Super-resolution STED imaging in the inner and outer whole-mount mouse retina
Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques a...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Ophthalmology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fopht.2023.1126338/full |
| _version_ | 1797851470120353792 |
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| author | Leon Kremers Leon Kremers Leon Kremers Leon Kremers Kseniia Sarieva Kseniia Sarieva Kseniia Sarieva Felix Hoffmann Zhijian Zhao Marius Ueffing Thomas Euler Thomas Euler Ivana Nikić-Spiegel Timm Schubert Timm Schubert |
| author_facet | Leon Kremers Leon Kremers Leon Kremers Leon Kremers Kseniia Sarieva Kseniia Sarieva Kseniia Sarieva Felix Hoffmann Zhijian Zhao Marius Ueffing Thomas Euler Thomas Euler Ivana Nikić-Spiegel Timm Schubert Timm Schubert |
| author_sort | Leon Kremers |
| collection | DOAJ |
| description | Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease. |
| first_indexed | 2024-04-09T19:17:17Z |
| format | Article |
| id | doaj.art-d787f5b94e2243bab941616a48a0b714 |
| institution | Directory Open Access Journal |
| issn | 2674-0826 |
| language | English |
| last_indexed | 2024-04-09T19:17:17Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Ophthalmology |
| spelling | doaj.art-d787f5b94e2243bab941616a48a0b7142023-04-06T04:44:28ZengFrontiers Media S.A.Frontiers in Ophthalmology2674-08262023-04-01310.3389/fopht.2023.11263381126338Super-resolution STED imaging in the inner and outer whole-mount mouse retinaLeon Kremers0Leon Kremers1Leon Kremers2Leon Kremers3Kseniia Sarieva4Kseniia Sarieva5Kseniia Sarieva6Felix Hoffmann7Zhijian Zhao8Marius Ueffing9Thomas Euler10Thomas Euler11Ivana Nikić-Spiegel12Timm Schubert13Timm Schubert14Institute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyWerner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, GermanyInstitute for Experimental Epileptology and Cognition Research, University of Bonn, Bonn, GermanyInternational Max Planck Research School for Brain and Behavior, Bonn, GermanyInstitute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyWerner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, GermanyHertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, GermanyInstitute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyInstitute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyInstitute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyInstitute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyWerner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, GermanyWerner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, GermanyInstitute for Ophthalmic Research, University of Tübingen, Tübingen, GermanyWerner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, GermanySince its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.https://www.frontiersin.org/articles/10.3389/fopht.2023.1126338/fullretinahorizontal cellsganglion cellsdiffraction limitsuper-resolutionsynapses |
| spellingShingle | Leon Kremers Leon Kremers Leon Kremers Leon Kremers Kseniia Sarieva Kseniia Sarieva Kseniia Sarieva Felix Hoffmann Zhijian Zhao Marius Ueffing Thomas Euler Thomas Euler Ivana Nikić-Spiegel Timm Schubert Timm Schubert Super-resolution STED imaging in the inner and outer whole-mount mouse retina Frontiers in Ophthalmology retina horizontal cells ganglion cells diffraction limit super-resolution synapses |
| title | Super-resolution STED imaging in the inner and outer whole-mount mouse retina |
| title_full | Super-resolution STED imaging in the inner and outer whole-mount mouse retina |
| title_fullStr | Super-resolution STED imaging in the inner and outer whole-mount mouse retina |
| title_full_unstemmed | Super-resolution STED imaging in the inner and outer whole-mount mouse retina |
| title_short | Super-resolution STED imaging in the inner and outer whole-mount mouse retina |
| title_sort | super resolution sted imaging in the inner and outer whole mount mouse retina |
| topic | retina horizontal cells ganglion cells diffraction limit super-resolution synapses |
| url | https://www.frontiersin.org/articles/10.3389/fopht.2023.1126338/full |
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