CryoFluorSEM – A new approach for fluorescence and EM imaging of cryofractured plant samples

Abstract

Abstract Cryo‐scanning electron microscopy (CryoSEM) permits the preparation and detailed imaging of bulky samples while keeping them in a hydrated state. For plant biology, cryofractures give information on cell ultrastructure and tissue organisation within a much larger context that is the whole organ or organism. To date, a method to locate fluorescence reporters on the cryofracture has not been reported. Our approach uses a stereofluorescence microscope with an 80 mm working distance and a high‐zoom ratio to image the fracture through a viewing port of the cryopreparation chamber while the sample is still frozen and under vacuum. We have applied this method to look at fluorescent reporters of auxin transport and signalling in plant shoot apices and seedlings, the expression of a poorly characterised gene in the young floral pedicel and nitrogen‐fixing rhizobial bacteria, expressing GFP, in root nodules. This method is applicable to any cryopreserved bulky sample that has a fluorescent output and paves the way for correlative light‐electron microscopy for cryoSEM‐based imaging.

Plant phenotyping relevance

植物試料の蛍光レポーターを凍結破断面上で位置特定・画像化する新規CryoFluorSEM法の開発であり、植物の構造・組織状態を取得する方法が中心である。

Code and data availability

The supplied blocks describe the CryoFluorSEM imaging method and Photoshop/Copilot overlay workflows but contain no data availability statement, no deposited phenotype datasets or images, and no author code repository or public URL. The only URLs present are citations to prior work, which are not paper-specific assets.

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