Light microscopy of proteins in their ultrastructural context

M’Saad, Ons; Bewersdorf, Joerg

doi:10.1038/s41467-020-17523-8

Cited by 112 publications

(199 citation statements)

References 39 publications

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“…In electron microscopy, non-specific stains for proteins and membranes are commonly used to provide structural detail at high spatial resolution. Recently, the use of non-specific NHS ester protein stains and other small molecule probes has been combined with ExM (M’Saad and Bewersdorf, 2020; Mao et al ., 2020; Yu et al ., 2020). Expansion allows visualization of intracellular detail in densely stained samples, which would not otherwise lead to meaningful contrast.…”

Section: Resultsmentioning

confidence: 99%

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Damstra

Eddison

Akhmanova

et al. 2021

Preprint

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Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the polymer gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors, for example using iterative expansion, but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.

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Section: Resultsmentioning

confidence: 99%

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Damstra

Eddison

Akhmanova

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…We anticipate that whole-ExM could be used to visualize the sub-10-nm details of protein structures over the whole organism. In this work, specimens were expanded only 4-fold, but a higher expansion factor, such as 10-or even 20-fold, has been demonstrated through iterative expansion 13,30,53,54 . A larger expansion factor does not always guarantee a higher resolution due to expansion inhomogeneity or signal blurring, but recent studies have reported that expansion microscopy conserves the sub-10-nm details of protein structures 13,55,56 .…”

Section: Discussionmentioning

confidence: 97%

“…We first investigated whether all anatomically relevant structures could be visualized using fluorophore NHS-ester staining. Recently developed expansion microscopy techniques visualize diverse cellular structures and cellular organelles by conjugating fluorophores to functional groups of specific molecular types [12][13][14]16 , such as the conjugation of fluorophore NHS esters to the primary amines of proteins [12][13][14] .…”

Section: Effect Of the Physicochemical Properties Of Fluorophores On The Range Of Labeled Structuresmentioning

confidence: 99%

“…Recently developed ExM techniques utilizing fluorophore labeling have enabled the unbiased visualization of nanoscale details of subcellular structures and organelles by labeling various types of molecules, such as proteins 12,14,24 , nucleic acids 12,16 , or lipids 15,16 , without antibodies [12][13][14]16 . However, the application of ExM to whole organisms has been limited to relatively small and simple organisms, such as bacteria [25][26][27] , fungi 28 , and parasitic micro-organisms 29 .…”

Section: Introductionmentioning

confidence: 99%

“…Expansion microscopy (ExM) combined with the labeling of functional groups of specimens with fluorophores would be an attractive candidate for satisfying such a need [12][13][14][15][16] . ExM can image proteins or mRNA in diverse specimens with a lateral resolution of 60-nm by physically expanding the specimens [17][18][19][20][21][22][23] .…”

Section: Introductionmentioning

confidence: 99%

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Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy

Sim

et al. 2021

Preprint

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Nanoscale imaging of all anatomical structures over whole vertebrates is needed for a systematic understanding of human diseases, but this has not yet been achieved. Here, we demonstrate whole-ExM, which enables nanoscale imaging of all anatomical structures of whole zebrafish larvae by labeling the proteins of the larvae with fluorophores and expanding them four-fold. We first optimize the fluorophore selection and labeling procedure to visualize a broader range of anatomical structures. We then develop an expansion protocol for zebrafish larvae having calcified body parts. Through this process, we visualize the nanoscale details of diverse larvae organs, which have corresponding organ counterparts in humans, over the intact larvae. We show that whole-ExM retains the fluorescence signals of fluorescent proteins, and its resolution is high enough to visualize various structures that can be imaged only with electron microscopy. Whole-ExM would enable the nanoscale study of the molecular mechanisms of human diseases.

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Super‐Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy

et al. 2022

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Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, a new technique called molecule anchorable gel‐enabled nanoscale Imaging of Fluorescence and stimulated Raman scattering microscopy (MAGNIFIERS) that integrates SRS microscopy with expansion microscopy (ExM) is described. MAGNIFIERS offers chemical‐specific nanoscale imaging with sub‐50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. MAGNIFIERS is used to visualize nanoscale features in a label‐free manner with CH vibration of proteins, lipids, and DNA in a broad range of biological specimens, from mouse brain, liver, and kidney to human lung organoid. In addition, MAGNIFIERS is applied to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, MAGNIFIERS is used to demonstrate 8‐color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super‐resolution label‐free chemical imaging, high‐resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

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Light microscopy of proteins in their ultrastructural context

Cited by 112 publications

References 39 publications

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy

Super‐Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy

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