A simple tissue clearing method for increasing the depth penetration of STED microscopy of fixed brain slices
| العنوان: | A simple tissue clearing method for increasing the depth penetration of STED microscopy of fixed brain slices |
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| المؤلفون: | U. Valentin Nägerl, Julie Angibaud, Christel Poujol, Patrice Mascalchi |
| المصدر: | Journal of Physics D: Applied Physics. 53:184001 |
| بيانات النشر: | IOP Publishing, 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | 0301 basic medicine, Materials science, Microscope, Acoustics and Ultrasonics, business.industry, STED microscopy, Cover slip, Condensed Matter Physics, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, law.invention, Embedding Medium, 03 medical and health sciences, Biological specimen, 030104 developmental biology, 0302 clinical medicine, Optics, law, Microscopy, business, Image resolution, Refractive index, 030217 neurology & neurosurgery |
| الوصف: | STED microscopy has been adopted by many research laboratories and imaging core facilities around the world. It offers biologists in principle a much higher spatial resolution than conventional light microscopy to reveal morphological and molecular details of cells beyond the diffraction barrier. However, this advantage is oftentimes hard for STED users to attain in the presence of optical aberrations, which can severely degrade microscope performance. A classic culprit is the mismatch in refractive index between the optics of the STED microscope and the biological specimen, which normally induces spherical aberrations. Inevitably, they smear out the focal intensity distributions of the excitation and STED lasers, and this problem increases rapidly with imaging depth. This means that STED microscopy has typically been restricted to distances of under 10 μm from the cover slip. However, for many neurobiological questions it is essential to image deeper into the tissue to avoid edge artifacts and to increase data throughput. Seeking to reduce such spherical aberrations and to improve the depth penetration of STED microscopy, we examined the embedding medium CFM3, which has the same refractive index as oil and is known to have a rapid clearing effect on biological tissue. We assessed CFM3 for STED imaging beyond the immediate surface of the biological sample and compared it with the common embedding medium Mowiol, using fluorescent beads and dendritic spines in fixed brain slices as test samples. We show that CFM3 makes it possible to properly resolve dendritic spines at a depth of around 40 μm with a standard commercial STED microscope, unlike Mowiol, where the spatial resolution is severely degraded. As CFM3 provides a cheap and easy way to reduce the refractive index mismatch as well as to optically clear brain tissue without distorting the micro-architecture of the tissue, we recommend its use as embedding medium for super-resolution imaging deep inside brain slices. |
| تدمد: | 1361-6463 0022-3727 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_________::a9a395827f9e5f3dcb134580cbdf80e0 https://doi.org/10.1088/1361-6463/ab6f1b |
| حقوق: | CLOSED |
| رقم الأكسشن: | edsair.doi...........a9a395827f9e5f3dcb134580cbdf80e0 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 13616463 00223727 |
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