Yang Li, Dong-Ryoung Lee, Edward S Allgeyer, Lena K Schroeder, Shijie Tu, Joerg Bewersdorf, Xiang Hao
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Abstract
In stimulated emission depletion (STED) nanoscopy, high 3D resolution requires harnessing a 4Pi architecture using two opposing objectives. Here, we provide the step-by-step process for the construction and alignment of a 4Pi-STED nanoscope, commonly referred to as an 'isoSTED nanoscope'. The procedure guides interested researchers through the assembly of the optomechanical components, the configuration of the electronic and control devices, the alignment of the optical beam path and the assessment of the instrument's performance. The protocol offers a detailed roadmap for constructing an isoSTED nanoscope with adaptive optics in about 12 months and is designed for users with expertise in optical instrumentation builds. Once the instrument is finely calibrated, researchers can expect to achieve 3D biological images with isotropic sub-50-nm resolution in thick samples ≤35 µm in depth.
期刊介绍：
Nature Protocols focuses on publishing protocols used to address significant biological and biomedical science research questions, including methods grounded in physics and chemistry with practical applications to biological problems. The journal caters to a primary audience of research scientists and, as such, exclusively publishes protocols with research applications. Protocols primarily aimed at influencing patient management and treatment decisions are not featured.
The specific techniques covered encompass a wide range, including but not limited to: Biochemistry, Cell biology, Cell culture, Chemical modification, Computational biology, Developmental biology, Epigenomics, Genetic analysis, Genetic modification, Genomics, Imaging, Immunology, Isolation, purification, and separation, Lipidomics, Metabolomics, Microbiology, Model organisms, Nanotechnology, Neuroscience, Nucleic-acid-based molecular biology, Pharmacology, Plant biology, Protein analysis, Proteomics, Spectroscopy, Structural biology, Synthetic chemistry, Tissue culture, Toxicology, and Virology.
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