Origin and compensation of imaging artefacts in localization-based super-resolution microscopy

Erdélyi, Miklós; Sinkó, József; Kákonyi, Róbert; Kelemen, András; Rees, Eric; Varga, Dániel; Szabó, Gábor

doi:10.1016/j.ymeth.2015.05.025

Cited by 11 publications

(10 citation statements)

References 52 publications

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“…In particular, SMLM methods tend to produce multiple localizations from the same molecule. The difficulty in properly assigning these localizations to the correct molecule or the grouping of localizations remains one of the most stubborn problems (Erdelyi et al, 2015). Without this crucial correction, it is impossible to perform a detailed molecular analysis of microclusters and the IS.…”

Section: Spatial Organization Of Microclustersmentioning

confidence: 99%

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Balagopalan

Raychaudhuri

Samelson

2021

Front. Cell Dev. Biol.

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When T cell receptors (TCRs) engage with stimulatory ligands, one of the first microscopically visible events is the formation of microclusters at the site of T cell activation. Since the discovery of these structures almost 20 years ago, they have been studied extensively in live cells using confocal and total internal reflection fluorescence (TIRF) microscopy. However, due to limits in image resolution and acquisition speed, the spatial relationships of signaling components within microclusters, the kinetics of their assembly and disassembly, and the role of vesicular trafficking in microcluster formation and maintenance were not finely characterized. In this review, we will summarize how new microscopy techniques have revealed novel insights into the assembly of these structures. The sub-diffraction organization of microclusters as well as the finely dissected kinetics of recruitment and disassociation of molecules from microclusters will be discussed. The role of cell surface molecules in microcluster formation and the kinetics of molecular recruitment via intracellular vesicular trafficking to microclusters is described. Finally, the role of post-translational modifications such as ubiquitination in the downregulation of cell surface signaling molecules is also discussed. These results will be related to the role of these structures and processes in T cell activation.

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Section: Spatial Organization Of Microclustersmentioning

confidence: 99%

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Balagopalan

Raychaudhuri

Samelson

2021

Front. Cell Dev. Biol.

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“…The difficulty in properly assigning multiple localizations to the correct molecule or the grouping of localizations remains one of the most stubborn problems in SMLM [67]. Without this crucial correction, it is impossible to perform a detailed molecular analysis of microclusters and the immune synapse.…”

Section: Notesmentioning

confidence: 99%

Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy

Barr

Samelson

2017

The Immune Synapse

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Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.

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“…A strictly controlled photoswitching process is required to keep the number of active fluorescent molecules at an optimum value during the data acquisition 7 . In case of a high labeling density, the spatially overlapping PSFs preclude the precise localization of independent molecules, while at a low density the increased acquisition time limits the final image quality 8 . SMLM is a photon number limited technique.…”

Section: Introductionmentioning

confidence: 99%

mmSTORM: Multimodal localization based super-resolution microscopy

Gajdos

Cserteg

Szikora

et al. 2019

Sci Rep

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Super-resolution localization microscopy provides a powerful tool to study biochemical mechanisms at single molecule level. Although the lateral position of the fluorescent dye molecules can be determined routinely with high precision, measurement of other modalities such as 3D and multicolor without the degradation of the original super-resolved image is still in the focus. In this paper a dual-objective multimodal single molecule localization microscopy (SMLM) technique has been developed, optimized and tested. The proposed optical arrangement can be implemented onto a conventional inverted microscope without serious system modification. The performance of the method was tested using fluorescence beads, F-actin filaments and sarcomere structures. It was shown that the proposed imaging method does not degrade the image quality of the original SMLM 2D image but could provide information on the axial position or emission spectra of the dye molecules.

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Origin and compensation of imaging artefacts in localization-based super-resolution microscopy

Cited by 11 publications

References 52 publications

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy

mmSTORM: Multimodal localization based super-resolution microscopy

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