Abstract Single particle tracking (SPT) of fluorescent molecules provides significant insights into the diffusion and relative motion of tagged proteins and other structures of interest in biology. However, despite the latest advances in high‐resolution microscopy, individual particles are typically not distinguished from clusters of particles. This lack of resolution obscures potential evidence for how merging and splitting of particles affect their diffusion and any implications on the biological environment. The particle tracks are typically decomposed into individual segments at observed merge and split events, and analysis is performed without knowing the true count of particles in the resulting segments. Here, we address the challenges in analyzing particle tracks in the context of cancer biology. In particular, we study the tracks of KRAS protein, which is implicated in nearly 20% of all human cancers, and whose clustering and aggregation have been linked to the signaling pathway leading to uncontrolled cell growth. We present a new analysis approach for particle tracks by representing them as tracking graphs and using topological events – merging and splitting, to disambiguate the tracks. Using this analysis, we infer a lower bound on the count of particles as they cluster and create conditional distributions of diffusion speeds before and after merge and split events. Using thousands of time‐steps of simulated and in‐vitro SPT data, we demonstrate the efficacy of our method, as it offers the biologists a new, detailed look into the relationship between KRAS clustering and diffusion speeds.

Researchers in the field of connectomics are working to reconstruct a map of neural connections in the brain in order to understand at a fundamental level how the brain processes information. Constructing this wiring diagram is done by tracing neurons through high-resolution image stacks acquired with fluorescence microscopy imaging techniques. While a large number of automatic tracing algorithms have been proposed, these frequently rely on local features in the data and fail on noisy data or ambiguous cases, requiring time-consuming manual correction. As a result, manual and semi-automatic tracing methods remain the state-of-the-art for creating accurate neuron reconstructions. We propose a new semi-automatic method that uses topological features to guide users in tracing neurons and integrate this method within a virtual reality (VR) framework previously used for manual tracing. Our approach augments both visualization and interaction with topological elements, allowing rapid understanding and tracing of complex morphologies. In our pilot study, neuroscientists demonstrated a strong preference for using our tool over prior approaches, reported less fatigue during tracing, and commended the ability to better understand possible paths and alternatives. Quantitative evaluation of the traces reveals that users' tracing speed increased, while retaining similar accuracy compared to a fully manual approach.