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キーワード:
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要旨:
The recently developed expansion microscopy method (ExM) allows for the resolution of structures below the diffraction limit of light not by sophisticated instrumentation, but rather by physically expanding the molecular structure of cells. This happens by crosslinking the protein in the sample to a hydrogel that is polymerized in situ and subsequently expanded, tearing the proteins apart in a nearly isotropic manner. In the resulting, larger facsimile of the original sample, the fluorescence-labeled molecules of interest can be optically separated by conventional fluorescence microscopy since the intermolecular distances are enlarged by a factor ranging from ~4 to 20 depending on the chemistry used for the hydrogel. The achieved improvement in resolution thus corresponds to the expansion factor. Further increase in resolution beyond this value may be achieved by combining ExM with established super-resolution microscopy methods. Indeed, this is possible using structured illumination microscopy (SIM) (Halpern et al., 2017; Wang et al., 2018), single molecule localization microscopy (SMLM) (Zwettler et al., 2020) and stimulated emission depletion (STED), as we and others have shown recently (Gambarotto et al., 2019; Gao et al., 2018; Kim, Kim, Lee, & Shim, 2019; Unnersjö-Jess et al., 2016). Here, we provide a protocol, for our method, called ExSTED, which enabled us to achieve an increase in resolution of up to 30-fold compared to conventional microscopy, well beyond what is possible with conventional STED microscopy. Our protocol includes a strategy to achieve very high intensity fluorescence labeling, which is essential for optimal signal retention during the expansion process for ExSTED.
