Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy
Open accessAbstract
Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescence imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here, we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed two-photon excitation and continuous wave stimulated emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor 594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3-fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located approximately 100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue.
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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 Ding et al., 2009
The Results ('Live-cell imaging of neuron dendrites and spines') report improved imaging of dendritic spines 30–110 μm below the surface, and 'Improved analysis of spine morphology' says lateral, but not axial, resolution improved. However, the experiments used acute mouse brain slices, not organotypic slices. The reported measurements also do not establish the claimed 60–150 nm lateral-resolution range; the Results report a 280 nm FWHM for imaged beads and thin filopodia. (LLM, conf=0.89)
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Cites
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Self-citation
- Authors
- Ding, J. B., Takasaki, K. T., & Sabatini, B. L.
- Affiliations
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- Corresponding author
- Sabatini, Bernardo L.
- Venue
- Neuron
- Publisher
- Elsevier BV
- Published
- 2009-08
- Volume
- 63
- Issue
- 4
- Pages
- 429-437
- ISSN
- 0896-6273 (ISSN-L)
- PMC
- PMC2756148
- OpenAlex
- W1982807939
- Cited by
- 177
- References
- 3
- Citations by year
- 2026: 4 · 2025: 5 · 2024: 4 · 2023: 2 · 2022: 5 · 2021: 5 · 2020: 4 · 2019: 8 · 2018: 8 · 2017: 8 · 2016: 9 · 2015: 16 · 2014: 21 · 2013: 16 · 2012: 27
- FWCI
- 12.28
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 1446 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Neuron (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Neurological Disorders and Stroke (R01NS046579, T32NS007484)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Engineering › Biomedical Engineering › Nonlinear Optical Materials Studies
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Keywords
- STED microscopy, two-photon microscopy, brain slices, dendritic spines, near-infrared excitation, neuronal structure
- MeSH terms
- Animals, Brain, Mice, Inbred C57BL, Microscopy, Confocal, Microscopy, Fluorescence, Multiphoton, Mice, In Vitro Techniques
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