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° and diameter 2.4 mm. It is a bright-field reflected-light illumination, achromatic objective with magnification of 25×/∞, 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.

Introduction

A major part of modern biological and physiological research involves the study of cellular mechanisms inside ``living'' tissue. When working with sections of tissue in which the cells of interest are located directly under a glass cover, optimal resolution can be obtained with conventional, high numerical aperture, oil-immersion objectives corrected for the cover glass. However, the resolution is severely reduced when using these oil-immersion objectives for focusing at depths more than several tenths of micrometers within the tissue; this is due to the difference in refractive indices of glass (1.515) and tissue (1.33–1.35). Under these circumstances, better results can be obtained with water-immersion objectives, which are available for working distances from several tenths of millimeters to several millimeters. Optically optimized long-range objectives of this kind allow sufficient space between the tissue and the objective for many experimental purposes (e.g. patch-clamping) and, in addition, their outer shape is often adapted to the in vitro or in situ situation. Examples of four commercially available water-immersion objectives are given in Fig. 1. However, for in vivo experiments the situation may be less than optimal because lateral access for recording electrodes, for example, is not possible or because optical access is hindered for anatomical reasons.

In our application, an objective is required for focusing the light from a laser Doppler vibrometer onto cellular structures in the cochlea. Although we (Gummer et al., 1996) were able to make successful vibration measurements in a temporal bone preparation of the cochlea using a commercially available objective (Zeiss Acroplan 40x/0.75 WPh2), in vivo vibration experiments proved impossible because, unlike the in vitro situation, it was not possible to remove sufficient overlying tissue to allow access with the Zeiss objective (Fig. 2A). The in vivo experiments, requiring the neuronal and systemic inputs to the inner ear to be left undisturbed, must be conducted with a much smaller ventral opening of the middle-ear cavity or bulla (Fig. 2). This paper describes the objective (Fig. 1C, Fig. 2B) that we designed for the in vivo vibration measurements, giving its theoretical and experimental performance.

Vibration measurements in the apical (low-frequency) region of the in vitro (Khanna et al., 1989, Ulfendahl et al., 1989) and in vivo (Khanna and Hao, 1996) cochlea have made use of long-range air-objectives in combination with a dipping cone. However, the dipping cone is not commercially available. Unfortunately, relevant structures can be discerned only occasionally when using commercially available water-immersion objectives. For vibration measurements, a long-range air-objective without a dipping cone requires a glass window at the interposed fluid-air interface. Generally, a decrease in the signal-to-noise ratio is produced by additional reflections and poor image quality due to the uncompensated change of the refractive index in the optical path. To compensate for a loss of image quality, the endolymphatic space must be opened and under normal in vivo conditions reflective beads must be placed on the vibrating surface (Cooper and Rhode, 1995). It is possible, however, to make vibration measurements of the highly reflecting lipid droplets of Hensen cells without introducing a reflector (Cooper and Rhode, 1996).

To design a dipping cone of high optical quality for a commercially built long-range objective, a knowledge of the detailed parameters of the objective is essential: Unfortunately, they are usually kept secret by the manufacturers. To avoid these disadvantages, we decided to develop a water-immersion objective with a narrow tip-diameter adapted to the special anatomical constraints of our experiments.

We present here an affordable way to develop and construct a custom-designed achromatic objective which is not only suited to our laser vibrometric experiments, but can also be used in other applications where space is a major constraint: for example, in certain types of patch-clamp experiment.