Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy

Lane, Ryan; Vos, Yoram; Wolters, Anouk H. G.; Kessel, Luc Van; Chen, S. Elisa; Liv, Nalan; Klumperman, Judith; Giepmans, Ben N. G.; Hoogenboom, Jacob P.

doi:10.1016/j.yjsbx.2021.100046

Cited by 7 publications

(7 citation statements)

References 35 publications

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“…Bouwer et al ( Bouwer et al, 2017 ) proposed applying a negative bias voltage to the sample to decrease the interaction volume for a given acceleration voltage and improve sectioning capabilities in block-face SEM, where the block face is imaged. More recently, Vos et al (2021) and Lane et al (2021) extended this approach to BSE-imaging of sections in a SEM. Although very useful, this condition is not available to many SEM-users.…”

Section: Discussionmentioning

confidence: 99%

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Baatsen

Gabarre

Vandael³

et al. 2021

Front. Cell Dev. Biol.

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Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

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Section: Discussionmentioning

confidence: 99%

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Baatsen

Gabarre

Vandael³

et al. 2021

Front. Cell Dev. Biol.

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“…Both low and high magnification imaging are performed at 2.5 keV primary beam energy with a −1 kV bias potential applied to the sample stage such that the landing energy is 1.5 keV, which proved optimal for ∼100 nm sections. The negative potential bias enhances the backscattered electron (BSE) signal, which is collected by the insertable concentric backscattered detector (Thermo Fisher Scientific) ( Lane et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

“…First, to prevent damaging or quenching of the fluorescence signal via electron-beam irradiation, each FM field of view must be acquired prior to EM exposure. Second, to compensate for the reduced application of contrast agents, backscattered electron (BSE) collection efficiency is enhanced via a negative stage bias, allowing for higher throughput ( Bouwer et al, 2016 ; Lane et al, 2021 ). Finally, a high precision EM-FM overlay is facilitated by the use of cathodoluminescent (CL) points, which eliminates the need for artificial fiducial markers ( Haring et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

Lane

Wolters

Giepmans

et al. 2022

Front. Mol. Biosci.

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Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs.

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“…The final but very important component of the image acquisition system is software that allows automatic imaging of large sample areas. Research has demonstrated that the signal-to-noise ratio can be significantly improved by applying a negative bias voltage to the sample (beam deceleration) with a simultaneous increase in the acceleration voltage [ 54 , 55 , 56 , 57 ]. The increase in the recorded signal occurs owing to the re-acceleration of BSE in the bias field toward the detector.…”

Section: Multiscale Imaging Of Large Sample Areasmentioning

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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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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Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy

Cited by 7 publications

References 35 publications

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

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

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