Varying label density allows artifact-free analysis of membrane-protein nanoclusters

Florian Baumgart; Andreas Arnold; Konrad Leskovar; Kaj Staszek; Martin Fölser; Julian Weghuber; Hannes Stockinger; Gerhard Schütz

doi:10.1038/NMETH.3897

Varying label density allows artifact-free analysis of membrane-protein nanoclusters

Florian Baumgart, Andreas Arnold, Konrad Leskovar, Kaj Staszek, Martin Fölser, Julian Weghuber, Hannes Stockinger, Gerhard Schütz

Research Center Wels

Research output: Contribution to journal › Article › peer-review

103 Citations (Scopus)

Abstract

We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.

Original language	English
Pages (from-to)	661-664
Number of pages	4
Journal	Nature Methods
Volume	13
Issue number	8
DOIs	https://doi.org/10.1038/NMETH.3897
Publication status	Published - 28 Jul 2016

Keywords

Animals
Antibodies, Monoclonal/chemistry
Artifacts
CHO Cells
Cell Membrane/metabolism
Cluster Analysis
Cricetulus
Fluorescent Dyes/chemistry
Humans
Jurkat Cells
Light
Membrane Proteins/chemistry
Microscopy, Fluorescence/methods
Nanostructures/chemistry

Access to Document

10.1038/NMETH.3897

Cite this

@article{870e06fc34594f439d203a451f69e923,

title = "Varying label density allows artifact-free analysis of membrane-protein nanoclusters",

abstract = "We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.",

keywords = "Animals, Antibodies, Monoclonal/chemistry, Artifacts, CHO Cells, Cell Membrane/metabolism, Cluster Analysis, Cricetulus, Fluorescent Dyes/chemistry, Humans, Jurkat Cells, Light, Membrane Proteins/chemistry, Microscopy, Fluorescence/methods, Nanostructures/chemistry",

author = "Florian Baumgart and Andreas Arnold and Konrad Leskovar and Kaj Staszek and Martin F{\"o}lser and Julian Weghuber and Hannes Stockinger and Gerhard Sch{\"u}tz",

year = "2016",

month = jul,

day = "28",

doi = "10.1038/NMETH.3897",

language = "English",

volume = "13",

pages = "661--664",

journal = "Nature Methods",

issn = "1548-7105",

publisher = "Nature Publishing Group",

number = "8",

}

TY - JOUR

T1 - Varying label density allows artifact-free analysis of membrane-protein nanoclusters

AU - Baumgart, Florian

AU - Arnold, Andreas

AU - Leskovar, Konrad

AU - Staszek, Kaj

AU - Fölser, Martin

AU - Weghuber, Julian

AU - Stockinger, Hannes

AU - Schütz, Gerhard

PY - 2016/7/28

Y1 - 2016/7/28

N2 - We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.

AB - We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.

KW - Animals

KW - Antibodies, Monoclonal/chemistry

KW - Artifacts

KW - CHO Cells

KW - Cell Membrane/metabolism

KW - Cluster Analysis

KW - Cricetulus

KW - Fluorescent Dyes/chemistry

KW - Humans

KW - Jurkat Cells

KW - Light

KW - Membrane Proteins/chemistry

KW - Microscopy, Fluorescence/methods

KW - Nanostructures/chemistry

UR - http://www.scopus.com/inward/record.url?scp=84979993369&partnerID=8YFLogxK

U2 - 10.1038/NMETH.3897

DO - 10.1038/NMETH.3897

M3 - Article

C2 - 27295310

SN - 1548-7105

VL - 13

SP - 661

EP - 664

JO - Nature Methods

JF - Nature Methods

IS - 8

ER -

Varying label density allows artifact-free analysis of membrane-protein nanoclusters

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Cite this