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TitleWaffle Method: A general and flexible approach for improving throughput in FIB-milling.
Journal, issue, pagesNat Commun, Vol. 13, Issue 1, Page 1857, Year 2022
Publish dateApr 6, 2022
AuthorsKotaro Kelley / Ashleigh M Raczkowski / Oleg Klykov / Pattana Jaroenlak / Daija Bobe / Mykhailo Kopylov / Edward T Eng / Gira Bhabha / Clinton S Potter / Bridget Carragher / Alex J Noble /
PubMed 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 ...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.
External linksNat Commun / PubMed:35387991 / PubMed Central
MethodsEM (tomography)
Structure data

EMDB-26396: CryoET of E. coli prepared with the Waffle Method
Method: EM (tomography)

Source
  • Escherichia coli (E. coli)

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