A Combined Single Molecule FRET/Magnetic Tweezers Instrument to Calibrate Molecular Tension - Based Fluorescence Probes

Abstract

Despite the importance of mechanical forces in biology, few methods are available to measure molecular forces in living systems. To address this issue, we developed molecular tension - based fluorescence microscopy (MTFM), a technique that allows one to visualize piconewton forces using a conventional fluorescence microscope. In MTFM, a flexible linker (polymer) is flanked by a fluorophore and a quencher, such that tension leads to extension of the linker and a 10-30 fold increase in fluorescence. Thus far, MTFM provides the only approach to map piconewton forces at the cell membrane.To accurately calibrate the conversion from fluorescence to tension, we integrate magnetic tweezers to a total internal reflection fluorescence (TIRF) microscope for single molecule Forster resonance energy transfer (smFRET) - force measurement. To minimize autofluorescence from paramagnetic beads, we use 4.4 micron lambda-DNA fragments to link a MTFM probe to each bead. The setup can apply forces ranging from 1-50 pN to calibrate the probes. Comparing its force-extension data to that of theoretical models, this direct measurement of force-induced extension change provides a good tool for determining cellular force with higher accuracy.