Mesoscale imaging with cryo‐light and X‐rays: Larger than molecular machines, smaller than a cell

Ekman, Axel; Chen, Jian‐Hua; Guo, Jessica; McDermott, Gerry; Gros, Mark A. Le; Larabell, Carolyn A.

doi:10.1111/boc.201600044

Cited by 36 publications

(38 citation statements)

References 111 publications

(244 reference statements)

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“…The past 20 years, however, have seen a rapid expansion in the application, utilization, and integration of X-raybased microscopy into workflows aimed to assay three-dimensional biological structures. Use of soft X-rays has been proven capable of studying minimally-perturbed cells, with resolutions nearing that of electron microscopy, and it holds the exciting prospect of being able to thoroughly explore the cellular mesoscale architecture (123). The development and application of metallic stains for XRM, and the ability to direct such staining to specific targets, offers the promise of exploring the means by which the spatial organization of any protein of interest effects the spatiotemporal dynamic properties of cells.…”

Section: Discussionmentioning

confidence: 99%

“…The development and application of metallic stains for XRM, and the ability to direct such staining to specific targets, offers the promise of exploring the means by which the spatial organization of any protein of interest effects the spatiotemporal dynamic properties of cells. The physical properties of both SXT and XRM hold unique quantification opportunities, with linear absorption coefficients offering the ability to identify sub-cellular compartments (123) and stain densities in XRM presenting a unique means of measuring protein aggregation. Application of X-ray microscopy techniques in EM correlative studies have demonstrated the increased efficacy the approach offers and the resultant increases in sample throughput.…”

Section: Discussionmentioning

confidence: 99%

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Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Bayguinov

Fisher

2020

Journal of Biological Chemistry

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Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes have been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, whilst insightful, are nonetheless limited by either restrictions in resolution or by the sheer complexity of generating multi-dimensional data. Recent advances in the use and application of X-rays to acquire micro- and nano-tomographic datasets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the sub-cellular analyses of unstained vitrified cells, three-dimensional localization of specific protein targets, and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles and applications of both soft X-ray tomography and X-ray microscopy, and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches, and detail how they have become integral in correlative microscopy workflows.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Bayguinov

Fisher

2020

Journal of Biological Chemistry

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“…Viruses Vaccinia [20,21], Herpes Simplex Virus 1 [22,23], Hepatitis C [24] Primary cells Erythrocytes [25,26], lymphocytes [9,12,22,27], olfactory sensory neurones [28,29] Immortalised lines EFN-R [30], HEK293 [31,32], G361 [33], RK13 [34], Cos7 [35], MCF-7 [36,37], Huh7 [38], RBL-2H3 [39], RAW264.7 [40], T-cell CEM [41], HT1080 [5], HT29 [15], J774 [42], PtK2 [20,43,44], 3T3 [42,45], DF1 [20], PC12 [19], mouse adenocarcinoma [25,46] Virus-host interactions (membrane remodelling) [48] Mitochondrial fission [35] Chromatin reorganisation [22,23,28] Phenotypic switching in yeast [45] Assembly of viral factories [20] Cholesterol crystal formation in macrophages [40] Plasmodium haeme detoxification [17] and egress…”

Section: Bacteriamentioning

confidence: 99%

“…Addressing this resolution gap, and as a direct result of a growing appetite for ever more diverse, cutting-edge imaging applications, cryo-soft X-ray tomography (cryo-SXT) has developed into a key player in the field. Cryo-SXT is a 3D imaging method for the visualisation of cellular ultrastructure at an intermediate resolution [5] and specifically addresses the need for detailed, 3D information on cellular features in thick specimens, such as whole cells, with little or no chemical or mechanical modification (Figure 2). It allows the observation of biological events on a range of scales, with imaging of details as fine as the internal structure of larger viruses and bacteria to the ultrastructure of yeast, protozoa and mammalian cells (Table 1A) and their associated components such as the nucleus, endoplasmic reticulum, mitochondria, parts of the cytoskeleton and more [47].…”

Section: Introductionmentioning

confidence: 99%

Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells

Harkiolaki

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et al. 2018

Emerging Topics in Life Sciences

100

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Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.

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Quantitative Analyzing the Spatial Organization of the Organelles in Cancer Cell Using Soft X-Ray Tomography

et al. 2017

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Using soft x-ray microscopy (SXM), we are able to image and quantitatively analyze the spatial organization of the organelles within cancer cells [1]. Crucial cellular activities, such as cancer invasion and metastasis, mostly take place within a three-dimensional extracellular matrix (ECM) [2]. Thus, the information of three-dimensional organization of organelles in cancer cells is required to tackle questions regarding those highly remodeling activities. Soft x-ray tomography (SXT), with 50nm spatial resolution [3], provides quantitative information based on the absorption differences from distinct constituents within cells. Image contrast also takes advantage from the property of "water window" of soft x-ray, which means water within cells will be seen as transparent. From the reconstruction tomography, each voxel was given a value, the linear absorption coefficient (LAC), based on the absorption ability. Each organelle has a specific LAC, for example the LAC of lipid droplets is about 0.7 µm -1 , while mitochondria has LAC ranges from 0.31 to 0.35 µmThe isotropic 3D tomographic image is reconstructed based on projections from different angles. To mimic the cellular growth microenvironment, HT1080, human fibrosarcoma cells, tagged with H2B-EGFP and cytoplasmic DsRed2, were cultured within tapered capillaries filled with a reconstructed collagen fiber mixed with growth media, DMEM [2]. The HT1080 cells were first detached from the flask by placing on the ice (4 degree) for ~20 minutes. Cell/collagen mixture was loaded into the tip of capillary using a micropipette and then placed in a conventional incubator (37 degree, 5% CO 2 ) for 60 minutes. This enabled us to collect images of cells in a condition that is closer to their 3D growth environment. Prior to imaging, cells were rapidly frozen by plunging into nitrogen-cooled propane liquid. This not only preserved the cellular structures in their most native state but also protected them from radiation damage.Our preliminary results have shown that we can quantitatively study the heterogeneity of cell volumes (few hundreds to thousands µm 3 ) as well as the morphology of the organelles. The volume of HT1080 cells is very diverse due to their rapid morphological remodeling for migration. Based on the LAC histogram, we can discern euchromatin from heterochromatin. To sum up, using soft x-ray microscopy, we are able to quantitatively study the spatial organization of organelles of cancer cells in their most native state [5].

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Mesoscale imaging with cryo‐light and X‐rays: Larger than molecular machines, smaller than a cell

Cited by 36 publications

References 111 publications

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches

Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells

Quantitative Analyzing the Spatial Organization of the Organelles in Cancer Cell Using Soft X-Ray Tomography

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