Fully refractive telecentric f-theta microscope based on adaptive elements for 3D raster scanning of biological tissues

Abstract

Various techniques in microscopy are based on point-wise acquisition, which provides advantages in acquiring sectioned images, for example in confocal or two-photon microscopy. The advantages come along with the need to perform three-dimensional scanning, which is often realized by mechanical movement achieved by stage-scanning or piezo-based scanning in the axial direction. Lateral scanning often employs galvo-mirrors, leading to a reflective setup and hence to a folded beam path. In this paper, we introduce a fully refractive microscope capable of three-dimensional scanning, which employs the combination of an adaptive lens, an adaptive prism, and a tailored telecentric f-theta objective. Our results show that this microscope is capable to perform flexible three-dimensional scanning, with low scan-induced aberrations, at a uniform resolution over a large tuning range of X=Y=6300 μm and Z=480 μm with only transmissive components. We demonstrate the capabilities at the example of volumetric measurements on the transgenic fluorescence of the thyroid of a zebrafish embryo and mixed pollen grains. This is the first step towards flexible aberration-free volumetric smart microscopy of three-dimensional samples like embryos and organoids, which could be exploited for the demands in both lateral and axial dimensions in biomedical samples without compromising image quality.