Machine Learning in Single-Molecule Tracking Analysis of Superresolution Optical Microscopy Data

Machine learning (ML) is transforming the analysis of biomolecular data, holding significant promise for improving the efficiency and accuracy of microscopy image analysis and for studying the dynamics of molecules in live cells. As data-driven approaches continue to evolve, they may eventually replace traditional statistical methods that rely on conventional analytical methods. This review examines and critically analyses the state of the art of ML techniques as applied to various levels of data supervision in the analysis of dynamic single-molecule datasets obtained using superresolution optical microscopy. Collectively encompassed under the umbrella of “nanoscopy”, these methods currently comprise targeted techniques such as stimulated emission depletion (STED) microscopy and stochastic techniques like single-molecule localization microscopies (SMLMs), comprising photoactivated localization microscopy (PALM), DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy, and minimal fluorescence photon flux (MINFLUX) microscopy. These techniques all enable the imaging of subcellular components and molecules beyond the diffraction limit, and some are additionally capable of studying their dynamics in real time, as reviewed here, using several ML techniques that facilitate motion analysis in two or three dimensions with qualitative and quantitative characterisation in the live cell. It is expected that the growing use of learning-based approaches in biological microscopy data processing will dramatically increase throughput and accelerate progress in this rapidly developing field.