CORRECTION OF AIR MICROSCOPE OBJECTIVES FOR USE IN SOLUTIONS FOR TISSUE CLEARING

Imaging of cleared tissue blocks, particularly whole mouse brains, has become widespread since the first demonstration of a cleared mouse brain (Dodt et al., Nature methods, 2007). High-resolution imaging in clearing solutions requires the use of specialized immersion objectives. Among these, the immersion objectives with the largest field of view are typically 10× lenses, which provide imaging fields of about 1 mm. Imaging larger specimens therefore necessitates extensive stitching, a process that is time-consuming and often produces uneven results. For light sheet microscopy of large samples low power objectives with large fields of view would be more suitable. When used with cleared samples, these are generally combined with simple immersion caps containing a plane glass window, which introduce significant aberrations. A simple, generally applicable solution for upgrading air objectives would thus be desirable. By developing dipping cones with special window optics we were able to eliminate effectively spherical aberration giving images with superb resolution. We evaluated this solution on two high-end zoom microscopes, the Nikon AZ100 and the Zeiss Axiozoom V16. In both systems, the meniscus lenses produced dramatically improved image quality compared to conventional plane-glass dipping cones. Furthermore, the working distance of the air objectives was preserved or even increased, depending on the lens design. Because such dipping cones can be retrofitted to existing air objectives, including large-diameter macroscope lenses with long working distances, this solution represents a practical and powerful tool for light sheet microscopy of large cleared samples.