Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Driel, Linda F. van; Valentijn, Jack A.; Valentijn, Karine M.; Koning, Roman I.; Koster, Abraham J.

doi:10.1016/j.ejcb.2009.07.002

Cited by 124 publications

(106 citation statements)

References 42 publications

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“…They were connected to the inner mitochondrial membrane only at a few points (video in supplementary material). This Wts with the "cristae junction model" originally proposed by Daems and Wisse (1966) and conWrmed in several newer studies based on electron tomography Zick et al 2009;van Driel et al 2009;Perkins et al 2010;Péranzi et al 2010).…”

Section: Discussionsupporting

confidence: 72%

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

Höhn

Sailer

Wang

et al. 2010

Histochem Cell Biol

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Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 μm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.

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Section: Discussionsupporting

confidence: 72%

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

Höhn

Sailer

Wang

et al. 2010

Histochem Cell Biol

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“…If cryo-LM imaging and sample grid transfers are performed with caution as described in the text and video, fully contamination free imaging can be accomplished. This method allows direct correlation of fluorescent and electron microscopy data of the same cell, organelle or macromolecular complex dispersed on the carbon support or contained within a thin tissue section 7 . It should be noted that the value of this cryogenic stage extends beyond cryo-LM/cryo-EM correlative studies to the field of light microscopy as a whole.…”

Section: Discussionmentioning

confidence: 99%

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

Carlson

Evans

2011

JoVE

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The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

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“…In correlative light and electron microscopy, proteins tagged with a fluorescent reporter, such as enhanced GFP (eGFP), or cell components stained with a selective dye can be directly identified on an EM grid and a tilt series acquired at the location of interest. This should enable mapping of cytoskeletal proteins onto high-resolution images created by electron microscopy, preferably in three dimensions (127)(128)(129)(130)(131). In addition, to catch dynamic structural changes, using a rapid-transfer system, samples can be cryoimmobilized once a physiological state has been observed in the cell (132).…”

Section: Future Perspectivesmentioning

confidence: 99%

Multidimensional View of the Bacterial Cytoskeleton

et al. 2013

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bThe perspective of the cytoskeleton as a feature unique to eukaryotic organisms was overturned when homologs of the eukaryotic cytoskeletal elements were identified in prokaryotes and implicated in major cell functions, including growth, morphogenesis, cell division, DNA partitioning, and cell motility. FtsZ and MreB were the first identified homologs of tubulin and actin, respectively, followed by the discovery of crescentin as an intermediate filament-like protein. In addition, new elements were identified which have no apparent eukaryotic counterparts, such as the deviant Walker A-type ATPases, bactofilins, and several novel elements recently identified in streptomycetes, highlighting the unsuspected complexity of cytostructural components in bacteria. In vivo multidimensional fluorescence microscopy has demonstrated the dynamics of the bacterial intracellular world, and yet we are only starting to understand the role of cytoskeletal elements. Elucidating structure-function relationships remains challenging, because core cytoskeletal protein motifs show remarkable plasticity, with one element often performing various functions and one function being performed by several types of elements. Structural imaging techniques, such as cryo-electron tomography in combination with advanced light microscopy, are providing the missing links and enabling scientists to answer many outstanding questions regarding prokaryotic cellular architecture. Here we review the recent advances made toward understanding the different roles of cytoskeletal proteins in bacteria, with particular emphasis on modern imaging approaches.

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Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Cited by 124 publications

References 42 publications

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

Multidimensional View of the Bacterial Cytoskeleton

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