National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
F32GM128303
United States
Simons Foundation
SF349247
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM103310
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
U24GM139171
United States
Citation
Journal: Nat Commun / Year: 2022 Title: Waffle Method: A general and flexible approach for improving throughput in FIB-milling. Authors: Kotaro 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 / 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 ...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.
EMPIAR-10981 (Title: CryoET of E. coli prepared with the Waffle Method / Data size: 94.0 Data #1: Unaligned multi-frame micrographs for each tilt image [micrographs - multiframe])
Instrument: OTHER Details: The value given for _emd_high_pressure_freezing.instrument is Wohlwend HPF Compact 01. This is not in a list of allowed values {'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM HPM100', 'OTHER', ...Details: The value given for _emd_high_pressure_freezing.instrument is Wohlwend HPF Compact 01. This is not in a list of allowed values {'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM HPM100', 'OTHER', 'LEICA EM PACT', 'BAL-TEC HPM 010', 'LEICA EM PACT2'} so OTHER is written into the XML file.
Sectioning
Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 3 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 70 K / Focused ion beam - Initial thickness: 10000 nm / Focused ion beam - Final thickness: 200 nm Focused ion beam - Details: Waffle Method. The value given for _emd_sectioning_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is ...Focused ion beam - Details: Waffle Method. The value given for _emd_sectioning_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 4.87 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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