Varying label density allows artifact-free analysis of membrane-protein nanoclusters.
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.
Institute of Applied Physics, TU Wien, Vienna, Austria.
Florian
F
Baumgart
Institute of Applied Physics, TU Wien, Vienna, Austria.
Andreas M
AM
Arnold
Institute of Applied Physics, TU Wien, Vienna, Austria.
Konrad
K
Leskovar
Institute of Applied Physics, TU Wien, Vienna, Austria.
Kaj
K
Staszek
Institute of Applied Physics, TU Wien, Vienna, Austria.
Martin
M
Fölser
School of Engineering and Environmental Sciences, University of Applied Sciences Upper Austria, Wels, Austria.
Julian
J
Weghuber
Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austria.
Hannes
0000-0001-6404-4430
H
Stockinger
Institute of Applied Physics, TU Wien, Vienna, Austria.
Gerhard J
0000-0003-1542-1089
GJ
Schütz
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016477:Artifacts; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D016000:Cluster Analysis; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D008565:Membrane Proteins