Development of a narrow water-immersion objective for laserinterferometric and electrophysiological applications in cell biology

Abstract

Laserinterferometric studies of the micromechanical properties of the organ of Corti using isolated temporal bone preparations are well established. However, there are relatively few measurements under in vivo conditions in the apical region of the cochlea because of its inaccessibility with commonly used techniques. Recently, optical-design programs have become affordable and powerful, so that the development of an optimized optical system is within the budget of physiologists and biophysicists. We describe here the development of a long-range water-immersion objective. To circumvent anatomical constraints, it has a narrow conical tip of taper 22 degrees and diameter 2.4 mm. It is a bright-field reflected-light illumination, achromatic objective with magnification of 25x/infinity, a working distance of 2.180 mm and a numerical aperture of 0.45. Chromatic errors are corrected at 546.1 and 632.8 nm, with emphasis on the latter wavelength which is used by the laser interferometer. The field curvature is relatively flat and a diffraction limitation (Strehl ratio better than 0.8) can be obtained in a field of 0.4 mm diameter. Using this objective, sound-induced vibrations of hair cells and Hensen cells could be recorded without placing a reflector on the target area. In addition, this objective was found to be diffraction-limited in the near infra-red (750-830 nm), with a slightly different working distance (2.186 mm), making it suitable for patch-clamp experiments using infra-red, differential interference contrast.