Supplementary Figure 4: Ultrathick sectioning apparatus and procedure. | Nature Methods

Supplementary Figure 4: Ultrathick sectioning apparatus and procedure.

From: Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics

Supplementary Figure 4

Current ultrathick sectioning apparatus and procedure. (a) Custom “Ultrathick Sectioning Testbed” utilizing nanometer precision linear stages (XMS50 and GTS30V, Newport) to move the tissue block in the horizontal plane across a stationary knife which is heated via the same jig as in Supplementary Figure 2a. The piezo of the ultrasonic knife is replaced with a high-load piezo (P-010.00H, Physik Instrumente) driven by a 200V amplifier (EPA-104, Piezo Systems). A laser vibrometer (CLV-2534, Polytec) is used to monitor the peak-to-peak vibrational amplitude of the knife (typically ~0.5 μm). Cutting is recorded via two video microscopes providing side and top views of the knife. Four force sensors (208C01, PCB Piezotronics) in the tissue block mount measure normal and transverse cutting forces during sectioning (typically 1000mN transverse peak). Force measurements are helpful in monitoring for chatter. (b) Example tissue block containing fly brain trimmed for ultrathick sectioning. Block is trimmed to have a rectangular face approximately 1.5 mm wide and 3 mm long, providing a large enough section with ample blank regions for manipulation with vacuum tweezers and forceps. The side of the block which will be the trailing edge during hot knife sectioning is strengthened by giving it a ~45° slope. (c) Side view of knife cutting a 20 μm thick section. (See Supplementary Video 1 for video of sectioning process.) (d) Same view but with block's profile outlined in yellow, ultrathick section outlined in blue, and knife angles shown. (e) Side view of knife showing how sections remain adhered to top surface of knife after sectioning. (Scale bar ~1 mm)

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