Super‐resolution imaging with stochastic single‐molecule localization: Concepts, technical developments, and biological applications

Oddone, Anna; Vilanova, Ione Verdeny; Tam, Johnny; Lakadamyali, Melike

doi:10.1002/jemt.22346

Cited by 33 publications

(29 citation statements)

References 64 publications

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“…To determine the events that take place at the synapse leading to this decrease of surface NMDAR levels, we used single-molecule localization microscopy, in particular stochastic optical reconstruction microscopy (STORM) (Oddone et al, 2014; Rust et al, 2006). STORM revealed that under control conditions (neurons cultured in the presence of CSF without NMDAR antibodies), the NMDARs are organized in nanosized clusters along the dendrite (Figure 1A), which we refer to as nano-objects.…”

Section: Resultsmentioning

confidence: 99%

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors

et al. 2018

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SUMMARY Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients’ antibodies cause crosslinking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons. Our findings show that, under control conditions, NMDARs form nanosized objects and patients’ antibodies increase the clustering of synaptic and extrasynaptic receptors inside the nano-objects. This clustering is subunit specific and predominantly affects GluN2B-NMDARs. Following internalization, the remaining surface NMDARs return to control clustering levels but are preferentially retained at the synapse. Monte Carlo simulations using a model in which antibodies induce NMDAR cross-linking and disruption of interactions with other proteins recapitulated these results. Finally, activation of EphB2 receptor partially antagonized the antibody-mediated disorganization of the nanoscale surface distribution of NMDARs.

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Section: Resultsmentioning

confidence: 99%

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors

et al. 2018

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“…The comparison and advantages of different superresolution microscopy techniques are reviewed extensively elsewhere 16,18,19,23,35,36 . The main advantage of STORM on biological tissue is its unsurpassed single-molecule localization accuracy, and, consequently, the possibility of high-resolution analysis of molecular distribution.…”

Section: Advantages and Limitationsmentioning

confidence: 99%

“…SMLM is one of the main superresolution approaches, and it relies on determining the precise position of isolated images of single fluorophores 19 . One of its branches, STORM 3 , exploits the photoswitching behavior of certain organic dyes to achieve temporal separation of otherwise overlapping emitters.…”

Section: Introductionmentioning

confidence: 99%

Correlated confocal and super-resolution imaging by VividSTORM

et al. 2015

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Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the life sciences as an efficient approach to visualize molecular distribution with nanoscale precision. However, it has been challenging to obtain and analyze such data within a cellular context in tissue preparations. Here we describe a 5-d tissue processing and immunostaining procedure that is optimized for SMLM, and we provide example applications to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated confocal and 3D-superresolution imaging on these sections, which allows the visualization of nanoscale protein localization within labeled subcellular compartments of identified target cells in a few minutes. Finally, we describe the use of VividSTORM (http://katonalab.hu/index.php/vividstorm), an open-source software for correlated confocal and SMLM image analysis, which facilitates the measurement of molecular abundance, clustering, internalization, surface density and intermolecular distances in a cell-specific and subcellular compartment-restricted manner. The protocol requires only basic skills in tissue staining and microscopy.

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“…In order to achieve this, we must venture beyond the lateral resolution obtainable by conventional LM and confocal microscopy (about 250 nm). By using super-resolution microscopes, such as photoactivated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), spatial resolution can be improved to 25-90 nm in order to obtain a more precise localisation of single molecules (Owen et al 2013;Klein et al 2014;Oddone et al 2014;Sibarita 2014). Resolution can be improved further, to 10 nm, by means of scanning electron microscopy (SEM) and to less than 1 nm by transmission electron microscopy (TEM).…”

Section: Introductionmentioning

confidence: 98%

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

Mayhew

2014

Cell Tissue Res

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Biological systems span multiple levels of structural organisation from the macroscopic, via the microscopic, to the nanoscale. Therefore, comprehensive investigation of systems biology requires application of imaging modalities that reveal structure at multiple resolution scales. Nanomorphomics is the part of morphomics devoted to the systematic study of functional morphology at the nanoscale and an important element of its achievement is the combination of immunolabelling and transmission electron microscopy (TEM). The ultimate goal of quantitative immunocytochemistry is to estimate numbers of target molecules (usually peptides, proteins or protein complexes) in biological systems and to map their spatial distributions within them. Immunogold cytochemistry utilises target-specific affinity markers (primary antibodies) and visualisation aids (e.g., colloidal gold particles or silver-enhanced nanogold particles) to detect and localise target molecules at high resolution in intact cells and tissues. In the case of post-embedding labelling of ultrathin sections for TEM, targets are localised as a countable digital readout by using colloidal gold particles. The readout comprises a spatial distribution of gold particles across the section and within the context of biological ultrastructure. The observed distribution across structural compartments (whether volume- or surface-occupying) represents both specific and non-specific labelling; an assessment by eye alone as to whether the distribution is random or non-random is not always possible. This review presents a coherent set of quantitative methods for testing whether target molecules exhibit preferential and specific labelling of compartments and for mapping the same targets in two or more groups of cells as their TEM immunogold-labelling patterns alter after experimental manipulation. The set also includes methods for quantifying colocalisation in multiple-labelling experiments and mapping absolute numbers of colloidal gold particles across compartments at specific positions within cells having a point-like inclusion (e.g., centrosome, nucleolus) and a definable vertical axis. Although developed for quantifying colloidal gold particles, the same methods can in principle be used to quantify other electron-dense punctate nanoparticles, including quantum dots.

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Super‐resolution imaging with stochastic single‐molecule localization: Concepts, technical developments, and biological applications

Cited by 33 publications

References 64 publications

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors

NMDA Receptor Autoantibodies in Autoimmune Encephalitis Cause a Subunit-Specific Nanoscale Redistribution of NMDA Receptors

Correlated confocal and super-resolution imaging by VividSTORM

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

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