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4 Janelia Publications

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    02/03/21 | Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)
    Hugo G.J. Damstra , Boaz Mohar , Mark Eddison , Anna Akhmanova , Lukas C. Kapitein , Paul W. Tillberg
    bioRxiv. 2021 Feb 03:. doi:

    Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the polymer gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors, for example using iterative expansion, but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.

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    10/06/19 | Expansion microscopy: scalable and convenient super-resolution microscopy.
    Tillberg PW, Chen F
    Annual Review of Cell and Developmental Biology. 2019 Oct 6;35:683-701. doi: 10.1146/annurev-cellbio-100818-125320

    Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 μm thick. Expected final online publication date for the , Volume 35 is October 7, 2019. Please see for revised estimates.

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    09/01/19 | BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples.
    Hörl D, Rojas Rusak F, Preusser F, Tillberg P, Randel N, Chhetri RK, Cardona A, Keller PJ, Harz H, Leonhardt H, Treier M, Preibisch S
    Nature Methods. 2019 Sep;16(9):870-74. doi: 10.1038/s41592-019-0501-0

    Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis.

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    08/02/18 | Expansion microscopy: protocols for imaging proteins and RNA in cells and tissues.
    Asano SM, Gao R, Wassie AT, Tillberg PW, Chen F, Boyden ES
    Current Protocols in Cell Biology. 2018 Aug 02;80(1):e56. doi: 10.1002/cpcb.56

    Expansion microscopy (ExM) is a recently developed technique that enables nanoscale-resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes via isotropic physical expansion of the specimens before imaging. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by ∼4.5 fold linearly when immersed in water. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged. Here we describe best-practice, step-by-step ExM protocols for performing analysis of proteins (protein retention ExM, or proExM) as well as RNAs (expansion fluorescence in situ hybridization, or ExFISH), using chemicals and hardware found in a typical biology lab. Furthermore, a detailed protocol for handling and mounting expanded samples and for imaging them with confocal and light-sheet microscopes is provided. © 2018 by John Wiley & Sons, Inc.

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