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

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.