Multiple-Beam Scanning Electron Microscopy

Eberle, Anna Lena; Schalek, Richard; Lichtman, Jeff W.; Malloy, Matthew; Thiel, B. L.; Zeidler, D.

doi:10.1017/s1551929515000012

Cited by 25 publications

(19 citation statements)

References 32 publications

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“…Imaging one square millimetre of tissue, in a single plane, takes just eight minutes, says Stephan Nickell, a product manager at Zeiss in Oberkochen, Germany. By tiling images together, users can get a picture representing a slice of brain that is several millimetres, and sometimes even centimetres, across, but can still zoom in for nanometrescale details 4 .…”

Section: Zooming Inmentioning

confidence: 99%

Connectomes make the map

Dance¹

2015

Nature

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Section: Zooming Inmentioning

confidence: 99%

Connectomes make the map

Dance¹

2015

Nature

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“…If that were to be developed, the available electron microscope time to acquire high resolution images of all such strains in a sample would quickly become limiting. In the future, such limitations could be overcome by increasing data collection speed using multi-beam EM (Lena Eberle et al, 2015) or by precise targeting of data collection using CLEM or immunogold labeling so that only regions of interest are imaged.…”

Section: Discussionmentioning

confidence: 99%

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

Bykov¹,

Cohen

Gabrielli³

et al. 2019

Preprint

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Genetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput.Here we overcome this obstacle and describe a robust method for screening by highthroughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

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“…In contrast, multi-beam scanning electron microscopy (mSEM) circumvents typical throughput limitations inherent to conventional single-beam scanning electron microscopes (sSEM) [6]. Its novel design enables the analysis of nanoscale morphologies across macroscopic specimens by implementing parallel electron beams and a multi-channel detector [10,11]. Multi-beam SEM is capable of reducing acquisition time by more than one order of magnitude and, therefore, of imaging larger surface areas with remarkable resolution [11], paving the path for seamless multiscale imaging of organ systems down to the cellular and even molecular scale [12].…”

Section: Introductionmentioning

confidence: 99%

“…Its novel design enables the analysis of nanoscale morphologies across macroscopic specimens by implementing parallel electron beams and a multi-channel detector [10,11]. Multi-beam SEM is capable of reducing acquisition time by more than one order of magnitude and, therefore, of imaging larger surface areas with remarkable resolution [11], paving the path for seamless multiscale imaging of organ systems down to the cellular and even molecular scale [12]. As a consequence, this technology has drawn interest within the scientific community, particularly in areas related to brain connectomics [13,14] and cross-scale musculoskeletal mechanobiology [2,10].…”

Section: Introductionmentioning

confidence: 99%

Creating High-Resolution Multiscale Maps of Human Tissue Using Multi-beam SEM

et al. 2016

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Multi-beam scanning electron microscopy (mSEM) enables high-throughput, nano-resolution imaging of macroscopic tissue samples, providing an unprecedented means for structure-function characterization of biological tissues and their cellular inhabitants, seamlessly across multiple length scales. Here we describe computational methods to reconstruct and navigate a multitude of high-resolution mSEM images of the human hip. We calculated cross-correlation shift vectors between overlapping images and used a mass-spring-damper model for optimal global registration. We utilized the Google Maps API to create an interactive map and provide open access to our reconstructed mSEM datasets to both the public and scientific communities via our website www.mechbio.org. The nano- to macro-scale map reveals the tissue’s biological and material constituents. Living inhabitants of the hip bone (e.g. osteocytes) are visible in their local extracellular matrix milieu (comprising collagen and mineral) and embedded in bone’s structural tissue architecture, i.e. the osteonal structures in which layers of mineralized tissue are organized in lamellae around a central blood vessel. Multi-beam SEM and our presented methodology enable an unprecedented, comprehensive understanding of health and disease from the molecular to organ length scale.

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Multiple-Beam Scanning Electron Microscopy

Cited by 25 publications

References 32 publications

Connectomes make the map

Connectomes make the map

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

Creating High-Resolution Multiscale Maps of Human Tissue Using Multi-beam SEM

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