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. 2022 Apr 6;13:1857. doi: 10.1038/s41467-022-29501-3

Fig. 1. Schematic of the general workflow for creating a waffle grid.

Fig. 1

Supplementary Movie 1 shows an animation of all steps. In Step 0, the waffle is prepared: a The planchette hats are polished to reduce lamellae curtaining, b The planchette hats and optional spacer are coated with 1-hexadecene to make easier to separate after high-pressure freezing, c ~25 nm of carbon is sputtered onto the non-grid bar side of the EM grid to rigidify it, d The high-pressure freezer (HPF) tip is cleaned with ethanol just prior to use, and e the EM grid is glow discharged/plasma cleaned just prior to sample application. In Step 1, the waffle is made: f The sample is applied to the EM grid with optional spacer on top of a planchette hat, g The waffle is vitrified with the HPF, h The HPF assembly is taken apart, the grid is carefully clipped, and ~5 nm of conductive platinum is sputtered onto the waffle. The researcher may want to screen their waffle grid in a cryo-FLM at this step. In Step 2, the waffle is milled inside of a cryo-FIB/SEM: i 1–4 μm of platinum Gas Injection System (GIS) is applied, (j) Grid bar lines are drawn onto the top of the waffled sample to expedite location identification during milling, k Two trenches per lamella are milled as perpendicular to the grid as possible and separated by tens of microns, l The grid is tilted to a shallow angle and all lamellae on the grid are milled in parallel while making sure the leading edge of the FIB beam always intersects platinum GIS first to reduce curtaining. It is recommended that a notch be milled into one side of each lamellae during the course of milling, as shown in Fig. 2. It is recommended to tilt the stage ±1° while milling the lamellae from 3 μm thick to 1.5 μm thick and ±0.5° while milling the lamellae from 1.5 μm thick to 0.5 μm thick prior to fine milling and polishing at the desired angle. Supplementary Figs. 3 and 4 show waffle milling FIB/SEM images. Supplementary Movie 2 shows most of Step 1 being performed.