A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

Handschuh, Stephan; Baeumler, Natalie; Schwaha, Thomas; Ruthensteiner, Bernhard

doi:10.1186/1742-9994-10-44

Cited by 98 publications

(96 citation statements)

References 49 publications

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“…The use of micro-CT microscopy proved a powerful tool for the accurate relocation of the ROI -a central vein that acted as a morphological landmark -as a consequence of the sample being rendered opaque to light, proceeding sample preparation for EM. This practice further ensured that a greater volume of the sample could be collected in the SBF-SEM, due to difficulty in deciding where to capture data from the sample block face{Karreman et al 49 #69; Handschuh et al 50 #67; Bushong et al 51 #66}. Whilst micro-CT was simply used as a relocation tool, future studies could plausibly exploit the technique to further extract relevant structural information.…”

Section: Discussionmentioning

confidence: 99%

3‐D EM exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning

Shami

Cheng

Huynh

et al. 2016

European Microscopy Congress 2016: Proceedings

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To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. Herein, we provide a comprehensive and rigorous appraisal of five different SBF-SEM sample preparation protocols for the large-volume exploration of the hepatic microarchitecture at an unparalleled X, Y and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a comprehensive SBF-SEM sample preparation protocol, based on the application of heavy metal fixatives, stains and mordanting agents. Employing the best-tested SBF-SEM approach, enabled us to assess large-volume morphometric data on murine parenchymal cells, sinusoids and bile canaliculi. Finally, we integrated the validated SBF-SEM protocol with a correlative light and electron microscopy (CLEM) approach. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions. 4 #19}, heralded a new era in analysing the complex geometric arrangement of biological cells and tissues at the nanometre scale. Throughout the infancy of EM techniques, including both transmission (TEM) and scanning electron microscopy (SEM), the structural information gained was limited to two-dimensional (2-D) space, providing a mere snapshot of innately three-dimensional (3-D) structures.Throughout the mid-1950s, advances in ultramicrotome design and specimen preparation procedures{Porter and Blum 5 #25} facilitated the generation of 3-D ultrastructural information via the superimposition of consecutive 2-D TEM micrographs that were acquired from a serially sectioned specimen -a technique dubbed serial-section TEM (ssTEM){#387 }. Currently, ssTEM is regarded as a partial effort in the generation of 3-D information, primarily due to the fact that it is a highly arduous and labour-intensive technique that is riddled with several sources of error, such as section compression and the loss of serial sections, which ultimately results in an incomplete data set. The tediousness of the technique is further compounded by the necessity to align image stacks in order to correct for misalignments that occur during the manual imaging of successive sections{Williams and Kallman 12 #15}. Moreover, the generation of high-resolution axial resolution is

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Section: Discussionmentioning

confidence: 99%

3‐D EM exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning

Shami

Cheng

Huynh

et al. 2016

European Microscopy Congress 2016: Proceedings

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“…Furthermore, combination with other techniques—for instance fluorescence microscopy—may allow to identify processes within the tissues for future studies on ecophysiology and ecotoxicology (e.g., angiogenesis, apoptosis, inflammation) (Gremse et al., 2015; Handschuh, Baeumler, Schwaha, & Ruthensteiner, 2013; Metscher, 2009a). Possible detrimental effects of radiation and stain ingestion on living organisms (especially invertebrates) remain to be elucidated.…”

Section: Perspectives and Final Considerationsmentioning

confidence: 99%

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

Gutiérrez

Ott

Töpperwien

et al. 2018

Ecology and Evolution

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Imaging techniques are a cornerstone of contemporary biology. Over the last decades, advances in microscale imaging techniques have allowed fascinating new insights into cell and tissue morphology and internal anatomy of organisms across kingdoms. However, most studies so far provided snapshots of given reference taxa, describing organs and tissues under “idealized” conditions. Surprisingly, there is an almost complete lack of studies investigating how an organism′s internal morphology changes in response to environmental drivers. Consequently, ecology as a scientific discipline has so far almost neglected the possibilities arising from modern microscale imaging techniques. Here, we provide an overview of recent developments of X‐ray computed tomography as an affordable, simple method of high spatial resolution, allowing insights into three‐dimensional anatomy both in vivo and ex vivo. We review ecological studies using this technique to investigate the three‐dimensional internal structure of organisms. In addition, we provide practical comparisons between different preparation techniques for maximum contrast and tissue differentiation. In particular, we consider the novel modality of phase contrast by self‐interference of the X‐ray wave behind an object (i.e., phase contrast by free space propagation). Using the cricket Acheta domesticus (L.) as model organism, we found that the combination of FAE fixative and iodine staining provided the best results across different tissues. The drying technique also affected contrast and prevented artifacts in specific cases. Overall, we found that for the interests of ecological studies, X‐ray computed tomography is useful when the tissue or structure of interest has sufficient contrast that allows for an automatic or semiautomatic segmentation. In particular, we show that reconstruction schemes which exploit phase contrast can yield enhanced image quality. Combined with suitable specimen preparation and automated analysis, X‐ray CT can therefore become a promising quantitative 3D imaging technique to study organisms′ responses to environmental drivers, in both ecology and evolution.

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“…4, 8). Staining is a crucial stage of micro-CT studies and is directly related to the final resolution of images of other soft-bodied organisms successfully stained with osmium tetroxide as a contrast enhancer (Handschuh et al, 2013;Fernández et al, 2014;Holst et al, 2016;Tessler et al, 2016), including other soft-bodied cnidarians (i.e., staurozoans, Holst et al, 2016). The core of Diadumene manezinha ( fig.…”

Section: Fig 12 (Continued) Diadumene Lineatamentioning

confidence: 99%

Sea Anemones through X-Rays: Visualization of Two Species ofDiadumene(Cnidaria, Actiniaria) Using Micro-CT

Gusmão

Grajales

Rodríguez

2018

American Museum Novitates

821

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Diadumenidae is a monogeneric family comprised of 10 valid species in the genus Diadumene. Although these species are distributed in all but the Southern Ocean, Diadumene lineata is a cosmopolitan species and one of the most widely distributed marine organisms. Recent phylogenetic analyses with multiple species have recovered Diadumenidae as a monophyletic group with high support. Although generic placement is straightforward, species-level identifications within Diadumene are difficult because species are defined by a mosaic of characters that vary in degree rather than kind. Two species of the genus have been recorded in the southwestern Atlantic: D. paranaensis along the south coast of Brazil and D. lineata along the southeast and northeast coasts of Brazil as well as in Argentina. Here we record D. leucolena for the first time in the southern hemisphere and describe D. manezinha, sp. nov., raising to four the number of species of Diadumene for Brazil and to 11 worldwide. We incorporated microcomputed tomography (micro-CT) into the description of both species in the first application of the method for Actiniaria. We evaluate the utility of micro-CT imaging and its potential to generate fast, low-cost, and high-resolution datasets despite the anemone's low-density soft tissue. We present a protocol for sample handling, chemical staining, and scanning parameters that resulted in satisfactory imaging of the two specimens examined. We also analyze advantages and limitations of using micro-CT over traditional techniques in the study of sea anemones.

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A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

Cited by 98 publications

References 49 publications

3‐D EM exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning

3‐D EM exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

Sea Anemones through X-Rays: Visualization of Two Species ofDiadumene(Cnidaria, Actiniaria) Using Micro-CT

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