Fig. 2: Concept of the centrifuge force microscope (CFM) and force clamp assay.
From: High-throughput single-molecule quantification of individual base stacking energies in nucleic acids
a The CFM is comprised of a video microscope that is centrifuged. Centrifugal force is applied to tethered microspheres and aligns with the imaging pathway to give a headon view of microspheres. b Images of the custom CFM module show the compact central optics, a clamshell style 3D printed housing, and supporting electronics, which fit inside a centrifuge bucket. c The CFM module operates in a benchtop centrifuge, which is controlled by an external computer that receives a live video stream by WiFi. d A sample microscopy image of ~100 tethered beads at a ~×40 magnification among thousands of such frames. e Concept and partial-frame images of tether dissociation observed in the force clamp assay. As the weak central duplex dissociates, tethered beads fall out of focus and disappear from view. f Custom MATLAB software tracks tethered beads over time and records dissociation times. Four examples shown with different colors correspond to a subset of beads in panel (e). g Decay plot obtained from the dissociation time analysis of the 16 tethers in sub-frame (e). The red line is a single-exponential fit to extract off rate.
