Hongqiang Ma, Jianquan Xu, Jingyi Jin, Yi Huang, Yang Liu

High-precision fluorescence microscopy such as superresolution imaging or single-particle tracking often requires an online drift correction method to maintain the stability of the three-dimensional (3D) position of the sample at a nanometer precision throughout the entire data acquisition process. Current online drift correction methods require modification of the existing two-dimensional (2D) fluorescence microscope with additional optics and detectors, which can be cumbersome and limit its use in many biological laboratories. Here we report a simple marker-assisted online drift correction method in which all 3D positions can be derived from fiducial markers on the coverslip of the sample on a standard 2D fluorescence microscope without additional optical components. We validate this method by tracking the long-term 3D stability of single-molecule localization microscopy at a precision of <2 and 5 nm in the lateral and axial dimension, respectively. We then provide three examples to evaluate the performance of the marker-assisted drift correction method. Finally, we give an example of a biological application of superresolution imaging of spatiotemporal alteration for a DNA replication structure with both low-abundance newly synthesized DNAs at the early onset of DNA synthesis and gradually condensed DNA structures during DNA replication. Using an isogenic breast cancer progression cell line model that recapitulates normal-like, precancerous, and tumorigenic stages, we characterize a distinction in the DNA replication process in normal, precancerous, and tumorigenic cells.