Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens
Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens
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DOI：
10.1038/s41598-019-53380-2
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发表时间：
2019-11-21
影响因子：
4.6
通讯作者：
Pampaloni, Francesco
中科院分区：
文献类型：
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作者：
Hoette, Katharina;Koch, Michael;Pampaloni, Francesco
在三维光学显微镜中，样品中光密度的不均匀性最终限制了可实现的穿透深度，从而限制了三维分辨率。最直接的方法来减少像差，提高对比度，并实现最佳的分辨率是最大限度地减少沿光路的折射率沿着的变化的影响。许多实施方式的光片荧光显微镜操作与一个大的腔室充满了水浸泡介质和进一步的内容器与标本嵌入在一个可能完全不同的非水介质。为了最大限度地减少后者对图像光学质量的影响，我们使用由真空形成的超薄氟碳（FEP）箔制成的多面比色皿。超薄FEP箔比色皿具有约10-12 μ m的壁厚。它们不透液体，但不透气体，惰性，耐用，机械稳定和灵活。重要的是，通常易碎的样本可以从接种到固定，清理和观察保持在同一个比色皿中，而不需要在任何这些步骤中移除或重新安装它。我们证实了超薄FEP箔比色皿的成像性能得到了改善，整个器官（如小鼠卵母细胞）、小鼠大脑和肾脏的厚组织切片以及致密胰腺和肝脏类器官簇的图像质量极佳。我们的超薄FEP箔比色皿在以下方面优于许多其他样品安装技术：将样品与浸没介质完全分离、与水性和有机清洁介质兼容、无需水凝胶包埋的快速样品安装以及适用于多视图成像和自动图像分割。此外，我们表明，超薄FEP箔比色皿适用于在至少十天的时间内接种和生长类器官。新的比色皿允许在保持器内固定和染色标本，保留脆弱的单层三维类器官等的精细形态。
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 to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10-12 mu m. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image 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 new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.