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Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina
Published on: February 13, 2021
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Super-resolution STED imaging in the inner and outer whole-mount mouse retina
Leon Kremers1,2,3,4, Kseniia Sarieva1,2,5, Felix Hoffmann1
1Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany.
Frontiers in Ophthalmology
|July 10, 2024
Summary
We developed a new STED microscopy protocol for whole-mount mouse retina imaging. This method enhances resolution for studying retinal synapses and cellular structures in deeper tissue layers.
Area of Science:
- Neuroscience
- Microscopy
- Cell Biology
Background:
- Super-resolution microscopy offers high-resolution imaging of subcellular structures.
- Previous super-resolution studies focused on thin samples, limiting deep tissue analysis.
- Mammalian whole-mount retinas remain underexplored with super-resolution techniques.
Purpose of the Study:
- To establish a Stimulated Emission Depletion (STED) microscopy protocol for whole-mount mouse retina.
- To enable super-resolution imaging in deeper retinal layers (30-50 µm).
- To investigate synaptic structures and protein composition in the retina.
Main Methods:
- Developed a sample preparation protocol including horizontal retinal slicing.
- Optimized immunolabeling with STED-compatible fluorophores.
- Adjusted image acquisition settings for enhanced resolution and depth penetration.
Main Results:
- Achieved resolution enhancement of two-fold or higher compared to confocal microscopy.
- Successfully visualized subcellular structures in retinal ganglion cells (somata, dendrites, axons).
- Enabled visualization of putative GABAergic horizontal cell synapses in the outer retina.
Conclusions:
- Established a reliable STED microscopy protocol for whole-mount mouse retina.
- The protocol allows imaging at depths of 30-50 µm.
- Facilitates research on retinal synaptic protein composition and cytoskeletal ultrastructure in health and disease.
