Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens

Abstract In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast, and achieve an optimal resolution is minimizing the impact of changes of the refractive index along an optical path. Many light sheet fluorescence microscopes operate with a large chamber that contains an aqueous immersion medium and an inner specimen holder that contains the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimize the impact of the specimen holder on the optical quality, we use multi-facetted cuvettes fabricated with vacuum-formed ultra-thin fluorocarbon (FEP) foils The ultra-thin FEP-foil cuvettes have a wall thickness of about 12 μm. They are resilient to fluidic exchanges, durable, mechanically stable and yet flexible. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs, thick tissue sections and dense organoid clusters. The cuvettes outperform many other sample-mounting techniques in terms of full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding, as well as their applicability for multiple-view imaging and automated segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The ultra-thin cuvettes allow the fixation and staining of the specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.