Whole-mount Retinal Organoid Visualization with Cellular Resolution

Our work focuses on developing and studying retinal organoids that serve as a human in vitro model to study how human retina forms, how diseases affect, and how we might treat them.

To advance the research in the regenerative medicine field, we use 3D retinal organoids combined with optical clearing and immuno labeling to visualize entire structures and to study the special organization of retinal cells with confocal microscopy.

Whole-mount imaging faces challenges, like uneven antibody penetration in dense tissues and spherical aberration in organoids, which cause surface bias labeling and focus loss during the tissue visualization.

Our method reveals retinal cell connections, cell types, under 3D organization, providing crucial insights into the underlying causes of retinal diseases.

[Instructor] To begin, obtain the fluorophore aliquots dissolved in anhydrous DMSO, dehydrated and frozen. Add 1 to 10 microliters of DMSO to an aliquot. Now, combine 50 microliters of secondary immunoglobulin G or primary antibody, six microliters of one molar sodium bicarbonate, and one to five microliters of fluorophore, and incubate on a rocking platform for 40 minutes at room temperature, protected from light. While the reaction is progressing, remove the lids of the purification size exclusion columns and allow the buffer to pass through. Equilibrate the column by running three rounds of two to three milliliters of PBS through the column. If the last equilibration finishes before incubation ends, put the lids back to avoid them drawing, and wait for the reaction to finish. After incubation, add 140 microliters of PBS to the labeling reaction to bring the volume to approximately 200 microliters, and vortex it. Add the solution to the center of the column and let it enter the column. After the last drop has eluded, push the solution with 550 microliters of PBS. When the liquid stops falling, elute with 300 microliters of PBS, and collect in a 1.5 milliliter microcentrifuge tube. Measure the absorbance of the sample at 280 nanometers, and at the fluorophore-specific wavelengths to calculate the antibody concentration and labeling ratios. Store the labeled antibodies at four degrees Celsius, protected from light, for up to six months. Fix the retinal organoids with 4% paraform aldehyde at room temperature for 45 minutes. After fixing, add antigen retrieval solution over the organoids, and incubate the dish at 60 degrees Celsius, with mild shaking at 30 revolutions per minute for one hour. Next, permeabilize the organoids with PBS containing 1% Triton X-100. Incubate the mixture at room temperature, with mild shaking for four hours. Now, block the organoids in 2% BSA with 0.1% Triton X-100 at room temperature overnight or for over one day. The next day, add diluted primary antibodies to the organoids. Incubate at four degrees Celsius for two days with mild shaking. Wash the organoids three times for 15 minutes each in washing solution at room temperature with mild shaking. Now, add the dilution solution secondary antibodies and incubate at four degrees Celsius for two days with mild shaking. After washing the organoids as demonstrated earlier, incubate the organoids with fluorescent dyes diluted in washing solution at room temperature for one hour with mild shaking. Then, wash again. Next, prepare 1-propanol solutions in ultrapure water at 15%, 30%, 45%, 60%, 75%, and 90% concentrations, and adjust each to pH 9.5 with trimethylamine. Dehydrate the samples sequentially in increasing gradients of 1-propanol solutions for two hours each at 30 degrees Celsius with mild shaking. Then, transfer the sample to 100% 1-propanol solution, and incubate overnight at 30 degrees Celsius with mild shaking. For sample clearing, prepare benzyl alcohol and benzyl benzoate mixture in a one to two ratio to make BABB solution. Immerse the samples in BABB at room temperature overnight. Refresh the BABB solution before imaging. With a glass pipette, position the organoid in a glass bottom Petri dish with a drop of BABB, ensuring it contacts the surface of the cover glass. Now, use an inverted confocal laser scanning microscope with low and high magnification objectives to acquire Z-stack images for 3D cellular resolution. Recalibrate the step size of the Z-stack acquisition by accounting for the refractive index mismatch between clearing solution and immersion media. Now, launch ImageJ, update the voxel depth by selecting Image and clicking on Properties to create image projections of the Z-stack. Inspect the Z-stack depth by selecting Image, then Stacks, and choosing Orthogonal Views to display XY, XZ, and YZ views of the retinal organoid. Save the desired regions by clicking on File and selecting Save As. Lastly, create an animation of the 3D render of the Z-stack using processing software. Fructose glycerol cleared retinal organoids showed the least improvement in transparency, hindering visualization of the organoid core. ECI clearing improved visualization of retinal layers, but still failed to reveal the organoid core due to persistent light scattering. Fluoclear BABB cleared organoids exhibited the highest transparency, clearly revealing both the cortex and core with consistent fluorescence. Both ECI and fluoclear BABB cleared organoids exhibited visible shrinkage in bright field images due to dehydration steps. BABB-induced shrinkage increased sample compactness, enabling the use of high-magnification objectives to image deeper structures. TUJ1 immuno labeling at 40 days in vitro showed a single thin neuronal layer without visible stratification. By 90 days, the cells densely populated the apical region, and at 170 days, the organoid exhibited clear signs of layered organization with a defined apical zone. At 200 days, elongated cell projections extended inward from the surface into the core, and the organoid developed into three distinct retinal layers at 250 days. Neuronal fibers extended from the organoid center toward the periphery across maturation, becoming thicker and more complex by 250 days in vitro. Cone photoreceptors expressing blue and green/red opsins appeared at late stages, and showed elongated morphologies with bright tips. Rod photoreceptors expressing rhodopsin were identified by their central nuclei and peripheral opsin distribution. Chx10-positive cells were initially distributed widely, but later localized specifically to the inner nuclear layer during organoid maturation. Endogenous GCaMP6s expression was preserved following BABB clearing, with GFP signal detectable in the neural retina after long-term fixation.