Super-Thin Serial Sectioning for High-Resolution 3-D Reconstruction of Cellular Structures

Extract

We are developing techniques for obtaining the thinnest possible serial sections of biological specimens. Our goal is to produce not just an occasional very thin section but large numbers of 5-15 nm serial sections suitable for 3-D reconstruction. We have worked out conditions that allow us routinely to cut serial sections ˜15 nm thick, and have achieved ribbons of sections with an average thickness of only 11 nm.

We have worked with the Leica Ultracut S and Ultracut UCT microtomes, which have proven to be exceptionally stable instruments, capable of very regular advance between cutting strokes. The resulting low variability in section thickness is essential for serial sections thinner than 15 nm. However, these machines have an inherent forward drift that increases the thickness of sections cut soon after a block has been mounted and prepared for cutting. This drift is initially 50-100 nm/min, but decreases to only 10-20 nm/min if the microtome is allowed to cycle for 0.5—1.5 hours.