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.
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 position2,3,4,5.
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 retina2,3,4,5,6. Intact whole retina imaging provides an unprecedented view of the retina for studying the pathogenesis of retinal vascular, degenerative, and inflammatory diseases2,3,4,5,6. 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 retinas3,4,6. 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 retina3,5. However, bleaching is not suitable for animal strains with endogenous fluorophores such as green fluorescent protein (GFP) or after in-vivo immunofluorescent stainings3,5,7. 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 immunolabeling9,10. 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 light2,4,6. 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 layers2,3,4,5. 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 cleared6,12,13,14,15,16,17,18.
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 preparation19,20,21,22,23. 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.
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
- 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.
- 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.
- First, transfuse 30–50 mL of ice-cold phosphate balanced saline solution (PBS) and then, 30–50 mL of freshly prepared 4% paraformaldehyde (PFA).
- 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
- 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.
- 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)
- 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).
- 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.
- 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.
- 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
- Permeabilize the tissue by immersing it in PBS containing 0.2% Tween-20 at room temperature for 20 min.
- Wash the sample with PBS 3 times on a shaker for 10 min.
- Incubate the sample with 5% normal goat serum (NGS) in PBS containing 0.25% Triton X-100 at room temperature for 1 h.
- 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).
- Wash 3 times with PBS, for 5 min per wash.
- 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).
- Wash with PBS 3 times, for 1 h each, and then proceed with tissue clearing steps.
5. Optical clearing with 2,2′-thiodiethanol (TDE)
- 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.
- 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).
- Stop the clearing process overnight, if needed, at any of the successive clearing exchange steps.
6. Whole eye imaging using a light sheet microscopy
- 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.
- Fill the microscope chamber with 60% TDE as clearing solution.
- Immerse the sample within the light sheet microscope chamber in 60% TDE solution (the final clearing concentration).
- 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.
- 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.
- 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.
- 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.
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 of intact whole eye imaging (Figure 3C) and flat mount preparations (Figure 3D). Microglia were quantified and compared in randomly selected regions of interest from intact whole retina and flat mount preparations. No statistically significant difference was noted between the flat mount and intact whole retina imaging in terms of microglia numbers (Figure 3E). All representative images in Figure 3 were captured from CX3CR1-GFP mouse retina.
GFP-tagged microglia (green channel) and the vascular network (red channel) in a CX3CR1-GFP mouse retina that was imaged using the current intact whole retina imaging protocol is further described in Supplemental Video 1. Endogenous and in vivo staining fluorophores would have been bleached if the pigment bleaching methods used by Henning et al.3 and Vigouroux et al.5 were used to remove choroid and RPE pigmentation. Supplemental Video 2 shows a view of the hyaloid vessels and retrolental vascular plexus in their natural state. It should also be noted that removing the anterior segment of the eye as described by Prahst et al.2 and Chang et al.4 would have disturbed the hyaloid vessels.
Figure 1: Dissection of sclera/choroid/retinal pigment epithelium. (A-F) Removal of outer opaque and pigmented layers allows high resolution optical imaging of retina and vitreous cavity without interference from surrounding pigmented structures. Please click here to view a larger version of this figure.
Figure 2: Sample preparation for intact whole eye imaging. (A) After sampling, the eyeball was covered with opaque sclera, choroid, and RPE. (B) Opaque sclera/choroid/RPE were dissected out but the retina tissue was still relatively optically opaque for microscopic imaging. (C) Successive immersion in increasing concentrations of TDE rendered the sample clear. After the sample was clear (D), it was mounted onto the light sheet microscope platform for high resolution imaging. (E) For the light sheet microscope system that was used in this experiment, the intact whole retina was mounted to the tip of a hypodermic needle on a syringe using cyanoacrylate glue. The sample was then placed inside the imaging chamber that was filled with the final concentration of the clearing agent. Please click here to view a larger version of this figure.
Figure 3: Representative results for intact whole retina imaging. (A) Zero angle projection of the peripapillary vascular network and microglia in an 8-month old female CX3CR1-GFP mouse intact whole retina. (B) Flat projection of the green channel showing microglia distribution in an intact whole retina from an 8-month old female CX3CR1-GFP mouse. Peripheral retina was distorted in this flat projection of the cup shaped intact retina. (C-D). Representative 250 µm x 250 µm square flat projection from an intact whole retina scans (C) and flat mount preparation (D), both from 8-months old female CX3CR1-GFP mice (scale bar 50 µm). (E) Retinal microglia enumeration in flat mount preparations versus intact whole retina imaging using light sheet fluorescent microscope: Three regions of interest (ROI) were randomly selected from the mid-peripheral retina for microglia quantification. Orthogonal projections of the entire retina thickness in green channel was prepared for each ROI. Microglia numbers were counted within ImageJ. A two tailed Student’s t-test did not show statistically significant difference in the number of microglia in flat mount and intact whole eye preparations. Please click here to view a larger version of this figure.
Supplemental video 1: Intact whole retina imaging of a CX3CR1-GFP mouse. Retinal vessels were visualized in their entirety. Imaging was performed without choroid/RPE pigment bleaching. In this GFP-tagged mouse (and similar strains with innate fluorophores), choroid/RPE bleaching would have removed the GFP-tagged microglia signals. In this video, the three-dimensional depiction of retinal microglia distribution highlights this method’s strength in detecting microglia distribution and activation. Please click here to download this video.
Supplemental video 2: Hyaloid vasculature and retrolental vascular plexus in a 4-day old mouse. Anterior segment of the eyeball, including the cornea, iris, and lens, was not removed, leaving the vitreous cavity and its vasculature untouched. Please click here to download this video.
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. removed the outer coatings of the eyeball as well as the cornea thereby possibly disrupting the vitreous body and hyaloid vasculature and peripheral retina2,4. As such, their method may not be suitable for studying cellular features of retina and vitreous development and pathologies involving hyaloid vasculatures. Alternatively, Henning et al. and Vigouroux et al. used pigment bleaching as a technique to render choroid and RPE transparent for microscopic imaging3,5. However, pigment bleaching removes GFP and may compromise the performance of other innate/endogenous fluorophores as well as the fluorophores used for in vivo staining as imaging probes7. In the method introduced here, no bleaching was used, and the sclera, choroid, and RPE were dissected out while the cornea, iris, and lens were left untouched allowing for the visualization of the anterior segment of the eye, peripheral retina and hyaloid vessels in their undisturbed state.
The existing methods described for visualization of hyaloid vessels in diseases such as persistent fetal vasculature (PFV) and retinopathy of prematurity (ROP) involve dissecting anterior segment of the eye that disturbs the vitreous body and inevitably hyaloid vasculature22,23. In contrast to these methods, the current protocol does not perturb hyaloid and retrolental vasculature thus allowing for the visualization of the intact hyaloid vasculature as shown in Supplemental Video 2.
All the existing methods for intact whole eye imaging, including the method outlined above, involve tissue optical clearing, a process that matches a tissue’s refractive index with the surrounding medium to minimize light scattering and absorption11,27. Three major tissue clearing approaches include hydrophobic, hydrophilic and hydrogel-based methods depending on the agent used to clear the tissue13,27,28,29,30,31. A variety of these tissue clearing techniques have been successfully used in the previously described intact whole eye imaging methods, but they have all been time consuming, taking 3 to 7 days4,5. The protocol detailed here uses a modification of the previously-described 2,2′-thiodiethanol (TDE) exchange method to suit retina tissue32. In the current protocol, the intact whole retina/vitreous was immersed in a successively increasing concentration of TDE to gradually equalize the tissue’s refractive index and to render it clear in less than two days. TDE is a glycol derivative hydrophilic clearing agent miscible with water in any ratio that makes it possible to create a gradient of concentration to allow penetration of high refractive index solution into the tissue. TDE does not quench the fluorescence of various fluorophores unlike certain other clearing methods such as CLARITY and CUBIC33. In addition, TDE does not have the potential to damage equipment, as is the case for organic solvent-based clearing agents such as benzyl benzoate/benzyl alcohol (BABB). It also prevents the growth of contaminating organisms that is observed with the fructose based SeeDB clearing method33.
Light Sheet Fluorescent Microscopy is an efficient tool with a high temporal and spatial resolution for imaging thick tissues2,3,4,5,6,34. In LSFM, a thin sheet of light is directed through the sample to excite fluorophores only in a thin imaging plane that in turn enables lower phototoxicity and photobleaching, faster imaging, and higher contrast due to minimal out of focus excitation. Imaging of the dissected and optically cleared whole murine eyes as described here may be performed utilizing one of the many commercially available LSFM or confocal microscopes. In the current method, LSFM offers a fast and detailed view of retinal neurons and vessels and enables a quantitative analysis of microglia density.
The current method is limited in showing the photoreceptor-RPE interaction because RPE is being removed during dissection. Such interaction may be better studied with methods that uses bleaching and skip the dissection3,5. Tissue shrinkage and possible distortion are inherent limitations of all the tissue fixation and clearing methods including the method described here. These limitations should be acknowledged when interpreting results obtained with the current and other similar methods. Two critical steps that contribute to the success of this method include good surgical dissection of the ocular coatings without damaging the retina in combination with the application of TDE as optical clearing agent. Dissecting sclera/choroid/RPE layers in a small rodent eye can be technically challenging and cutting through peripheral retina or the anterior segment of the eye should be avoided. Mastering this technique requires the same set of skills utilized in retina flat mount preparation and, in our experience, essential dexterity can be acquired after performing the dissection steps described above on three to five eyes. The incubation period for each clearing step may be modified if the method needs to be applied on larger animal (such as rabbit) eyes. Whether hyaloid vessel stain better with a modified immunolabeling technique by injecting the primary and secondary antibodies into the vitreous cavity needs to be explored in future.
In summary, the method described here is based on further development of previously described techniques for high resolution whole retina imaging enabling imaging of the molecular and structural targets including innate and in-vivo staining fluorophores in an intact whole retina and vitreous. This method addresses several of the limitations of existing intact whole retina preparation methods while offering a new approach for an unprecedented visualization of the hyaloid vasculature in intact retina.
No relevant commercial conflict of interest.
This work has been done at the University of Texas Medical Branch. The authors appreciate Harald
Junge, PhD, Debora Ferrington, PhD, and Heidi Roehrich, University of Minnesota for their help in preparing Figure 1 and movie 2. LO was supported by NIEHS T32 Training Grant T32ES007254.
|CX3CR1-GFP Mouse||The Jackson Laboratory||5582|
|Tissue harvesting, fixation, and sample dissection|
|cardiac perfusion pump||Fisher scientific||NC9069235|
|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|
|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|
|2,2′-thiodiethanol (TDE)||Fluka analytica||STBD7772V|
|Rocking shaker||Fisher scientific||02-217-765|
|Light sheet fluorescent microscope (LSFM)||Zeiss||Z1|
|ImageJ||National Institue of Health|
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