Waffle Method: A general and flexible approach for improving throughput in FIB-milling

Abstract

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 with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called 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. 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 manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

A cryogenic, coincident fluorescence, electron, and ion beam microscope

“…Currently, the system presented is solely compatible with samples in an AutoGrid form factor. With minor modifications, other planar sample types, such as samples prepared by the Waffle method ( Kelley et al, 2022 ), would fit inside the sample shuttle. Identifying grid bars and sample navigation can be done directly using the LM.…”

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for Label-Free Detection of Molecules across Entire Cell Sections

“…While data collection throughput on the TEM is fundamentally limited by the exposure time, this amount of data could be collected within 12 hours by improving the data acquisition scheme to perform all necessary calculations in parallel with actual exposure of the camera. Sample preparation using a FIB/SEM is also currently a bottleneck, but preparation of large lamellae with multiple cellular cross-sections using methods like WAFFLE [28] might allow sufficient throughput. As stated in the results, at least for 2DTM computation will remain challenging and approximately 17,000 GPU hours would be required for a 100 lamellae dataset.…”

Cryomicroscopy in situ: what is the smallest molecule that can be directly identified without labels in a cell?

“…Empirically, it has been noted that the milled sections are under tensile stress and various stress-relief geometries (such as notches) have been proposed. 137,138 Understanding the changes that occur in amorphous ice under irradiation with high-energy ions will be instrumental for eliminating artefacts, instabilities and movement in FIB milled sections. Careful comparisons of the difference between ionic species (e.g.…”