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M. Schneider, A. Arnold, F. Baumgart, R. Sablatnig, C. Hüsson, M. Brameshuber, G. Schütz:
"2-Color Localization Microscopy and Significance Testing Approach (2-CLASTA)";
Biophysical Journal, 118 (2020), 3, Supplement 1; 167a.

Knowledge about the organization of proteins at the plasma membrane is key to our understanding of cellular signaling processes. Studies using singlemolecule localization microscopy (SMLM) have led to the notion that protein nanoclustering is a prevalent feature of membrane organization. However, doubts have been raised: blinking of fluorescent labels leads to multiple observations of the same molecule, resulting in clustered localizations that can easily
be mistaken for molecular nanoclustering.
2-CLASTA offers an elegant analytical method for overcoming the erroneous
detection of clustering due to fluorophore blinking. The approach is based on
2-color localization data from SMLM experiments and targets the underlying
distribution of biomolecules rather than the mere distribution of localizations.
First, the biomolecule of interest is labeled with a mixture of two distinguishable
fluorescent probes. This allows for analyzing distance distributions between localizations from the two channels. In case of real molecular clusters, localizations of the two color channels are correlated and distances show a characteristic bias towards shorter lengths. Applying toroidal shifts to the localization data of one channel breaks correlations between them and provides control data sets. These controls allow for statistical significance tests of the null
hypothesis of randomly distributed biomolecules.
The proposed approach offers a parameter-free, highly sensitive method for the
investigation of biomolecular distributions while circumventing imaging artifacts
due to overcounting. As a result, it yields a p-value for the null hypothesis
of a random distribution of labeled biomolecules. The performance and reliability of the method were tested extensively in simulations and experiments.
2-CLASTA is readily applicable to even a single recorded 2-color image
and does not require any additional measurements. Further advantages include its independence of fluorophore blinking statistics and cluster shape. The provided ImageJ plugin 2-CLASTA makes the method easily accessible to other scientists.

http://dx.doi.org/10.1016/j.bpj.2019.11.1026