The last three decades have seen major breakthroughs in single-molecule biophysical techniques, which have advanced our understanding of biological processes across various scales from molecules to cells and tissues. The need to manipulate, control and monitor the dynamics of biomolecules simultaneously motivated the combination of complementary single-molecule methods. One of the most successful examples of such a combination is integrated optical tweezers (OT) force-fluorescence microscopy. OT is capable of immobilizing individual molecules, typically DNA, using optically trapped micron-sized beads, applying precisely controlled stretching forces, and monitoring kinetic processes in real time. Integration with fluorescence microscopy allows simultaneous monitoring of the identity and dynamics of molecules interacting with tethered DNA. A research field that particularly benefits from advanced OT force–fluorescence microscopy is the investigation of DNA replication, a crucial biological process involving complex protein‒DNA and protein‒protein interactions. In this review, we summarize the recent technical developments in OT force–fluorescence spectroscopy and associated advancements in biochemical sample preparation; briefly introduce the viral, bacterial and eukaryotic DNA replication machinery; and discuss the applications of OT force–fluorescence spectroscopy in the investigations of DNA replication mechanisms.
Liu et al. (2026) studied this question.