Single Molecule Laser Trap Assay
A three-bead single molecule laser trap assay. An actin filament attached to two glass beads held in optical traps are brought in to contact with a third bead sparsely coated with myosin. A quadrant photo-diode detects nanometer displacements of the filament caused by myosin.
Trapping Beads
The video above displays how the trapping laser can "grab" a free-falling bead out of the solution and then hold it in a precise location.
To construct the three bead assay we use both brightfield and fluorescence imaging. The left image depicts a bright field image of two 1um beads held in two optical traps with a third 3um bead serving as a pedestal for a single myosin molecule (a top down view). A fluorescently-labeled actin filament is attached to the optical trapped beads and visualized in epi-fluorescence (Right image).
Single Actomyosin Binding Events
Sample displacement vs. time recording from the QD during a three bead assay. The binding events (red arrows) are characterized by a drop in signal variance caused by the formation of the actomyosin bond and a displacement of the actin filament caused by the swinging of myosin’s lever arm.
Force Generation by a Mini-Ensemble of Myosin
Sample data record from a three-bead assay in which the myosin concentration has been increased to a level where ~8 molecules of myosin interact with a single actin filament. Under these conditions multiple myosin molecules bind to the actin filament displacing it far from the center of the trap. Since the optical trap behaves like a linear spring the force on the filament increases with the distance moved. When this force is greater than the myosin molecules can generate the filament detaches from the myosin and returns to baseline. Using the characteristic stiffness of the trap the displacement records can be converted into force vs time record and the force generating capacity of a mini-ensemble of myosin can be quantified.
Fun with the AOD controlled laser trap
Below is a video showing the precise control afforded by the laser trap which allows us to move the beads in intricate patterns.
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