Name: Ex Vivo Confocal Imaging of Mouse Retina
Uploaded: 2023-06-30T13:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Confocal Imaging of Mouse Retina at JoVE.com

ex vivo confocal imaging of mouse retina

Mus Retina OCT Fibergram Justering med Konfokale Billeder

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 models5,6,7,8,9. Early detection of these morphological changes in RGCs is thus essential for timely intervention to prevent RGC and vision loss.
We have recently developed a new clinical-ready imaging technology called visible-light optical coherence tomography (vis-OCT) to satisfy the need for in vivo monitoring of RGC damage. Vis-OCT improved the axial resolution, reaching 1.3 &#181;m in the retina10, 11, allowing for the visualization of individual RGC axon bundles in the RNFL. Subsequently, vis-OCT fibergraphy (vis-OCTF) was established to track and quantify RGC damage at the single axon bundle level in mice11,12,13. However, ex vivo confocal imaging of the same retina as the gold standard is often necessary to validate the in vivo findings. Therefore, this study will demonstrate how to align in vivo images acquired by vis-OCTF with ex vivo confocal images of the same mouse retina. The protocol aims to validate the in vivo findings by ex vivo confocal imaging and establish a foundation for examining the molecular and cellular changes underlying RGC damage in diseased conditions.

Forfatter Spotlight: Fremskridt inden for in vivo og ex vivo retinal billeddannelse til forbedret glaukomdiagnose og behandling 

Justering af optisk kohærenstomografi i synligt lys med konfokale billeder af samme musenethinde

We present a protocol for aligning in vivo VIS-OCT images with ex vivo immunostained retinal nerve fibers to validate our novel imaging technology. VIS-OCT 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 confirm findings.The process is applicable to mouse models 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 neuro damage in humans, which can significantly improve glaucoma diagnosis and treatment in the future.

Watch this Scientific Journal Video about Ex Vivo Confocal Imaging of Mouse Retina at JoVE.com

Ex Vivo Confocal Imaging of Mouse Retina  

Ex vivo konfokal billeddannelse af musenethinden

Enucleate øjnene på en euthaniseret mus, der markerer den tidsmæssige side til orienteringsformål. Fjern det forreste kammer. Fjern derefter linsen og glaslegemet og læg øjenkopper i paraformaldehyd i 30 minutter.Vask øjenkopper med PBS i 30 minutter. Udskiftning af opløsningen tre gange. Vask derefter med 0,5% Triton X-100 i 30 minutter.Inkuber øjenkopper i blokeringsbufferen i to timer ved stuetemperatur. Efter immunfarvning isoleres nethinden fra øjenkopperne ved hjælp af et mikroskop. Opdel nethinden i fire blade og fladmonter dem med retinale ganglioncellelag opad.Sørg for, at mærket på den tidsmæssige side forbliver fastgjort til orientering. Dæk nethinderne med monteringsmedium. Placer dækselsedler.Og forsegl diasene ved hjælp af neglelak. Tænd for det konfokale mikroskop, og find interesseområdet under Find-tilstand ved hjælp af okularet. Skift til anskaffelsestilstand, og konfigurer fliser til at dække hele nethinden sammen med Z-Stack-udsnit til alle lag af information.Forestil dig mindst 25 fliser på tværs af hele nethinden for at opnå en samlet lydstyrke. Projicer Z-Stack-skiverne for at generere todimensionelle konfokale mikroskopibilleder på ansigtet. Det sammensatte vis-OCT-fibergram sammenlignes med det tilsvarende konfokale billede af fladmonteret nethindeimmunfarvet med TuJ1 for RGC-axoner.Blodkar udviser skelnelige forgreningsstrukturer, som kan matches med ICAM-2-mærkede blodkar på det konfokale billede. Side-by-side sammenligning mellem ex vivo konfokal mikroskopi og in vivo vis-OCT afslørede identiske RGC axon bundle netværk og omgivende retinal vaskulatur.

Research

JoVE Journal

Neuroscience

Ex Vivo Confocal Imaging of Mouse Retina

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

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...