Fabian F. Voigt, Anna Maria Reuss, Thomas Naert, Sven Hildebrand, Martina Schaettin, Adriana L. Hotz, Lachlan Whitehead, Armin Bahl, Stephan C. F. Neuhauss, Alard Roebroeck, Esther T. Stoeckli, Soeren S. Lienkamp, Adriano Aguzzi, Fritjof Helmchen, University of Zurich, and Voigt, Fabian F
Imaging of large, cleared samples in diverse media is achieved using a mirror objective.Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.