Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.

Bordeaux Imaging Center UMS 3420 CNRS Université de Bordeaux US4 INSERM 33077 Bordeaux France

Department of Biophysical Chemistry J Heyrovsky Institute of Physical Chemistry Czech Academy of Sciences 182 23 Prague Czech Republic

Department of Biotechnology and Biophysics Biocenter University of Wuerzburg D 97074 Wuerzburg Germany

Department of Radioelectronics FEE Czech Technical University Prague 166 27 Prague Czech Republic

Imaging Methods Core Facility BIOCEV 252 50 Vestec u Prahy Czech Republic

Interdisciplinary Institute for Neuroscience UMR 5297 CNRS Université de Bordeaux 33077 Bordeaux France

Laboratoire d'Optique Biomédicale École Polytechnique Fédérale de Lausanne STI IBI CH 1015 Lausanne Switzerland

Laboratory of Leukocyte Signalling Institute of Molecular Genetics Czech Academy of Sciences 142 20 Prague Czech Republic

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