AbstractHow do you get from a block of resin containing your biological sample to a beautiful 3D isotropic dataset collected at a Focused Ion Beam Scanning Electron Microscopy (FIB-SEM)? The dual beam technology enables the collection of isotropic volumes, which are on average 50x50x50µm3, from simple cultured cell monolayers up to complex organisms or tissues. For any multicellular specimen though, the targeted volume can be located at a significant depth relative to the block surface, and the FIB-SEM should not be used as a trimming tool. My aim is to always apply the simplest approach that answers the biological questions at hand. Using examples of aquatic organisms (microalgal symbiont Phaeocystis cordata within its acantharian host and free living form; and the freshwater Demosponge, Spongilla lacustris), I will outline the steps taken to trim and target specific ROIs within a resin block. In both cases, macroscopic inspection of the specimen before trimming is key to define and target the ROI. Each sample can require a very different approach and not all instances require great lengths to ready them for the microscope but when care is taken to properly prepare the sample, the results you can achieve are worth all the effort. In all cases a 90° trimming diamond knife and an ultramicrotome are indispensable. I hope to convince you that the work leading up to a session at a FIB-SEM is just as important, if not, even more so, as the time spent to collect the final dataset.
Bio available soon