Two-photon excitation STED microscopy in two colors in acute brain slices
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Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ~350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-…
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Two-photon STED has been successfully applied to dendritic spines in organotypical slices, resulting in 60-150 nm lateral resolution at 50-100 μm depths, but without improving the axial resolution.
“Nevertheless, it has been successfully applied to dendritic spines in organotypical slices, resulting in 60–150 nm lateral resolution at 50–100 μm depths, but without improving the axial resolution (Ding et al., 2009; Bethge et al., 2013; Takasaki et al., 2013).”
From Barriers in the brain: resolving dendritic spine morphology and compartmentalization (2014) · cites this paper as Bethge et al., 2013
Two-color detection has been established using spectral unmixing of either pairs of organic dyes or the fluorescent proteins YFP and GFP.
“In addition, two-color detection has been established using spectral unmixing of either pairs of organic dyes (Tønnesen et al., 2011) or the fluorescent proteins YFP and GFP (Bethge et al., 2013).”
From Barriers in the brain: resolving dendritic spine morphology and compartmentalization (2014) · cites this paper as Bethge et al., 2013
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- Authors
- Bethge, P., Chéreau, R., Avignone, E., Marsicano, G., & Nägerl, U. V.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Université de Bordeaux (FR)
- Corresponding author
- Nägerl, U. Valentin
- Venue
- Biophysical Journal
- Publisher
- Elsevier BV
- Published
- 2013-02
- Volume
- 104
- Issue
- 4
- Pages
- 778-785
- ISSN
- 0006-3495 (ISSN-L)
- PMC
- PMC3576543
- OpenAlex
- W2118583944
- Cited by
- 139
- References
- 37
- Citations by year
- 2026: 4 · 2025: 5 · 2024: 4 · 2023: 9 · 2022: 6 · 2021: 15 · 2020: 9 · 2019: 15 · 2018: 17 · 2017: 11 · 2016: 8 · 2015: 9 · 2014: 22 · 2013: 5
- FWCI
- 8.89
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Biophysical Journal (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Physical Sciences › Engineering › Biomedical Engineering › Nonlinear Optical Materials Studies
- Keywords
- STED microscopy, two-photon excitation, super-resolution imaging, acute brain slices, dendritic spines, microglial cells, glial processes, dual-color imaging, time-lapse imaging, sub-diffraction resolution, microscope design
- MeSH terms
- Animals, Brain, Mice, Inbred C57BL, Microscopy, Confocal, Microscopy, Fluorescence, Multiphoton, Dendritic Spines, Mice
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