Three-dimensional confocal microscopy of the mammalian inner ear

Abstract Objective: Control the refractive index in fixed specimens of mammalian inner ear to reduce spherical aberrations that limit our ability to obtain 3-dimensional images of fluorescently labeled inner ear specimens by conventional laser scanning confocal microscopy. Study Design: Mouse inner ear specimens were fixed with minimal dissection, rapidly decalcified and fluorescently labeled by immunohistochemistry then impregnated by epoxy resin or a clearing agent composed of 5 parts methyl salicylate:3 parts benzyl benzoate. The specimens were imaged by both confocal microscopy and by widefield epi-fluorescent microscopy, with additional processing by deconvolution. Results: Rapid decalcification preserved tissue morphology and antigenicity for the antibodies tested. Although the epoxy allowed some reduction of spherical aberration, the clearing agent enabled optical volumes of high quality and resolution to be collected through the inner ear. The conditions for immunolabeling are important to ensure adequate perfusion of the immuno-labeling reagents throughout the specimen. Conclusion: Spherical aberration reduces signal intensity, contrast and resolution in optical microscopy. Creating a homogeneous refractive index throughout the inner ear to reduce spherical aberration allowed optical volumes to be collected through an intact, fluorescently labeled cochlea in a manner limited by the working distance of the objective lens rather than by spherical aberration. Optical volumes collected by this method from the mammalian inner ear promise to be useful for applications such as tracing innervation patterns, counting sensory cells or other structures over large regions of the sensory epithelium, and characterization of the inner ear in animal models of human deafness disorders.