Optical Tweezers is formed with a continuous wave Titanium-sapphire laser tuned to 830 nm and pumped by a 532 nm solid-state frequency-doubled Nd: YVO4 laser (maximum power: 6 W). The beam is then steered via an optical path to fill the back aperture of an objective lens (numerical aperture of 1.4) housed in an inverted microscope. The laser power and position of the beam are controlled by two acousto-optic deflectors (AODs). The position of the trapped fluorescent (e.g., Texas red fluorophore) bead is monitored with a quadrant photodiode. The bead fluorescence is excited with a TLED lamp and the emission is filtered with a bandpass filter and detected by the quadrant photodiode. The quadrant photodiode monitors the change in the axial position (X, Y) of the bead and the sum of fluorescence intensity of the bead.
To correct for photobleaching the position of the bead is normalized with respect to sum of fluorescence intensity. The biosensor is controlled, and the signal is monitored with a virtual instrument (LabVIEW, v.12). Voltage signals are generated with a field-programmable gate array and are acquired with a digital acquisition card with a maximum input voltage of ± 0.1 V. The control of the laser beam and movement of piezoelectric stage is determined by the resolution of FPGA and exhibits a precision of ± 2 nm. The trap behaves like a Hookean spring and damped harmonic oscillator with a time constant 23.4 s-1mW-1.
Position detection of the QPD is linear up to bead radius with a force range of plus/minus 400 pN. Quadrant photodiode voltage outputs are monitored at a collection frequency of 200 kHz and averaged 25-100×. The resolution of the displacement and force is 10 nm and 1 pN. During an experiment designed to monitor actin-based forces the time constant of the trap is typically 3000 s-1 and exhibits a spring constant of 0.115 pN nm-1. This facilitates measurement rates up to 300-600 s-1 5 to 10 × less than the instrument. During experiments that monitor membrane teth