Ligand binding of membrane proteins triggers many important cellular signaling events by the
lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma
membrane. This local clustering can lead to the co-enrichment of molecules that create an
intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium
towards the execution of a signaling event. In this way, clustering can serve as a cellular switch.
The underlying uneven distribution and local enrichment of the signaling cluster’s constituting
membrane proteins can be used as a functional readout. This information is obtained by combining
single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly
distinguish clusters from fluctuations in the background noise to generate quantitative data on
this complex process.
Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative
field, and several algorithms and software solutions have been put forward. However, in most
cases, such cluster algorithms require multiple analysis parameters to be defined by the user,
which may lead to biased results. Furthermore, most cluster algorithms neglect the individual
localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it
has entailed high computational cost, increasing runtime drastically. Finally, most software is
challenging to use as they require advanced technical knowledge to operate.
Here we combined three advanced cluster algorithms with the Bayesian approach and
parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing
than for previous approaches. Our work will simplify access to a well-controlled analysis of
clustering data generated by SMLM and significantly accelerate data processing. The inclusion
of a simulation mode aids in the design of well-controlled experimental assays