Enabling Hours-Long Drift Correction with Nanometer Resolution in Optical Microscopy through Reflection Interferometry of Fiducial Beads

Optical microscopy excels at revealing the dynamics of biological systems in real time. However, focal drift limits most microscopy approaches to only several minutes in the absence of drift-mitigation strategies. Here we introduce <u>f</u>ocus <u>re</u>adjustment for <u>e</u>nhanced <u>v</u>ertical <u>r</u>esolution (FREVR), a method which uses micrometer-sized fiducial beads alongside the specimen. By tracking the interference fringes generated by these beads, FREVR can detect changes in focus position with nanometer precision. We showcase this approach on a microscope equipped with a high-speed CMOS camera used for bead tracking and a highly sensitive EMCCD camera tasked to quantify fluorescence with spectral resolution. Using this approach, we measured samples in focus for several hours on several single-molecule systems, as well as on mammalian cells having their actin filaments or membrane proteins fluorescently labeled. FREVR has the potential to dramatically increase the temporal and spatial resolution and the long-term stability of microscopy techniques.