(a) SEM cross section of a vibrotome cut surface showing extensive ultrastructure damage. Scale bar = 5 μm. (b, c) Diamond knife boat heater clamp used for hot knife thick sectioning. (d) Schematic showing how hot knife cut sections were mounted for SEM imaging of their sectioned surfaces. (e) SEM BSE (backscatter electron) image (taken at 6kV) of a sequential pair of 20 μm thick hot knife sections mounted so that their matching faces are facing up. Scale bar = 100 μm. (f) BSE image zooming in on the tip region of one of the sections shown in (e). This BSE image may appear to be damage free, but when the same region is imaged using SE (secondary electron) imaging (g) considerable surface damage is evident. Scale bars = 1 μm. (h,i) BSE and SE images respectively of a separate hot knife cut section. Chatter damage is visible at bottom. Surface damage is clearly seen in the SE image (i) across the entire section with the exception of a triangular region at the very center of the tissue. This same triangular region is seen to contain less stain contrast in (h) supporting the hypothesis that some surface damage was due to the overly brittle nature of this ROTO prepared tissue in those regions of the tissue where this ROTO procedure fully penetrated. (see Online Methods for more details.) Scale bars = 10 μm.