Waffle Method: A general and flexible approach for improving throughput in FIB-milling
10.1038/s41467-022-29501-3
Crossref journal-article
Springer Science and Business Media LLC
Nature Communications (297)
Abstract

AbstractCryo-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.

Bibliography

Kelley, K., Raczkowski, A. M., Klykov, O., Jaroenlak, P., Bobe, D., Kopylov, M., Eng, E. T., Bhabha, G., Potter, C. S., Carragher, B., & Noble, A. J. (2022). Waffle Method: A general and flexible approach for improving throughput in FIB-milling. Nature Communications, 13(1).

Authors 11
  1. Kotaro Kelley (first)
  2. Ashleigh M. Raczkowski (additional)
  3. Oleg Klykov (additional)
  4. Pattana Jaroenlak (additional)
  5. Daija Bobe (additional)
  6. Mykhailo Kopylov (additional)
  7. Edward T. Eng (additional)
  8. Gira Bhabha (additional)
  9. Clinton S. Potter (additional)
  10. Bridget Carragher (additional)
  11. Alex J. Noble (additional)
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Dates
Type When
Created 3 years, 4 months ago (April 6, 2022, 6:08 a.m.)
Deposited 2 years, 8 months ago (Nov. 24, 2022, 11:44 a.m.)
Indexed 58 minutes ago (Aug. 20, 2025, 10:43 p.m.)
Issued 3 years, 4 months ago (April 6, 2022)
Published 3 years, 4 months ago (April 6, 2022)
Published Online 3 years, 4 months ago (April 6, 2022)
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@article{Kelley_2022, title={Waffle Method: A general and flexible approach for improving throughput in FIB-milling}, volume={13}, ISSN={2041-1723}, url={http://dx.doi.org/10.1038/s41467-022-29501-3}, DOI={10.1038/s41467-022-29501-3}, number={1}, journal={Nature Communications}, publisher={Springer Science and Business Media LLC}, author={Kelley, Kotaro and Raczkowski, Ashleigh M. and Klykov, Oleg and Jaroenlak, Pattana and Bobe, Daija and Kopylov, Mykhailo and Eng, Edward T. and Bhabha, Gira and Potter, Clinton S. and Carragher, Bridget and Noble, Alex J.}, year={2022}, month=apr }