Background

Studying biological systems in close-to-native or in situ states opens wide frontiers towards precise contextual understandings of the mechanistic behaviors of molecular machines. In cryo-electron tomography (cryo-ET) pipelines, the sample is vitrified, which 1) captures and preserves biological processes in their close-to-native state, 2) maintains sample hydration, and 3) protects it from the vacuum of the electron microscope (Saibil, 2022). Vitrification is commonly followed by sample thinning with cryo-focused ion beam (cryo-FIB) milling (Marko et al., 2007) before cryo-ET data acquisition, providing the highest resolution in situ three dimensional (3D) views into the structures of individual protein complexes and cell parts in native contexts (Wang et al., 2022). Cryo-ET also allows for the assessment of the overall morphology of more complex biological systems, including cellular organelles, whole cells, intact organisms, and tissues (Bauerlein and Baumeister, 2021). However, vitrification with common plunge-freezing approaches is limited to samples up to ~5–10 μm thick. For thicker samples, a high-pressure freezer (HPF) must be used, which vitrifies specimens up to at least 60 μm thick (Harapin et al., 2015). HPF vitrification has been successfully applied to multicellular biological samples (Harapin et al., 2015), and its applicability has been extended with the waffle method (Kelley et al., 2022). The waffle method allows for 1) vitrification of samples with thicknesses of tens of microns, 2) elimination of preferred orientation issues, 3) production of large (between 10 μm × 10 μm and 70 μm × 25 μm in x,y) and specimen-dense cryo-lamellae, and 4) an increase in cryo-FIB lamellae production throughput.

Specimens vitrified with an HPF cannot be directly imaged by cryo-ET and need to be thinned down to <300 nm to be transparent enough to the electron beam. During the last decade, cryo-FIB has gained substantial traction for thinning vitrified biological specimens, overtaking the promising but technologically challenging method of cryo-ultramicrotomy (McDowall et al., 1983; Marko et al., 2007). Until several years ago, HPF vitrification of large (10+ μm thick) specimens followed by cryo-FIB/SEM and cryo-ET has typically been demonstrated on multicellular C. elegans organisms (Harapin et al., 2015; Mahamid et al., 2015; Schaffer et al., 2019). Direct cryo-FIB milling of these samples requires a significant amount of continuous milling (>1 day) and has a low success rate. To increase the throughput, Mahamid et al. (2015) and Schaffer et al. (2019) developed a cryo-liftout (cryo-LO) approach, where a section of the specimen is extracted with a micro-manipulator and transferred to a special half-moon grid, for further thinning. Cryo-LO allows for region of interest (ROI) extraction, and decreases milling times per single lamella of ~150 nm thickness and a suitable imaging area of ~8 μm × 7 μm to ~10 h (Schaffer et al., 2019); however, it requires specialized milling equipment and expertise.

To address these issues, we developed the waffle method, as described in Kelley et al. (2022). Here, we present a detailed protocol to support the waffle method, including sample vitrification (Procedure A), and waffle sample milling (Procedure B), that is applicable to a variety of biological samples. Samples prepared with Procedure A are generally tens of microns thick. Milling of these waffle samples does not require specialized instrumentation and can be performed manually or semi-automatically. Procedure B describes semi-automated milling, where milling takes ~2 h per lamellae site, with target thickness of 150–200 nm (as defined by the AutoTEM Cryo software), and approximate dimensions of 12 μm in width × 15–20 μm in length. Procedure B utilizes the MAPS software for defining ROIs and lamellae sites, then TFS AutoTEM Cryo automates the majority of the waffle milling process similarly to freely-available milling software packages (Buckley et al., 2020; Klumpe et al., 2021). Waffle lamellae are typically larger in cross-sectional area than lamellae generated with conventional plunge-frozen cryo-FIB-milling, and may be produced at a higher rate (~2,800 μm² per 24 h for waffle lamellae versus ~600 μm² per 24 h for conventional lamellae; see Notes for calculations).

To increase the quality and efficiency of the protocol, and to ensure that the resulting cryo-ET tomograms contain objects of interest, ROI localization is optionally done by correlating fluorescent images of the sample before or after freezing. Cryo-fluorescent light microscopy (cryo-FLM) is used to illuminate fluorescently-tagged objects in the vitrified sample and may be performed with a stand-alone cryo-FLM, or with an FLM integrated inside the milling instrument (iFLM, Gorelick et al., 2019). Cryo-FLM can be done before (as screening), during (for FIB-milling navigation), and after FIB-milling (for guiding data collection) (Watanabe et al., 2020; Gupta et al., 2021). However, it is important to account for autofluorescence for certain samples (Carter et al., 2018). In addition to fluorescence-based approaches, two other methods for localizing or obtaining statistical information of objects of interest in situ exist, but fall outside the scope of this protocol: samples with EM-tags for localization in cryo-ET data (Silvester et al., 2021; Tan et al., 2021), and structural mass spectrometry (Klykov et al., 2022).