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Variable immersion microscopy with a high numerical aperture.

Keita Ishida,Kanta Naruse,Yuta Mizouchi,Y. Ogawa,M. Matsushita,T. Shimi,H. Kimura,S. Fujiyoshi

Published 2021 in Optics Letters

ABSTRACT

Three-dimensional (3D) optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. We developed a variable immersion lens (VIL) to passively minimize these aberrations. A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror). Wave-optics simulation at a wavelength of 488 nm showed that a VIL microscope enables diffraction-limited 1.2-NA imaging in water (refractive index of 1.34) at a depth of 0.3 mm by minimizing aberrations due to refraction of a sample interface. Another aberration due to the refractive index mismatching between a mounting medium, and an object can also be corrected by the VIL system, because various fluids with different refractive indices can be used as mounting media for the VIL. As a result of correcting the two aberrations at the same time, we experimentally demonstrated that a 6 µm diameter fluorescent bead can be imaged to the true dimensions in 3D.

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