Enhanced nanoscale molecular dynamics mapping in live cells by galvo-accelerated single-molecule displacement analysis

Alterations in the biophysical properties of subcellular compartments, particularly in dynamic structures, are fundamental to various cellular processes. However, methods for probing fast nanoscale changes in live cells remain limited. Single-molecule displacement mapping (SMdM) measures the diffusion coefficients of freely diffusing molecules using paired laser pulses, but its implementation is constrained by camera limitations and depends on specific hardware for precise timing control. To address this, we introduce galvo-accelerated single-molecule displacement mapping (G-SMdM), which overcomes the limitations of SMdM and achieves multi-fold performance enhancement. By utilizing a fast-scanning galvo mirror to spatially multiplex stroboscopic measurements, G-SMdM provides a generalized optical strategy for measuring rapid single-molecule displacements, significantly increasing acquisition efficiency while decoupling SMdM from specialized hardware constraints. Through simulation and experimental validation, we demonstrate improved accuracy and precision in diffusivity measurements using G-SMdM. In live-cell imaging, G-SMdM enabled high-fidelity mapping of fast-diffusing molecules with nanoscale resolution. Biological application of G-SMdM is demonstrated by revealing stress-induced alterations in endoplasmic reticulum membrane and lumen dynamics. Thus, G-SMdM provides a versatile, camera-independent, and modular platform for investigating nanoscale dynamics under physiological and pathological conditions.