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

• DOI: 10.1038/s41467-022-29501-3
• Publication Date: 2022-04-06T10:08:13Z
• Authors: Kotaro Kelley; Ashleigh M. Raczk...; Oleg Klykov; Pattana Jaroenlak; Daija Bobe; Mykhailo Kopylov; Edward T. Eng; Gira Bhabha; Clinton S. Potter; Bridget Carragher; Alex J. Noble
• Keywords: Electron Microscope Tomography; Food; Science; Q; Cryoelectron Microscopy; Freezing; Article; Workflow

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as method for obtaining highest resolution structural information complex biological samples in-situ in native and non-native environments. However, challenges remain conventional cryo-FIB/SEM workflows, including milling thick specimens vitrification issues, preferred orientation, low-throughput when small and/or low concentration specimens, that distribute poorly across grid squares. Here we present a general approach called 'Waffle Method' which leverages high-pressure freezing to address these challenges. We illustrate mitigation by applying Waffle Method reveal macrostructure polar tube microsporidian spores multiple complementary orientations, was previously not possible due orientation. demonstrate broadness it three additional cellular single particle sample using variety cryo-FIB-milling hardware, manual automated approaches. also unique critical stress-relief gap designed specifically waffled lamellae. propose way achieve many advantages cryo-liftout on specimen while avoiding long, challenging, technically-demanding process required cryo-liftout.