Single-molecule microscopy for precision medicine and diagnostics

Abstract

The advent of precision medicine has created an urgent need for detecting disease at its molecular origins. Conventional bioanalytical methods, including immunoassays and mass spectrometry, rely on ensemble averaging and often obscure rare events, molecular heterogeneity, and dynamic processes that are critical to disease initiation and progression. Recent advances in fluorescence-based single-molecule microscopy address these limitations by enabling ultrasensitive, molecule-resolved analysis in complex biological environments. This review provides a conceptual overview of fluorescence-based single-molecule detection strategies and their emerging applications in precision diagnostics. We outline the basic principles of single-molecule fluorescence detection, introduce recent advances in key methodological classes, including single-molecule counting for absolute quantification, single-molecule localization microscopy for nanoscale spatial mapping, and single-molecule dynamic tracing approaches for probing interactions and conformational states, as well as their potential applications in disease diagnosis. Finally, we discuss current challenges and future opportunities for integrating single-molecule microscopy into clinically workflows.