Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina
June 30th, 2023
The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.
We present a protocol for aligning in vivo vis-OCTF images with ex vivo immunostained retinal nerve fibers to validate our novel imaging technology. Vis-OCTF enables visualization of retinal nerve fibers in vivo, and this process confirms the accuracy of our findings. Our protocol combines in vivo and ex vivo imaging to provide a real-time visualization of retina structure and confirmed findings.
The process is applicable to most modals of eye diseases, provided the anterior eye portion allows for optical imaging. This includes diabetic retinopathy and retinal ischemia models. These studies lay the foundation for establishing an objective evaluation of neural damage in humans, which can significantly improve glaucoma diagnosis and treatment in the future.
Start by positioning the anesthetized mouse on the animal holder and securing it with two Velcro straps. Turn on the computer, and open the software to activate the laser. Adjust the animal holder to center the laser in the mouse's eye.
Visualize the posterior part through an on-face preview, the field of view of the superficial vascular plexus, a B-scan, and the retina cross-section within the field of view. Acquire a visible-light optical coherence tomography or vis-OCT volume after making minor adjustments to the optical focus. Align the optical nerve head in each of the four corners of the field of view to cover different retinal areas.
Use an intensity-based threshold method to detect the retina surface and generate vis-OCT fiber grams from the volume. Crop the retinal nerve fiber layer or RNFL by selecting the first 16 micrometers depth. Next, calculate the mean intensity projection along the axial direction to generate the fiber gram image composed of retinal ganglion cell axon bundles and surrounding vasculature.
Align the blood vessels using a graphics editor to montage the four images, post-processing. The composite vis-OCT fiber gram is compared with the corresponding confocal image of flat-mounted retina immunostained with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures, which can be matched with the ICAM II labeled blood vessels on the confocal image.
Side by side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC axon bundle networks and surrounding retinal vasculature. Nucleate the eyes of a euthanized mouse, marking the temporal side for orientation purposes. Remove the anterior chamber, then remove the lens and vitreous, and place eye cups in paraldehyde for 30 minutes.
Wash eye cups with PBS for 30 minutes, replacing the solution three times. Then wash with 0.5%Triton X-100 for 30 minutes. Incubate eye cups in the blocking buffer for two hours at room temperature.
After immunostaining, isolate the retinas from the eye cups using a microscope. Divide the retinas into four leaves and flat-mount them with the retinal ganglion cell layer facing up. Ensure the mark on the temporal side remains attached for orientation.
Cover the retinas with mounting medium, place cover slips, and seal the slides using nail polish. Turn on the confocal microscope, and under Locate mode, find the area of interest using the eyepiece. Switch to Acquisition mode, and set up tiles to cover the entire retina along with Z-stack slices for all layers of information.
Image at least 25 tiles across the whole retina to achieve a total volume. Project the Z-stack slices to generate two-dimensional on-face confocal microscopy images. The composite vis-OCT fiber gram is compared with the corresponding confocal image of a flat-mounted retina immunostained with TUJ1 for RGC axons.
Blood vessels exhibit distinguishable branching structures, which can be matched with the ICAM II labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC axon bundle networks and surrounding retinal vasculature. Start by creating a composite image using the four obtained images from each mouse.
Utilize a graphics editor to align all the blood vessels. Employ the optic nerve head and blood vessel patterns as landmarks for aligning the composite fiber gram. Overlap the composite OCT images and the confocal images of the same retina stained with ICAM II to achieve in vivo and ex vivo alignment.
The composite vis-OCT fiber gram is compared with the corresponding confocal image of flat-mounted retina immunostained with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures, which can be matched with the ICAM II labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC axon bundle networks and surrounding retinal vasculature.
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Overview
This protocol outlines how to align in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of mouse retinas. It aims to verify the retinal ganglion cell axon bundle morphology observed in the in vivo images, using a combination of optical imaging techniques applicable to various eye diseases.
Key Study Components
Area of Science
- Neuroscience
- Optical Imaging
- Retinal Research
Background
- In vivo imaging techniques are essential for evaluating retina structure in real time.
- This study leverages vis-OCTF and confocal microscopy for anatomical validation.
- The methodology allows for assessment of retinal nerve fibers and their morphological changes.
- Important for understanding diseases like diabetic retinopathy and glaucoma.
Purpose of Study
- To establish a reliable alignment method between in vivo and ex vivo imaging techniques.
- To confirm the anatomical accuracy of retinal ganglion cell axon bundles in vivo.
- To provide a comprehensive visualization of retinal structures that aids in disease evaluation.
Methods Used
- Utilizes visible-light optical coherence tomography (vis-OCT) and confocal imaging techniques.
- Employs a mouse model to assess retinal nerve fiber structures, focusing on the retinal ganglion cell layer.
- The protocol involves anesthetizing mice, positioning them, and activating imaging devices.
- Steps include cropping and aligning images using landmark features such as blood vessels and optic nerve head.
- Confocal immunostaining is used to visualize RGC axons for comparison with the vis-OCT fiber gram.
Main Results
- The protocol successfully validates the morphology of RGC axon bundles as identified in the vis-OCT images.
- Confocal and vis-OCT images reveal identical networks of retinal vasculature and axons.
- This alignment enhances the reliability of in vivo imaging for evaluating retinal pathologies.
Conclusions
- The study demonstrates a robust method for correlating in vivo and ex vivo retinal imaging.
- Increases the potential for applying this technique to understanding and diagnosing retinal diseases.
- Establishes pathways for improved glaucoma diagnosis and treatment based on objective evaluation.
Frequently Asked Questions
Executive Industry Relevance
Reliable alignment of in vivo vis-OCTF and ex vivo confocal retinal images enables objective, quantitative validation of neural damage in preclinical eye disease models. This capability strengthens predictive confidence in non-invasive imaging biomarkers and supports translational continuity from animal studies to human disease evaluation. The approach is directly relevant for de-risking early discovery and advancing portfolio decisions in ophthalmic R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rigorous validation of in vivo imaging biomarkers against gold-standard ex vivo confocal data.
- Supports mechanistic de-risking by confirming neural structure integrity in disease models.
- Facilitates objective assessment of retinal ganglion cell axon damage for target validation.
Screening & Assay Development
- Provides standardized, reproducible imaging outputs for quantitative comparison across experimental conditions.
- Prepares validated biological systems for downstream compound screening in retinal disease models.
- Enables high-content, scalable imaging workflows for robust assay development.
Translational & Preclinical Research
- Aligns in vivo imaging readouts with ex vivo molecular markers for translational biomarker development.
- Ensures continuity of neural damage assessment from preclinical models to potential clinical endpoints.
- Supports risk-adjusted advancement of ophthalmic candidates based on objective neural integrity metrics.
Pipeline & Workflow Integration
This alignment protocol bridges early discovery imaging with preclinical validation, supporting workflows from hypothesis testing to lead identification in ophthalmic research.
- Discovery Biology: Confirms biological relevance of in vivo imaging findings through direct comparison with immunostained ex vivo tissue.
- Screening: Delivers reproducible, quantitative imaging outputs suitable for compound evaluation and assay standardization.
- Analytics: Provides high-resolution, statistically comparable measurements of retinal nerve fiber integrity.
- Translational Research: Facilitates biomarker alignment and continuity between animal models and human disease studies.
- Enterprise Reuse: Establishes a reusable imaging-validation workflow for diverse retinal disease models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neural damage assessment.
- Operational Value: Standardizes imaging validation, improving reproducibility and scalability across studies.
- Strategic Value: Enables more informed go/no-go decisions and reduces late-stage biological risk in ophthalmic portfolios.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of discovery and preclinical assets.
Implementation Considerations
- Requires expertise in both in vivo optical imaging and ex vivo confocal microscopy.
- Demands access to advanced imaging instrumentation and image analysis software.
- Necessitates cross-team standardization of imaging protocols and alignment procedures.
- Must be adapted for specific retinal disease models and animal strains as needed.
- Alignment precision and image quality are critical for robust validation and downstream analysis.
