In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method
Oleg Klykov,
Daija Bobe,
Mohammadreza Paraan,
Jake Johnston,
Clinton Potter,
Bridget Carragher,
Mykhailo Kopylov,
Alex Noble
Affiliations
Oleg Klykov
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, USA
Daija Bobe
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA
Mohammadreza Paraan
New York Structural Biology Center, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA
Jake Johnston
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USADepartment of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA
Clinton Potter
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USADepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
Bridget Carragher
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USADepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
Mykhailo Kopylov
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, USA
Alex Noble
National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA
Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness. However, this approach may have several disadvantages, including low throughput for cells that are small, or at low concentration, or poorly distributed across accessible areas of the grid, as well as for samples that may adopt a preferred orientation. Here, we present a detailed description of the “Waffle Method” protocol for vitrifying thick specimens followed by a semi-automated milling procedure using the Thermo Fisher Scientific (TFS) Aquilos 2 cryo-FIB/scanning electron microscope (SEM) instrument and AutoTEM Cryo software to produce cryo-lamellae. With this protocol, cryo-lamellae may be generated from specimens, such as microsporidia spores, yeast, bacteria, and mammalian cells, as well as purified proteins and protein complexes. An experienced lab can perform the entire protocol presented here within an 8-hour working day, resulting in two to three cryo-lamellae with target thicknesses of 100–200 nm and dimensions of approximately 12 μm width and 15–20 μm length. For cryo-FIB/SEMs with particularly low-contamination chambers, the protocol can be extended to overnight milling, resulting in up to 16 cryo-lamellae in 24 h.Graphical abstract:
