Background

In the conventional in-situ cryo-electron tomography (cryo-ET) workflow, initially cultured cells expressing a fluorescent marker gene are required to adhere to an electron microscopy grid before they are cryo-fixed by plunge freezing (Iancu et al., 2007; Rigort et al., 2012; Mahamid et al., 2016). To navigate through the complex environment of the frozen-hydrated cell, correlative light and electron microscopy (CLEM) approaches were introduced into the workflow to localize the regions of interest (ROIs) (van Driel et al., 2009; Schorb and Briggs, 2014; Klein, Wachsmuth-Melm, et al., 2021). To this end, the grids are typically transferred to a cryo-fluorescence microscope to acquire spatial information on various features of interest using fluorescence (Arnold et al., 2016; Chakraborty et al., 2020). Following a second cryo-transfer step to a cryo focused-ion-beam–scanning electron microscopy (cryo FIB–SEM), the cells are thinned down to ~150 nm-thick lamellae using a focused ion beam, making them amenable to cryo-ET or cryo-EM imaging (Marko et al., 2007; Rigort et al., 2012; Villa et al., 2013; Lucas et al. 2021).

The workflow provides natively preserved insight into the structure-dependent functionality of cellular components at molecular or even close-to-atomic resolution (Xing et al., 2023). However, the complexity of the workflow poses challenges that frequently compromise the throughput and success rate (Hylton and Swulius, 2021). To name a few: i) conventional cell culture procedures lack proper control over cell shape and cell positioning on the grids. This limitation often results in cell clustering and cells positioned on grid bars rather than in the center of the grid squares, both of which make them unsuitable for FIB milling (Wagner et al., 2020). ii) The time-consuming nature of cryo FIB milling requires long residence time of the lamellae within the vacuum chamber. Consequently, the risk of in-chamber ice contamination significantly increases during batch milling sessions, making it impossible to mill multiple lamellae in an overnight session (Buckley et al., 2020; Tacke et al., 2021). iii) Combining CLEM approaches with cryo FIB milling allows for targeted preparation of lamellae at each intended region of interest (ROI). However, the cryo-transfer steps to and from the cryo-fluorescence microscope significantly increase the chance of ice contamination and devitrification of the sample. iv) The conventional way of lamella preparation and target determination is time consuming and complicated. Therefore, the error-prone procedure prior to lamella milling can easily lead to a mismatch in correlation and result in the loss of the ROI (Fukuda et al., 2014; Klein, Wimmer, et al., 2021).

In this protocol, we describe the details of a combined workflow with several newly available technological upgrades. i) Micropatterning the electron microscopy (EM) grids prior to cell culture using PRIMO: the customized patterns result in improved control over cell shape and spatial distribution over the entire grid (Engel et al., 2019; Swistak et al., 2021). By covering the EM grid in an anti-fouling layer and selectively removing it through UV light, the cell can only grow on selected areas of the grid. Provided the applied mask of the UV light is not too big, the cell will adapt to the mask shape, allowing one to reposition neuronal axons, study stress fibers in a specific area of the cell periphery, or adjust the cell shape in some other way. Consequently, more millable sites will be available per grid, making an overnight milling session worthwhile (Toro-Nahuelpan et al., 2020; Sibert et al., 2021). ii) Cryo FIB milling in the presence of the CERES ice shield: the CERES ice shield eliminates the parasitic growth of an amorphous ice layer on top of the lamellae. Without it, lamella can be rendered unusable due to excessive ice contamination at the end of an overnight batch milling session (Tacke et al., 2021; Lau et al., 2022). iii) Continuous, in-chamber fluorescence detection using METEOR: by integrating the METEOR cryo-fluorescence microscope into the vacuum chamber of the cryo FIB–SEM, one major cryo-transfer step is eliminated from the workflow, which significantly reduces the chances of sample devitrification, contamination, and mishandling (Smeets et al., 2021). iv) The fluorescence integration in the FIB–SEM further allows for continuous monitoring of the ROI within the lamella during the milling process, thus eliminating the need for separate signal correlation to find back the ROI. This protocol has been proven to substantially increase the number of produced usable lamellae per milling session and hence surpass the conventional achievable success rate and throughput for high-resolution imaging in a cryo-transmission electron microscope (cryo-TEM). One can normally expect to produce 3–7 lamella in a fully manual milling session, and an estimated 25 lamella in automated session using micropatterned grids and the technological improvements described in this protocol, which are summarized in Table 1.

Table 1. Estimation of the effect of different workflow improvements on the output