Retarding Field Integrated Fluorescence and Electron Microscope

Vos, Yoram; Lane, Ryan; Peddie, Christopher J.; Wolters, Anouk H. G.; Hoogenboom, Jacob P.

doi:10.1017/s1431927620024745

Cited by 5 publications

(4 citation statements)

References 44 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%

“…In the past, other groups have obtained results with SEM-imaging of similar preparations using different accelerating voltages and instruments, while information on other parameters was sparse or lacking ( Peddie et al, 2014 ; Markert et al, 2016 ; Bouwer et al, 2017 ). More recently, it was shown that biasing the sample can ameliorate SEM-imaging of sections ( Vos et al, 2021 ). In this study, we show how different imaging parameters affect image quality and propose guidelines on how to optimize basic SEM settings in function of section thickness for high-resolution thin-section imaging.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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%

Section: Introductionmentioning

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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“…Excitation wavelengths of 405 and 555 nm are used to excite Hoechst and AF594. The SECOM is equipped with a CFI S Plan Fluor ELWD 60XC (0.70 NA) objective (Nikon), which allows for long working distance imaging (1.8–2.6 mm), to prevent electrical breakdown in vacuum, which must be accounted for due to the presence of high electric fields induced by the stage bias ( Vos et al, 2021 ).…”

Section: Methodsmentioning

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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“…Retarding (or deceleration) fields applied between electron objective lens and sample have been shown to improve imaging contrast in SEM and FIB-SEM, and therefore allow the reduction of heavy metal staining (Lane et al, 2021). Lately, Vos et al (2020) have successfully applied a retarding (or deceleration) field in an integrated fluorescence and SEM setup. Besides technological improvements, new labelling approaches are being explored.…”

Section: Future Perspectivesmentioning

confidence: 99%

One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM

Heiligenstein¹,

Lucas

2022

Front. Cell Dev. Biol.

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Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an “all in one” sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.

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Retarding Field Integrated Fluorescence and Electron Microscope

Abstract: Abstract

Cited by 5 publications

References 44 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

One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM

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