In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.
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Subtomogram averages were deposited in the Electron Microscopy Data Bank under accession codes EMD-33834, EMD-33115, EMD-33116, EMD-33117, EMD-33118 and EMD-33833. Raw tilt series frames were deposited in the Electron Microscopy Public Image Archive under accession codes EMPIAR-11111, EMPIAR-10985, EMPIAR-10986 and EMPIAR-10987.
SerialEM python scripts for auxiliary functions, target selection and PACE-tomo are available at https://github.com/eisfabian/PACEtomo.
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We are grateful to Y. Fujiyoshi and H. Suzuki for stimulating discussions and valuable feedback. We thank K. Nakamura and Y. Sakamaki for the management and support of the Graduate School of Medicine cryoEM facility. We are grateful for support of cryoFIB milling and instrument maintenance by Y. Fukuda and for comments on the manuscript by I. Selvam. This research was partially supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant number JP21am0101115j0005. F.E. is an International Research Fellow of the Japan Society for the Promotion of Science (JSPS, #P20764) and received a Grant-in-Aid for Scientific Research (KAKENHI, 21F20764). H.Y. and M.K. were supported by Grant-in-Aid for Transformative Research Areas A (JSPS, 21H05248). S.T. received funding from Grant-in-Aid for Specially Promoted Research (JP19H05468). R.D. was supported by Takeda Science Foundation 2019 Medical Research Grant and Japan Science and Technology Agency PRESTO (18069571).
The authors declare no competing interests.
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a. Shown are defocus values estimated by CTF fitting for two tilt series collected on the same lamella using PACE-tomo. One tilt series was recorded without applying a defocus ramp (dashed line). The other tilt series (solid line) was recorded while applying a defocus ramp of 0.03 µm/degree. Outliers at extreme tilt angles were not considered for the linear fit of the defocus slope. b. Shown are defocus values estimated by CTF fitting for two tilt series collected on amorphous carbon support using PACE-tomo. One tilt series was recorded using the tilt axis offset determined by the fine eucentricity routine of SerialEM (dashed line). The other tilt series (solid line) was recorded using the tilt axis offset determined using the PACEtomo_measureOffset.py script.
a. Specimen shifts in µm along x and y (parallel and perpendicular to the tilt axis respectively). The behavior of the negative tilt angle branch differs from the positive tilt angle branch. b. Defocus throughout the tilt series measured by beam tilt. While defocus remains relatively constant for the positive tilt angle branch, it shows a significant slope for the negative tilt angle branch.
a. An overview image of a holey carbon sample area indicating the positions at which tilt series were collected. Targets are numbered according to acquisition order. Red frame indicates the position of the tracking tilt series. The tilt axis is along Y = 0. Collection time was 118 minutes for 25 tilt series with an angular range of ± 30°. b, c. Measured specimen shift errors by cross correlation-based alignment to first tilt image at 0° in each tilt series in x- (b) and y-direction (c), parallel and perpendicular to the tilt axis, respectively. Tilt series 1 is the tracking tilt series and points are coloured grey. d. Defocus values for each tilt series estimated by CTF fitting. e. Defocus values for each tilt series estimated by CTF fitting of the 0° tilt image plotted against the target coordinates in X and Y. The 3D plot shows that targets are approximately on a plane indicating significant tilt of the sample support film.
a. Tilt series of dataset 1 were divided into 5 groups parallel (red groups) and perpendicular (blue groups) to the tilt axis, respectively, and reconstructed independently. Resolutions were plotted against the relative distance to the central group. Neither parallel nor perpendicular groups to the tilt axis show a significant resolution dependency on the distance from the centre. b. Acquisition pattern for dataset 2 – PACE-tomo with 81 targets in a regular 9 by 9 pattern. Targets are numbered according to acquisition order. The tracking tilt series (red frame) is at the origin and all other targets are acquired by relative image shift along the tilt axis (x-axis) and perpendicular to the tilt axis (y-axis). Collection time was 6 hours. c. Vectors representing beam tilt determined by RELION Tomo CTF refinement using 1 optics group per tilt series. Origins of vectors are at their respective image shifts relative to the tracking tilt series at 0° tilt angle. Magnitude scale of vectors is indicated by the scale bar (top right). d. Tilt series of dataset 2 were divided into 5 groups parallel (red groups) and perpendicular (blue groups) to the tilt axis, respectively, and reconstructed independently. Resolutions were plotted against the relative distance to the central group. Parallel groups to the tilt axis show a resolution dependency on the distance from the centre while perpendicular groups do not.
a. Shown is a 0.66 nm thick slice through one of the 21 cryo electron tomograms (acquisition area 3) collected on the lamella shown in Fig. 4b. The subtomogram average was mapped into the tomogram using the refined coordinates and orientations. Membrane associated and cytoplasmic ribosomes were coloured red and blue, respectively. Scale bar: 100 nm. b. Shown is a 0.66 nm thick slice through one of 7 cryo electron tomograms collected on the bottom lamella shown in c. The subtomogram average of actin was mapped into the tomogram using the refined coordinates and orientations. Scale bar: 100 nm. c. Overview montages of two (out of 41) cryoFIB-milled lamellae indicating the positions at which tilt series were collected. Red frame indicates the position that was used for the tracking tilt series. White arrowheads mark targets that would not have been accessible by conventional cryoET collection schemes. Collection times were 23 minutes for 6 tilt series (top) and 30 minutes for 7 tilt series (bottom, used in dataset 5).
a. Scanning electron microscopy image of a representative sample of EpH4 cells grown on a 150-mesh gold grid. A total of eight grids were used to prepare 41 lamellae. Scale bar: 1 mm. b. CryoFIB image after premilling trenches at 38° that mark targets for lamella milling and limit lamella length. Shown are four examples of the 41 prepared lamellae. Scale bar: 100 µm. c. Scanning electron microscopy image of finished lamella with micro-expansion joints that was used for a PACE-tomo acquisition (Extended Data Fig. 5c top). Shown is one of the 41 prepared lamellae. Scale bar: 10 µm.
Shown are the essential steps of the PACE-tomo data acquisition scheme.
Shown are the processing workflows for datasets 1–5 using RELION-4.0-beta. Blue and red boxes indicate steps done in dynamo and PEET, respectively. Final maps were filtered and coloured according to the local resolution distribution.
Supplementary Table 1, and captions for Supplementary Videos 1 and 2.
Slices through tomogram from dataset 4 collected on the cryoFIB-milled lamella shown in Fig. 4b (tilt series 3). The subtomogram average was mapped into the tomogram using the refined coordinates and orientations. Membrane-associated and cytoplasmic ribosomes were coloured red and blue, respectively.
Cross-correlation aligned tilt series from dataset 4 collected on the cryoFIB-milled lamella shown in Fig. 4b (tilt series 3). Scale bar, 100 nm.
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Eisenstein, F., Yanagisawa, H., Kashihara, H. et al. Parallel cryo electron tomography on in situ lamellae. Nat Methods 20, 131–138 (2023). https://doi.org/10.1038/s41592-022-01690-1