What we talk about when we talk about nanoclusters

Baumgart, Florian; Arnold, A.; Rossboth, Benedikt K.; Brameshuber, Mario; Schütz, Gerhard J.

doi:10.1088/2050-6120/aaed0f

Cited by 26 publications

(21 citation statements)

References 137 publications

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“…Taken together, we believe that the 2-CLASTA approach is well suited for a qualitative assessment of spatial two-dimensional biomolecular distributions, before more sophisticated methods are used to characterize the clustering quantitatively 6 . It thereby addresses an interest by the community to include hypothesis testing in SMLM analysis 24 .…”

Section: Resultsmentioning

confidence: 91%

Verifying molecular clusters by 2-color localization microscopy and significance testing

Arnold

Schneider

Hüsson

et al. 2020

Sci Rep

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While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential has not yet been fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the qualitative analysis of two-dimensional SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers. Single Molecule Localization Microscopy (SMLM) has boosted our insights into cellular structures below the diffraction limit of light microscopy 1. Common to all SMLM variants is the stochastic switching of single dye molecules between a bright and a dark state. Conditions are chosen such that only a marginal portion of the molecules is in the bright state, so that single molecule signals are well separated on each frame. The final superresolution image is reconstructed from the localizations of all single molecule signals. Researchers have been particularly intrigued by the possibility to determine the spatial distribution of biomolecules in their natural environment, in most cases the intact cell. For example, models for cellular signaling are crucially affected by the spatial organization of receptor and downstream signaling molecules at the plasma membrane 2,3. Application of SMLM to various plasma membrane proteins revealed the presence of nanoclusters to different degrees 4. More recently concerns were raised that the stochastic activation process of the fluorophores, along with the presence of more than one dye molecule per labeled biomolecule, may lead to multiple observations of the same biomolecule in the superresolution image 5,6. Different attempts were undertaken to approach this problem 5,7-11 , e.g. by merging localization bursts into one localization 12 , by analyzing the number of blinking events per localization cluster 10,11 , or by evaluating the spatial spread of the localization clusters 7. A disadvantage of existing methods is the requirement of user-defined parameters 7,12 or additional experiments to characterize the blinking statistics of the chosen fluorophores 10,11. We recently developed a parameter-free method to identify global protein clustering based on a label titrati...

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Section: Resultsmentioning

confidence: 91%

Verifying molecular clusters by 2-color localization microscopy and significance testing

Arnold

Schneider

Hüsson

et al. 2020

Sci Rep

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“…in refs. 20,21 . As first step, we group the localizations of each oligomer based on the obtained intensities N x and N y .…”

Section: Resultsmentioning

confidence: 99%

A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

Schneider

Telschow

Mercier

et al. 2020

Preprint

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Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

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“…However, the existence of TCR clusters in antigen experienced "resting" cells is less clear, and new analysis suggests that preclustered TCR may be an artifactual symptom of molecular overcounting [8,16]. Alternatively, such clusters may represent ligand-independent triggering of the TCR that happens when T cells come into contact with various surfaces commonly used for in vitro assays, including poly-l-lysine [17].…”

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confidence: 99%

Bridging the Nanoscopy-Immunology Gap

Shannon

Owen

2019

Front. Phys.

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Bridging the gap between traditional immunology and nanoscale biophysics has proved more difficult than originally thought. For cell biology applications however, super-resolution microscopy has already facilitated considerable advances. From neuronal segmentation to nuclear pores and 3D focal adhesion structure-nanoscopy has begun to illuminate links between nanoscale organization and function. With immunology, the explanation must go further, relating nanoscale biophysical phenomena to the manifestation of specific diseases, or the altered activity of specific immune cell types in a bodily compartment. What follows is a summary of how nanoscopy has elucidated single cell immunological function, and what might be achieved in the future to link quantifiable, nanoscale, biophysical phenomena with cell and whole tissue functionality. We explore where the gaps in our understanding occur, and how they might be addressed.

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What we talk about when we talk about nanoclusters

Cited by 26 publications

References 137 publications

Verifying molecular clusters by 2-color localization microscopy and significance testing

Verifying molecular clusters by 2-color localization microscopy and significance testing

A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

Bridging the Nanoscopy-Immunology Gap

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