Integrated Correlative Light Electron Microscopy for Automated Array Tomography

Lane, Ryan; Boer, Pascal de; Giepmans, Ben N. G.; Hoogenboom, Jacob P.

doi:10.1017/s1431927618002350

Cited by 2 publications

(3 citation statements)

References 4 publications

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“…The term array tomography, depending on application, comprises three different techniques: (i) fluorescence microscopy array tomography, which delivers volumetric, high-resolution data on the distribution of molecules and enables the detection of several antigens in the same section [ 59 , 64 ], (ii) electron microscopy array tomography, which enables the capturing of ultrathin sections for 3D ultrastructural studies [ 65 ], and (iii) correlative array tomography [ 66 , 67 , 68 ], which combines fluorescence imaging and electron microscopy imaging to obtain voxel-level associations between structure and chemistry. The new concept is the use of array tomography to locate targets for z -axis high-resolution imaging through FIB-SEM [ 69 ].…”

Section: Array Tomographymentioning

confidence: 99%

“…However, note that the best results are obtained after the intensive infiltration of samples with heavy metals because of the lack of charging and section contrasting artifacts. Array tomography enables correlative light and electron microscopy studies, and their combination with 3D analyses [ 66 , 68 , 74 ]. The disadvantages of this method are the time-consuming preparation of sections for imaging and the alignment of sections into z-stacks.…”

Section: Array Tomographymentioning

confidence: 99%

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Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

Lewczuk

Szyryńska

2021

Animals

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The development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.

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Section: Array Tomographymentioning

confidence: 99%

Section: Array Tomographymentioning

confidence: 99%

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

Lewczuk

Szyryńska

2021

Animals

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“…Given that on iLEMs, typical ultrathin sections are imaged, these devices are the ideal candidate for correlative AT [17]. However, so far, correlative AT using an iLEM system has not been widely adopted [17,23].…”

Section: Introductionmentioning

confidence: 99%

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography

Gabarre

Vernaillen²,

Baatsen

et al. 2021

BMC Biol

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Background Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. Results We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. Conclusions Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

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Integrated Correlative Light Electron Microscopy for Automated Array Tomography

Cited by 2 publications

References 4 publications

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography

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