Development of methods to mechanically stimulate hair cells

Abstract

The cochlea is one of the most sensitive organs in the human body. It can detect atomic-scale vibrations, amplify sound energies just above thermal noise, and discriminate frequencies with a resolution of 0.2% of its frequency spectrum, up to twenty thousand cycles per second. These astonishing capabilities of the cochlea are the work of the hair cells, the sensory receptors of sound pressure waves. Hair cells convert mechanical energy from sound to electrical energy, a process known as mechanoelectrical transduction, or mechanotransduction. This process causes the release of neurotransmitters that trigger afferent neurons to the brain. Since the discovery of the mechanotransduction process in the 1970s, methods for direct stimulation of hair cells to study mechanotransduction mechanisms have been limited to about 10% of the human frequency spectrum. Thus, the lack of a direct stimulation method at high frequencies has limited our understanding of key processes about human hearing. First, how does the ear adapt and reset itself, a process known as adaptation, in a matter of microseconds at high frequencies? And how do hair cells in the high-frequency regime amplify the stimulus? In this thesis, I describe two methods that were developed in parallel to mechanically stimulate hair cells, and that have the potential to stimulate at frequencies up to hundreds of kilohertz. In the first method, we use photonic pressure from a laser pulse to deliver piconewton- level force onto the hair bundle. The hair bundle is a collection of stereocilia atop the hair cell. First, we developed the theoretical formalism using Maxwell’s electromagnetic radiation concepts and analyzed the range of force needed to deflect the hair bundles by 140 nm, the full physiological range of the hair bundle movement in normal hearing. The idea is to place an optical fiber with a microlens at the tip a few microns away from the hair bundle on its symmetric axis and pulse the laser to apply photonic pressure. Because the hair cell is about 15 times smaller than the smallest commercially-available optical fiber (125 μm), we chemically etched the optical fiber to match the required spot size and melted a 4 μm diameter ball lens at the tip of the optical fiber to focus the laser beam onto the hair bundle. In addition to developing the tapered fiber-optic (TFO) probe, we developed a high-resolution photometric system to measure the nanometer scale deflections. We illuminate the hair-cell preparation with a bright LED from the basal side of the hair cell and image the bright spot of stereocilia on the apical side of the cell with a microscope. This bright image is further magnified, externally to the microscope, and projected on to a dual-photodiode. The deflection of the hair bundle changes the light levels between the two sides of the dual-photodiode. This differential output gives rise to the signal that is calibrated in volts per nanometer. In order to understand the quality of the laser beam spot and the divergence characteristics of the laser beam, we developed an optical setup. We characterize the light output from the TFO by measuring the numerical aperture of the focusing optic. This allows us to place the TFO at a known distance from the hair bundle during experimentation. Using the TFO we localize the photonic pressure to the apex of the hair bundle. We stimulate both frog and rat hair cells and measured the mechanical response of the hair bundle on the order of a few hundred microseconds. We found that, on average, the beam diameter of the TFO is approximately 10 μm when the fiber-tip is 10 μm away from the target. We show that the photonic force produced with 30 mW of laser power directed onto a hair bundle is capable of deflecting a hair bundle of a frog approximately 50nm, and a hair bundle from the second apical turn of the rat cochlea 40nm. This method opens up the exploration of fast-mechanotransduction kinetics of hair cells that have been elusive for over forty years. In the second method, I developed a micro-spring that has the stiffness to match the mechanical impedance (500 μN/m - 2 mN/m) of the hair bundle and use the spring to apply force (push) on the hair bundle using a piezo-electric actuator. For this, I made a PDMS polymer spring that is 5 μm in length, end area of 1 μm-square, and stiffness of 500 μN/m using lithography techniques. I used soft-material synthesis techniques to derive a 1 μm thickness polymer layer, a novel thickness for PDMS layers. I used electron-beam lithography and dry reactive ion etching (DRIE) to pattern an accordion-type spring on the polymer layer. However, this project was set aside before it was tested because we realized the photonic-force method is more advantageous.