Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

Lane, Ryan; Wolters, Anouk H. G.; Giepmans, Ben N. G.; Hoogenboom, Jacob P.

doi:10.3389/fmolb.2021.822232

Cited by 7 publications

(6 citation statements)

References 54 publications

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“…Furthermore, the sections can be imaged at the light level prior to electron microscopy, to collect molecular information from immunofluorescence, neural tracers or genetically encoded probes 70 , 71 , 107 , 108 . Integrated light and scanning electron microscopes are a promising solution for efficient acquisition and registration of fluorescence microscopy and electron microscopy images from array tomography specimens 109 , 118 , 119 .…”

Section: Experimentationmentioning

confidence: 99%

Volume electron microscopy

Peddie

Genoud

Kreshuk

et al. 2022

Nat Rev Methods Primers

102

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Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.

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Section: Experimentationmentioning

confidence: 99%

Volume electron microscopy

Peddie

Genoud

Kreshuk

et al. 2022

Nat Rev Methods Primers

102

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“…The latter being transparent, makes it advantageous for LM ( Lucas et al, 2012 ). These arrays can be stored and re-investigated, as well as further stained or contrasted, making array tomography highly suited to localize rare cellular structures or events in complex tissue samples ( Micheva et al, 2010 ; Oberti et al, 2010 ; Burel et al, 2018 ; Lane et al, 2022 ). Analogous to approaches described in the previous subchapter, Höhn et al (2015) detected mCherry-labeled virus particles by en bloc CLSM, prior to acquisition of volume data by FIB-SEM.…”

Section: One Sample For All Imaging Modes or The Ideal Clem Sample: M...mentioning

confidence: 99%

“…Nucleic stains such as DAPI or Hoechst can be easily applied on sections, both on SEM and TEM sample supports. These specific, intercalating dyes can still interact with chromatin in resin-embedded samples, and yield a bright and stable fluorescent signal ( Lucas et al, 2012 ; Delevoye et al, 2016 ; Lane et al, 2022 ).…”

Section: One Sample For All Imaging Modes or The Ideal Clem Sample: M...mentioning

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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“…AT can be combined with other imaging techniques in what’s called conjugate AT [ 50 , 76 , 77 ]. For example, careful sample processing allows the combination of AT with EM.…”

Section: Recent Advances In Array Tomographymentioning

confidence: 99%

Array tomography: 15 years of synaptic analysis

Avila

Henstridge

2022

Neuronal Signaling

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Synapses are minuscule, intricate structures crucial for the correct communication between neurons. In the 125 years since the term synapse was first coined, we have advanced a long way when it comes to our understanding of how they work and what they do. Most of the fundamental discoveries have been invariably linked to advances in technology. However, due to their size, delicate structural integrity and their sheer number, our knowledge of synaptic biology has remained somewhat elusive and their role in neurodegenerative diseases still remains largely unknown. Here, we briefly discuss some of the imaging technologies used to study synapses and focus on the utility of the high-resolution imaging technique array tomography. We introduce the array tomography technique and highlight some of the ways it is utilised with a particular focus on its power for analysing synaptic composition and pathology in human post-mortem tissue. We also discuss some of the benefits and drawbacks of techniques for imaging synapses and highlight some recent advances in the study of form and function by combining physiology and high-resolution synaptic imaging.

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Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

Cited by 7 publications

References 54 publications

Volume electron microscopy

Volume electron microscopy

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

Array tomography: 15 years of synaptic analysis

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