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      Cryo-soft X-ray tomography: a journey into the world of the native-state cell

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          Abstract

          One of the ultimate aims of imaging in biology is to achieve molecular localisation in the context of the structure of cells in their native state. Here, we review the current state of the art in cryo-soft X-ray tomography (cryo-SXT), which is the only imaging modality that can provide nanoscale 3D information from cryo-preserved, unstained, whole cells thicker than 1 μm. Correlative cryo-fluorescence and cryo-SXT adds functional information to structure, enabling studies of cellular events that cannot be captured using light, electron or X-ray microscopes alone.

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          Cryo-electron tomography: The challenge of doing structural biology in situ

          Vladan Lučić, Alexander Rigort, Wolfgang Baumeister (2013)
          Electron microscopy played a key role in establishing cell biology as a discipline, by producing fundamental insights into cellular organization and ultrastructure. Many seminal discoveries were made possible by the development of new sample preparation methods and imaging modalities. Recent technical advances include sample vitrification that faithfully preserves molecular structures, three-dimensional imaging by electron tomography, and improved image-processing methods. These new techniques have enabled the extraction of high fidelity structural information and are beginning to reveal the macromolecular organization of unperturbed cellular environments.
          Article 0 comments Cited 127 times – based on 0 reviews     Review now
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            Nuclear aggregation of olfactory receptor genes governs their monogenic expression.

            Carolyn Larabell, E Clowney, A Legros … (2012)
            Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally synchronous with the downregulation of lamin b receptor (LBR) and can be reversed by ectopic expression of LBR in mature olfactory neurons. LBR-induced reorganization of nuclear architecture and disruption of OR aggregates perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography.

              Wolfgang Baumeister, Jürgen M. Plitzko, R Gatz … (2007)
              Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.
              Article 0 comments Cited 100 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                : +44-1235-778057 , elizabeth.duke@diamond.ac.uk
                Journal
                Journal ID (nlm-ta): Protoplasma
                Journal ID (iso-abbrev): Protoplasma
                Title: Protoplasma
                Publisher: Springer Vienna (Vienna )
                ISSN (Print): 0033-183X
                ISSN (Electronic): 1615-6102
                Publication date (Electronic): 22 November 2013
                Publication date PMC-release: 22 November 2013
                Publication date (Print): 2014
                Volume: 251
                Pages: 449-458
                Affiliations
                [ ]Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, WC2A 3LY UK
                [ ]Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE UK
                Author notes

                Handling Editor: David Robinson

                Article
                Publisher ID: 583
                DOI: 10.1007/s00709-013-0583-y
                PMC ID: 3927064
                PubMed ID: 24264466
                SO-VID: 39acdcf3-1fa9-47bb-a5ab-33190be38fb2
                Copyright © © The Author(s) 2013
                License:

                Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                History
                Date received : 28 October 2013
                Date accepted : 30 October 2013
                Categories
                Subject: Special Issue: New/Emerging Techniques in Biological Microscopy
                Custom metadata
                issue-copyright-statement © Springer-Verlag Wien 2014

                ScienceOpen disciplines: Molecular biology
                Keywords: correlative,cells,native state,cryo-soft x-ray tomography,cryo-fluorescence
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: correlative, cells, native state, cryo-soft x-ray tomography, cryo-fluorescence

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