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Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

Published: June 30th, 2023



1Department of Biology, University of Virginia, 2Department of Biomedical Engineering, Northwestern University, 3Department of Ophthalmology, University of Virginia, 4Program in Fundamental Neuroscience, University of Virginia, 5Department of Psychology, University of Virginia

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.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the "gold standard" to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Retinal ganglion cells (RGCs) play a critical role in visual information processing, receiving synaptic inputs through their dendritic trees in the inner plexiform layer (IPL) and transmitting the information via their axons in the retinal nerve fiber layer (RNFL) to the brain1,2,3,4. In diseased conditions such as glaucoma, early RGC degeneration may result in subtle changes in the RNFL, the ganglion cell layer (GCL), the IPL, and the optic nerve in both patients and rodent models5,

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All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Virginia and conformed to the guideline on Use of Animals from the National Institute of Health (NIH). See the Table of Materials for details related to all materials, reagents, and instruments used in this protocol.

1. In vivo vis-OCT imaging

  1. The vis-OCT system
    1. Image the mice eyes using a small animal vis-OCT system that.......

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The composite vis-OCT fibergram is compared with the corresponding confocal image of flat-mounted retina immunostained with Tuj-1 for RGC axons (Figure 1D, top panel). Axon bundles imaged by vis-OCTF can be matched with the Tu-j1-labeled axon bundles on the confocal image. Blood vessels usually exhibit distinguishable branching structures compared with surrounding axon bundles in fibergram images, which can be matched with the ICAM-2-labeled blood vessels on the confocal image (

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There are two steps in this protocol that require attention. First, it is necessary to ensure that the animal is under deep anesthesia and that their eyes are fully dilated before vis-OCT imaging. If the mice are not adequately anesthetized, their fast breathing may lead to unstable movements of the en face images, which can adversely affect the quality of the fibergram. Moreover, insufficient dilation can also have a negative impact on image quality since the iris may obstruct the light, preventing it from reac.......

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This study is supported by the Glaucoma Research Foundation Shaffer Grant, 4-CA Cavalier Collaborative Award, R01EY029121, R01EY035088, and Knights Templar Eye Foundation. 


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NameCompanyCatalog NumberComments
Halo 100Opticent Health, Evanston, IL
Zeiss LSM800 microscopeCarl Zeiss
Drugs and antibodies
4% paraformaldehyde (PFA)Santz Cruz Biotechnology, SC-2816921-2 drops
Bovine serum albumin powderFisher Scientific, BP9706-1001:10
Donkey anti Mouse Alexa Fluor 488 dyeThermo Fisher Scientific, Cat# A-212021:1,000
Donkey anti rat Alexa Fluor 594 dyeThermo Fisher Scientific, Cat# A-212091:1,000
Euthasol (a mixture of pentobarbital sodium (390 mg/mL) and phenytoin sodium (50 mg/mL))Covetrus, NDC 11695-4860-115.6 mg/mL
KetamineCovetrus, NADA043304114 mg/kg
Mouse anti-Tuj1A gift from Anthony J. Spano, University of Virginia1:200
Normal donkey serum(NDS)Millipore Sigma, S30-100 mL1:100
Phosphate-buffered saline (PBS, 10x), pH 7.4
(Contains 1370 mM NaCl, 27 mM KCl, 80 mM Na2HPO4, and 20 mM KH2PO4)
Thermo Fisher Scientific, Cat# J62036.K31:10
Rat anti-ICAM-2BD Pharmingen, Cat#5533251:500
Tropicamide drops Covetrus, NDC17478-102-12
Triton X-100
(Reagent Grade)
VWR, CAS: 9002-93-11:20
Vectashield mounting mediumVector Laboratories Inc. H2000-10
XylazineCovetrus, NDC59399-110-2017 mg/kg

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