Figure 1.

Setup and procedures of the optical trapping technique for measuring membrane mechanics of dorsal root ganglion neurons. (A) Main components of optical trapping setup. The optical trapping technique uses infrared laser to trap a bead whose position relative to the center of the laser beam is detected by the position-sensing device quadrant photodiode. (B) The bead in the optical trap is used as a handle to hold cell membranes and to allow pulling a membrane tether. Membrane tether exerts a force to shift bead position in the laser trap. By measuring the position shift, tether force (F₀) can be determined and parameters about membrane mechanics can then be derived mathematically. These parameters include in-plane membrane tension (T_m), tension due to the plasma membrane-cytoskeletons adhesion (γ), and membrane bending stiffness (B). The measured total plasma membrane tension T = T_m + γ = F₀/4πR_t and the membrane bending stiffness B = F₀ × R_t/2π, where R_t is the radius of membrane tether. (C) Stage arrangement for the application of the optical trapping technique to dorsal root ganglion neurons in culture. The stage is controlled be a piezo device that precisely moves cells on the stage in x-y-z directions. (D) Image shows a cultured DRG neuron and an optically trapped bead in a recording chamber viewed under a 100× objective. (E–G) Schematic diagrams illustrate optical trapping procedures for pulling a membrane tether from a DRG neuron by a laser-trapped bead. The cell is positioned 10 μm away from the bead and kept in this position for 5 s (BL, baseline; E). The cell is then moved toward the bead at the speed of 1 μm/s until it touches the bead (10 s), and the cell is kept at this position for 5 s (F). The cell is then pulled away from the bead, returned to original position (10 s), and held in this stationary position for 5 s (G). During these procedures, the bead position in the trap is recorded continuously by the quadrant photodiode. (H–J) An example shows an actual optical trapping experiment on a cultured DRG neuron. O’clock time is shown in each procedure from (H) to (J). A membrane tether was pulled out from the neuron (arrow indicated in J). Each vertical red line in (E)–(J) is the center of the laser trap and each white asterisk indicates the center of the bead.