Abstract
Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.
Highlights
Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors
Our group has developed an array of molecular tension fluorescence microscopy (MTFM) probes for quantifying and visualizing the pN-scale molecular forces transmitted by cell-surface receptors[4,8,9,10,11,12,13,14,15,16,17,18,19]
The structured illumination microscopy (SIM) technique fundamentally depends on polarization modulation, which conveniently coincides with the optical requirements for performing molecular force microscopy (MFM) (Fig. 1a)
Summary
Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Our group has developed an array of molecular tension fluorescence microscopy (MTFM) probes for quantifying and visualizing the pN-scale molecular forces transmitted by cell-surface receptors[4,8,9,10,11,12,13,14,15,16,17,18,19]. The intensity (I) of a fluorophore excited by plane-polarized excitation light (which has a fixed polarization vector denoted by E) can be described by the relationship: I
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