Paper

Waffle Method: A general and flexible approach for FIB-milling small and anisotropically oriented samples

Raczkowski, Ashleigh M, Kelley, Kotaro, Potter, Clinton S, Noble, Alex J, Jaroenlak, Pattana, Eng, Edward T, Carragher, Bridget, Bhabha, Gira, Klykov, Oleg, Kopylov, Mykhailo, Bobe, Daija

2021

Overview

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the 'Waffle Method' which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout. Competing Interest Statement The authors have declared no competing interest. Footnotes * Journal revisions. Includes a new recommended part of the workflow: incubate the polished planchette hats with a drop of 1-hexadecane for 15 minutes immediately before assembling a waffle.

• Publisher: Cold Spring Harbor Laboratory Press
• Subject: Freezing / Lamellae / Spores