Coordinate-based colocalization analysis of single-molecule localization microscopy data

Malkusch, Sebastian; Endesfelder, Ulrike; Mondry, Justine; Gelléri, Márton; Verveer, Peter J.; Heilemann, Mike

doi:10.1007/s00418-011-0880-5

Cited by 179 publications

(214 citation statements)

References 25 publications

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“…2A) and developed an analysis strategy that combines cluster detection and colocalization analysis. The degree-of-colocalization (DoC) analysis (29) determines the local density for each molecule at increasing radii, providing a density gradient for each channel (Fig. 2B).…”

Section: Cd3ζ Molecules Exist In Nanoclusters That Increase In Molecularmentioning

confidence: 99%

“…Two-Color Colocalization Analysis. The two-color data were analyzed using a modified version of the coordinate-based colocalization method (29). The first step in the analysis is to remove molecules which are isolated, by excluding points with a local density below a random distribution (total density = total number of molecules/total area of region of interest).…”

Section: Primary Mouse Ot-i Cd8mentioning

confidence: 99%

“…To identify the functional role of TCR nanoclusters, we used two-color SMLM data and integrated a cluster detection method, density-based spatial clustering of applications with noise (DBSCAN) (28) with a customized colocalization analysis (29). This process allowed us to distinguish phosphorylated from nonphosphorylated TCR-CD3 complex clusters in intact T cells and identify the spatial organization at which individual TCR-CD3 complexes had the highest signaling efficiency.…”

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confidence: 99%

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Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Pageon

Tabarin

Yamamoto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

207

235

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Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

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Section: Cd3ζ Molecules Exist In Nanoclusters That Increase In Molecularmentioning

confidence: 99%

Section: Primary Mouse Ot-i Cd8mentioning

confidence: 99%

mentioning

confidence: 99%

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Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Pageon

Tabarin

Yamamoto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

207

235

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“…Fluorescence images produced by stochastic optical reconstruction microscopy (STORM) allows superior subcellular localization of proteins and visualization of interacting proteins as adjacent particles at nanometer resolution (21). Direct STORM (dSTORM) (22) relies on the reversible photoswitching of fluorophores.…”

Section: K Atp Channel Subunits Associate With Plakoglobin Andmentioning

confidence: 99%

Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes

Hong

Kefaloyianni

et al. 2012

Journal of Biological Chemistry

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Ventricular KATP channels link intracellular energy metabolism to membrane excitability and contractility. We identified plakoglobin (PG) and plakophilin‐2 (PKP2) as KATP channel associated proteins and investigated whether the association of KATP channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co‐immunoprecipitation experiments confirmed physical interaction between KATP channels and PKP2 and PG in rat heart. Immunolocalization experiments demonstrated that KATP channel subunits are expressed at a higher density at the intercalated disk (ICD) in hearts, where they colocalized with PKP2 and PG. Super‐resolution microscopy demonstrate that KATP channels are clustered within nanometer distances from junctional proteins. The local KATP channel density was larger at the cell end when compared to local currents recorded from the cell's center. The KATP channel unitary conductance, block by MgATP and activation by MgADP did not differ between these two locations. Whole‐cell KATP channel current density was ~40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of KATP channels was absent in the PKP2 deficient mice, but the KATP channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of KATP channel distribution within a cardiac myocyte. The higher KATP channel density at the ICD implies a possible role at the intercellular junctions during cardiac ischemia. Funding from NIH HL085820.

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“…It turns out that the most commonly used parameter, the so-called Pearson's Correlation Coefficient (PCC) is superior to the Manders Overlap Coefficient [13,14] (MOC) which is currently implemented in most commercial software packages for image processing [15]. A closer look at the PCC, however, reveals that it has some frequently cited major drawbacks which significantly limit its utility for colocalization analysis in certain cases [16][17][18][19][20]. For example, it was found that the PCC, which can adopt values between þ1 and À1 as a measure of the similarity of channels (+1: perfect colocalization; values between 0 and À1: no colocalization), is very sensitive to strong intensity fluctuations or threshold variations.…”

Section: Biophotonicsmentioning

confidence: 99%

Quantifying molecular colocalization in live cell fluorescence microscopy

Humpert

Yahiatène

Lummer

et al. 2013

Journal of Biophotonics

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One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal‐to‐noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live‐cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)

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Coordinate-based colocalization analysis of single-molecule localization microscopy data

Cited by 179 publications

References 25 publications

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes

Quantifying molecular colocalization in live cell fluorescence microscopy

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