors and cut circumferentially until the sclera/choroid/RPE can be peeled off from the outer surface of the retina (Figure 1D,E).
   NOTE: It is important to perform this step slowly and gently to avoid puncturing the retina. The first few cuts are particularly critical to avoid cutting through retina.
3. If needed, make radial relaxing cuts on the sclera/choroid/RPE to facilitate the process of circumferential cutting and the subsequent peeling of the optic nerve and sclera/choroid/RPE. Remove small patches of RPE (Figure 1F) using a size 1 painting brush soaked in PBS.
4. Transfer the whole intact eyeball to a tube containing PBS. Proceed immediately to the next step or preserve in 4 ˚C for immunolabeling.
   NOTE: Marks may be placed on the eyeball after enucleation and then, after dissection to preserve the orientation of the eye if needed. The protocol may be paused here, and the samples may be preserved overnight in a 4 °C fridge before proceeding to the next steps.

4. Vascular staining

1. Permeabilize the tissue by immersing it in PBS containing 0.2% Tween-20 at room temperature for 20 min.
2. Wash the sample with PBS 3 times on a shaker for 10 min.
3. Incubate the sample with 5% normal goat serum (NGS) in PBS containing 0.25% Triton X-100 at room temperature for 1 h.
4. Incubate with the primary antibody at 4 ˚C overnight. Here, an anti-mouse Collagen IV antibody was used (final concentration was prepared in PBS containing 0.2% Tween-20).
5. Wash 3 times with PBS, for 5 min per wash.
6. Incubate the sample with fluorescent-labeled secondary antibodies. Here, an anti-rabbit Alexa Fluor 568 was used for 12 h at 4 ˚C (1:200 dilution in PBS containing 0.2% Tween-20).
7. Wash with PBS 3 times, for 1 h each, and then proceed with tissue clearing steps.

5. Optical clearing with 2,2′-thiodiethanol (TDE)

1. Prepare working TDE concentrations using stock TDE solution with PBS for a final concentration of 10%, 20%, 30%, 40%, 50%, and 60% volume to volume (v/v). Prepare at least 2 mL of solution for each eye sample to allow enough excess volume to penetrate the tissue.
2. Incubate the samples in a 6 or 12 well plate well at increasing concentration of TDE. Start by immersing the intact whole eyeballs in 10% TDE solution for 2–4 h on a shaker at room temperature. Successively, transfer the sample to a higher TDE concentration for 2–4 h in each TDE concentration (Figure 2C).
   NOTE: Retina starts to clear at concentrations of 40%–50%, but maximum clearing occurs after incubation in a 60% solution. Retina becomes less transparent at concentrations of 70% and higher (Figure 2D).
3. Stop the clearing process overnight, if needed, at any of the successive clearing exchange steps.

6. Whole eye imaging using a light sheet microscopy

1. Mount the intact whole eye samples considering the configuration of the light sheet microscope platform being used. Follow the microscope and acquisition software instructions to set up acquisition parameters including light sheet alignment and the illumination and detection of optical paths.
   NOTE: The samples used in this experiment were glued from the cornea side to the tip of a hypodermic needle on an insulin syringe (Figure 2E). The sample was then suspended inside the microscope chamber.
2. Fill the microscope chamber with 60% TDE as clearing solution.
3. Immerse the sample within the light sheet microscope chamber in 60% TDE solution (the final clearing concentration).
4. Image the cleared eye by means of a variety of commercial or custom-built confocal and light sheet microscopes. In this protocol, a dual-side illumination light sheet microscope is used.
5. Use low resolution and low magnification imaging (5x, NA 0.16) to image cellular morphology and cellular process tracing especially when combined with tiling. Use high resolution and magnification imaging (20x, NA 1.0) to image both cellular morphology and large sub-cellular organelles such as nuclei and mitochondrial clusters.

7. Post-acquisition image processing

NOTE: Post-acquisition processing depends on the type of file and software compatible with the imaged files.

1. Apply deblurring or deconvolution to further augment the raw images prior to stitching the imaged tiles. A Weiner filter can be applied to deblur the images. Alternatively, images can be iteratively deconvolved after denoising with the Richardson-Lucy deconvolution and a theoretical or experimentally measured PSF using modelling tools such as the ImageJ PSF generator plugin²⁵.
2. Perform the stitching of pre-processed z-stacks and an affine and non-rigid volume transformations followed by multi-view volume registration and fusion using a variety of commercial or public-domain software packages (ImageJ – BigStitcher plugin)²⁶.

النتائج

A zero-angle projection of peripapillary vascular network and microglia is shown in Figure 3A. Also, intact whole retina microglia distribution in a CX3CR1^-GFP mouse is presented in Figure 3B. A major advantage of the method presented here, is its ability to image innate fluorophores. Figure 3C,D show microglia in representative Z projections (green channel) from samples prepared with the current method o...

Discussion

Retina and vitreous development and pathologies are best studied with intact whole retina imaging techniques in which the retina is not cut for sections or for flat mount preparations. Existing intact whole eye imaging methods either incorporate pigment bleaching, which removes innate fluorophores, or involve physical removal of the opaque coatings of the eyeball (RPE, choroid, and sclera) along with the anterior segment of the eye, which may disturb peripheral retina and vitreous body. Chang et al. and Prahst et al. rem...

Materials

Name	Company	Catalog Number	Comments
Experimental animal
CX3CR1-GFP Mouse	The Jackson Laboratory	5582
Anesthetic
Dexmedetomidine	Par Pharmaceutical	42023-146-25
Ketamine	Fresenius Kabi
Tissue harvesting, fixation, and sample dissection
cardiac perfusion pump	Fisher scientific	NC9069235
Cyanoacrylate superglue	amazon.com
Fine scissors-sharp	Fine Science Tools	14160-10
Fine tweezers	Fine Science Tools	11412-11
Paraformaldehyde (PFA)	Electrone microscopy sciences	15710-S
Phosphate buffered saline (PBS)	Gibco	10010049
size 1 painting brush	dickblick.com
straight spring scissors	Fine Science Tools	15000-03
syringe, needle tip, 27 gauge x 1.25"	BD
Tubes 1.5 ml, 15 ml, 50 ml	Thermo sceintific
Tween-20	ThermoFisher	85114
Immunofluorescent staining
Anti-mouse collagen IV antibody	Abcam	ab19808	1:200 dilution
Anti-rabbit Alexa Fluor 568	Invitreogen	A-11011	1:200 dilution
Normal goat serum	ThermoFisher	50062Z	10% concentration
Tissue clearing
2,2′-thiodiethanol (TDE)	Fluka analytica	STBD7772V
Rocking shaker	Fisher scientific	02-217-765
Microscopy
Fluorescent microspheres	TetraSpeck	T14792
Light sheet fluorescent microscope (LSFM)	Zeiss	Z1
Microglia enumeration
ImageJ	National Institue of Health