Abstract
Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization microscopy by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical and biochemical processes, including the organization of actin networks, movement of molecular motors, conformations of DNA strands, growth and remodeling of amyloid aggregates, and composition changes within lipid membranes. In this review, we discuss recent innovations in SMOLM and cover three key aspects: (1) biophysical insights enabled by labeling strategies that endow fluorescent probes to bind to targets with orientation specificity; (2) advanced imaging techniques that leverage the physics of light-matter interactions and estimation theory to encode orientation information with high fidelity into microscope images; and (3) computational methods that ensure accurate and precise data analysis and interpretation, even in the presence of severe shot noise. Additionally, we compare labeling approaches, imaging hardware, and publicly available analysis software to aid the community in choosing the best SMOLM implementation for their specific biophysical application. Finally, we highlight future directions for SMOLM, such as the development of probes with improved photostability and specificity, the design of “smart” adaptive hardware, and the use of advanced computational approaches to handle large, complex datasets. This review underscores the significant current and potential impact of SMOLM in deepening our understanding of molecular dynamics, paving the way for future breakthroughs in the fields of biophysics, biochemistry, and materials science.