Photostimulation for cell physiology

Photostimulation of IHCs was achieved using a blue 473 nm laser (MBL 473, CNI Optoelectronics). Irradiance and duration of the light pulses were controlled using the EPC-9 amplifier via a custom controller unit, allowing the transformation of a particular voltage to a particular laser power (i.e. photostimulation during 5, 10, or 50 ms from 2 to 5 V with different increasing steps). A FITC filter set was used to direct the stimulating blue light to the sample. Radiant flux (mW) was measured before each experiment with a laser power meter (LaserCheck; Coherent Inc or Solo2 Gentec-eo) placed under the 40× objective lens. The diameter of the illumination spot was estimated using a green fluorescent slide and a stage micrometer, and it was used to calculate the irradiance in mW/mm². Photocurrents were measured in voltage-clamp mode and the photodepolarization in current-clamp mode. Only the first series of evoked photocurrents and photodepolarizations were analyzed in order to rule out potential changes in the photoresponses due to inactivation of ChR2-H134R-EYFP (Lin et al., 2009).