Membrane Architecture in the Spotlight of Correlative Microscopy

Ganeva, Iva; Kukulski, Wanda

doi:10.1016/j.tcb.2020.04.003

Cited by 17 publications

(14 citation statements)

References 75 publications

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“…Correlative light and electron microscopy (CLEM), the coupling of fluorescent light microscopy (FLM) and transmission electron microscopy (TEM), adds temporal and spatial information from FLM to the high-resolution structural information of EM, facilitating identification of rare or dynamic events during host-virus interactions ( Afzelius & Maunsbach, 2004 ; Bharat & Kukulski, 2019 ; Bykov et al, 2016 ; Ganeva & Kukulski, 2020 ; Laue, 2010 ; Romero-Brey, 2018 ). To preserve cellular structures and overall specimen integrity, biological samples undergo steps of fixation prior to EM imaging ( McDonald, 2009 ; Passmore & Russo, 2016 ; Romero-Brey, 2018 ; Romero-Brey and Bartenschlager, 2015 ; Studer et al, 2008 ; Thompson et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

Yang

Larson

Sibert

et al. 2021

Journal of Structural Biology

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Section: Introductionmentioning

confidence: 99%

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

Yang

Larson

Sibert

et al. 2021

Journal of Structural Biology

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“…From an exquisitely demanding technique practised by a few experts who occasionally, and with a great deal of effort, produced high-resolution structures, it has now become a mainstay of molecular cell biology in countless laboratories around the world. CryoEM spans the wide range from single-particle protein structures at truly atomic resolution (Nakane et al ., 2020; Yip et al ., 2020) to large assemblies in cells and tissues investigated by electron cryo-tomography (Nievergelt et al ., 2019), sub-tomogram averaging (Pfeffer and Mahamid, 2018) and correlative light and electron microscopy (Ganeva and Kukulski, 2020; Walter et al ., 2020). For these reasons, cryoEM is particularly promising as a technique for the future of structural biology.…”

Section: Discussionmentioning

confidence: 99%

Current limitations to high-resolution structure determination by single-particle cryoEM

D’Imprima

Kühlbrandt

2021

Quart. Rev. Biophys.

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CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam-induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. We examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution.

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“…In a narrow sense, correlative microscopy refers to correlative (also referred to as correlated) light and electron microscopy (CLEM) (de Boer et al, 2015), a method that integrates imaging information from light and electron microscopy of the same specimen (Ganeva and Kukulski, 2020;. One of the early reports of such an approach appeared (Bopp-Hassenkamp, 1959) soon after electron microscopy was first applied to biological specimens.…”

Section: Correlative Microscopymentioning

confidence: 99%

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

Kawano

Kakazu

Iwabuchi

et al. 2020

Preprint

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ABSTRACTBackgroundCorrelation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems.New methodHere we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets.ResultsWe imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level.Comparison with existing methodsThis method aligned images with minimal change or investment in the experimental systems.ConclusionsThis method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.

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Membrane Architecture in the Spotlight of Correlative Microscopy

Cited by 17 publications

References 75 publications

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy

Current limitations to high-resolution structure determination by single-particle cryoEM

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

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