Technical Reference #218

218.

Membrane Tether Formation from Outer Hair Cells with Optical Tweezers Zhiwei Li; Bahman Anvari; Masayoshi Takashima; Peter Brecht; Jorge H. Torres; and William E. Brownell, Rice University, Biophysical Journal, 82(218), (2002)
Link To Paper

Abstract:
Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasmamembrane by pulling tethers with 4.5-m polystyrene beads. Tether formation force and tether force were measured in staticand dynamic conditions. A

Materials & Methods:
OHC isolation Pigmented guinea pigs of either sex weighing 200 to 250 g were decapitated. The temporal bones were removed and the organ of Corti was isolated from the cochlea. The organ of Corti was incubated in trypsin for 5 min and transferred to a microwell petri dish (MatTek Ashland MA). The petri dish was coated with poly-D-lysine to ensure firm attachment of the OHCs to the coverslip. The OHCs were maintained in a bathing solution consisting of 155 mM NaCl 4 mM KCl 1 mM MgCl2 2 mM CaCl2 and 10 mM HEPES. The solution was adjusted to a pH of 7.2 and an osmolarity of 290 to 300 mOsm/kg. The cells were selected for experimentation if they exhibited a uniformly cylindrical shape a basally located nucleus and limited osmotic swelling or Brownian motion in the cytoplasm. All OHCs were used within 4 h after the animal sacrifice. Experimental setup The optical tweezers setup used a continuous wave tunable (650–1100 nm) Titanium-Sapphire laser (Spectra-Physics Model 3900S Mountain View CA) pumped by a 5-W solid state frequency doubled Nd:YVO4 laser (Spectra-Physics Millennia V) (Fig. 2). The Titanium-Sapphire laser was tuned to 830 nm where no or minimal damage to cells has been reported (Liang et al. 1996; Neuman et al. 1999). In our experiments there was no evidence of damage to the OHCs nor were thermally induced length changes observed. The laser beam was expanded 5 times by a beam expander (Chroma Technology cwbx-7.0-s-670/1064 Brattleboro VT) to fill the back aperture of a 100 microscope objective with a numerical aperture of 1.3 thereby achieving a high convergence angle required for strong trapping. An attenuator (Newport 925B Irvine CA) was placed in the beam path to change the light level during experimentation (if needed). The laser beam was directed by two mirrors toward the bottom port of an inverted microscope (Zeiss Axiovert S100TV Jena Germany). A dichroic mirror placed before the bottom port transmitted the laser light into the microscope and reflected the emitted light ( 650 nm) from the sample toward a beam splitter which in turn transmitted 10% of that light to a CCD camera (DAGE-MTI CCD100 Michigan City IN) for image collection and reflected the remaining 90% to a quadrant photodetector (Hamamatsu S4349 Somerville NJ) mounted on a micrometer stage for bead displacement measurements. The shadow of a 4.5- m diameter polystyrene trapped bead which was used as a “handle” to form plasma membrane tethers from the OHC lateral wall was projected onto the photodetector surface by a 15 eyepiece. The analog photodetector displacement signals resulting from the movement of a trapped bead by the plasma membrane tether were amplified by a circuit and digitized with an A-D converter (Iotech Wavebook512 Cleveland OH). The digital signals were subsequently analyzed by a LabView program.

Microscopic Technique
Electron Microscopy, Transmission Electron microscopy, Confocal Microscopy

Cell Type(s)
OHC