On the road to large volumes in LM and SEM: New tools for Array Tomography

BackgroundImaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM) large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited on solid substrates like silicon wafers or glass coverslips.ResultsTo ensure reliable retrieval of multiple ribbons from the boat of a diamond knife we introduce a substrate holder with 7 axes of translation or rotation specifically designed for that purpose. With this device we are able to deposit hundreds of sections in an ordered way in an area of 22 × 22 mm, the size of a coverslip. Imaging such arrays in a standard wide field fluorescence microscope produces reconstructions with 200 nm lateral resolution and 100 nm (the section thickness) resolution in z.By hierarchical imaging cascades in the scanning electron microscope (SEM), using a new software platform, we can address volumes from single cells to complete organs. In our first example, a cell population isolated from zebrafish spleen, we characterize different cell types according to their organelle inventory by segmenting 3D reconstructions of complete cells imaged with nanoscale resolution. In addition, by screening large numbers of cells at decreased resolution we can define the percentage at which different cell types are present in our preparation. With the second example, the root tip of cress, we illustrate how combining information from intermediate resolution data with high resolution data from selected regions of interest can drastically reduce the amount of data that has to be recorded. By imaging only the interesting parts of a sample considerably less data need to be stored, handled and eventually analysed.ConclusionsOur custom-designed substrate holder allows reproducible generation of section libraries, which can then be imaged in a hierarchical way. We demonstrate, that EM volume data at different levels of resolution can yield comprehensive information, including statistics, morphology and organization of cells and tissue. We predict, that hierarchical imaging will be a first step in tackling the big data issue inevitably connected with volume EM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0122-8) contains supplementary material, which is available to authorized users.

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“…Some preliminary, technical details of this workflow were already presented as abstracts to a specialized microscopy audience [16, 17]. …”

Section: Discussionmentioning

confidence: 99%

Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues

et al. 2016

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“…For manual preparation of a small to medium number of sections, a micromanipulator setup with an advanced substrate holder [ASH (Spomer et al, 2015)] is sufficient. This is especially useful with small samples and for the preparation of ribbons of sections (Wacker et al, 2015). Sections can be placed directly onto a silicon wafer as substrate, which is advantageous for imaging in a SEM, or onto indium tin oxide coated coverslips, such that imaging with light microscopes is possible as well.…”

Section: Overview Of Different Approaches To Volume Electron Microscopymentioning

confidence: 99%

Multi-Beam Scanning Electron Microscopy for High-Throughput Imaging in Connectomics Research

Eberle

Zeidler

2018

Front. Neuroanat.

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Major progress has been achieved in recent years in three-dimensional microscopy techniques. This applies to the life sciences in general, but specifically the neuroscientific field has been a main driver for developments regarding volume imaging. In particular, scanning electron microscopy offers new insights into the organization of cells and tissues by volume imaging methods, such as serial section array tomography, serial block-face imaging or focused ion beam tomography. However, most of these techniques are restricted to relatively small tissue volumes due to the limited acquisition throughput of most standard imaging techniques. Recently, a novel multi-beam scanning electron microscope technology optimized to the imaging of large sample areas has been developed. Complemented by the commercialization of automated sample preparation robots, the mapping of larger, cubic millimeter range tissue volumes at high-resolution is now within reach. This Mini Review will provide a brief overview of the various approaches to electron microscopic volume imaging, with an emphasis on serial section array tomography and multi-beam scanning electron microscopic imaging.

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“…Use of FM/EM-AT (Figs. 2 , 6 and 7 ) offers unique opportunities to combine the molecular discrimination strengths of fluorescence microscopy with the unrivaled structural resolution of electron microscopy [ 1 , 2 , 6 , 11 , 14 , 15 , 17 , 27 – 37 ]. While other approaches to correlative light and electron microscopy of individual specimens have proven extremely useful [ 38 ], AT alone offers a path to perfect registration of fluorescence and SEM voxels over extended volumes.…”

Section: When Should One Consider Using At?mentioning

confidence: 99%

“…Much improved FM-AT and EM-AT image quality and array stability are now obtained by replacing the gelatin with a thin, transparent layer of evaporated carbon laid down upon a silanized coverslip surface [ 31 ]. Flexible polymer substrates, such as carbon-coated Kapton tape, have enabled the use of a simple tape-transport method for automated, high-throughput array section collection [ 2 , 10 , 29 , 49 , 55 ]. Continuous tapes of array sections may be readily cut into shorter segments and glued to small silicon wafers in multi-row arrays for SEM imaging.…”

Section: How Are Arrays Constructed?mentioning

confidence: 99%

Q&A: Array tomography

Smith

2018

BMC Biol

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Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0560-1) contains supplementary material, which is available to authorized users.

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On the road to large volumes in LM and SEM: New tools for Array Tomography

Cited by 6 publications

References 3 publications

Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues

Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues

Multi-Beam Scanning Electron Microscopy for High-Throughput Imaging in Connectomics Research

Q&A: Array tomography

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