Introducing Diinamic, a flexible and robust method for clustering analysis in single-molecule localization microscopy.

Super-resolution microscopy allowed major improvements in our capacity to describe and explain biological organization at the nanoscale. Single-molecule localization microscopy (SMLM) uses the positions of molecules to create super-resolved images, but it can also provide new insights into the organization of molecules through appropriate pointillistic analyses that fully exploit the sparse nature of SMLM data. However, the main drawback of SMLM is the lack of analytical tools easily applicable to the diverse types of data that can arise from biological samples. Typically, a cloud of detections may be a cluster of molecules or not depending on the local density of detections, but also on the size of molecules themselves, the labeling technique, the photo-physics of the fluorophore, and the imaging conditions. We aimed to set an easy-to-use clustering analysis protocol adaptable to different types of data. Here, we introduce Diinamic, which combines different density-based analyses and optional thresholding to facilitate the detection of clusters. On simulated or real SMLM data, Diinamic correctly identified clusters of different sizes and densities, being performant even in noisy datasets with multiple detections per fluorophore. It also detected subdomains ("nanodomains") in clusters with non-homogeneous distribution of detections.