adapted to a commercial cryogenic vacuum
transfer system (Quorum Technologies, PP3010T;
fig. S6 and text S3). SIM images were pro-
cessed as described ( 95 ); SMLM processing
is described in text S15.
EM sample preparation
After optical imaging, samples were transfer-
red back to cryo-storage before being freeze-
substituted, resin-embedded, and re-embedded
(text S6b and S6c). Desired regions of interest
(ROIs) were identified in the plasticized speci-
mens (fig. S12) using an XRadia 510 Versa
micro X-Ray system (CarlZeissX-rayMicros-
copy Inc.) and then trimmed to expose small
(~100mm × 100mm×60mm) stubs (text S6d).
FIB-SEM imaging
Standard (8 nm × 8 nm × 8 nm isotropic voxel)
FIB-SEM datasets were generated using a cus-
tomized Zeiss Merlin crossbeam system ( 12 )
and further modified as specified in text S16.
TheSEMimagestackswereacquiredat500kHz
per pixel with an 8-nmx-ypixel using a 2-nA
electron beam at 1.2-kV landing energy for
imaging and a 15-nA gallium ion beam at
30 kV for FIB milling. Similarly, 4 nm × 4 nm ×
4 nm voxel datasets were generated using a
customized Zeiss GeminiSEM 500-Capella
crossbeam system. The block face was imaged
by a 250-pA electron beam with 0.9-kV landing
energy at 200 kHz. The final image stacks were
registered using a SIFT-based algorithm ( 96 ).
Computing resources
For most of the data analysis, except initial
SMLM peak detection and fitting, we used a
stand-alone Windows 10 x64 workstation with
dual Xeon Gold 5122 CPUs (3.60 GHz) and
1 TB of RAM. For SMLM peak detection and
fitting, we used up to 256 nodes on the Janelia
cluster.
REFERENCES AND NOTES
- D. W. Fawcett,The Cell(Saunders, ed. 2, 1981).
- H. D. Ouet al., ChromEMT: Visualizing 3D chromatin structure
and compaction in interphase and mitotic cells.Science 357 ,
eaag0025 (2017). doi:10.1126/science.aag0025;
pmid: 28751582 - J. A. Briggs, Structural biology in situ—The potential of
subtomogram averaging.Curr. Opin. Struct. Biol. 23 , 261– 267
(2013). doi:10.1016/j.sbi.2013.02.003; pmid: 23466038 - A. Al-Amoudiet al., Cryo-electron microscopy of vitreous
sections.EMBO J. 23 , 3583–3588 (2004). doi:10.1038/
sj.emboj.7600366; pmid: 15318169 - A. Al-Amoudi, L. P. O. Norlen, J. Dubochet, Cryo-electron
microscopy of vitreous sections of native biological cells and
tissues.J. Struct. Biol. 148 , 131–135 (2004). doi:10.1016/
j.jsb.2004.03.010; pmid: 15363793 - A. Al-Amoudi, D. Studer, J. Dubochet, Cutting artefacts and
cutting process in vitreous sections for cryo-electron
microscopy.J. Struct. Biol. 150 , 109–121 (2005). doi:10.1016/
j.jsb.2005.01.003; pmid: 15797735 - B. Liuet al., Three-dimensional super-resolution protein
localization correlated with vitrified cellular context.Sci. Rep. 5 ,
13017 (2015). doi:10.1038/srep13017; pmid: 26462878 - M. Schafferet al., Optimized cryo-focused ion beam sample
preparation aimed at in situ structural studies of membrane
proteins.J. Struct. Biol. 197 ,73–82 (2017). doi:10.1016/
j.jsb.2016.07.010; pmid: 27444390
9. S. Pfeffer, J. Mahamid, Unravelling molecular complexity in
structural cell biology.Curr. Opin. Struct. Biol. 52 , 111– 118
(2018). doi:10.1016/j.sbi.2018.08.009; pmid: 30339965
10. G. Knott, H. Marchman, D. Wall, B. Lich, Serial section
scanning electronmicroscopy of adult brain tissue using
focused ion beam milling.J. Neurosci. 28 , 2959–2964 (2008).
doi:10.1523/JNEUROSCI.3189-07.2008; pmid: 18353998
11. K. Narayan, S. Subramaniam, Focused ion beams in biology.
Nat. Methods 12 , 1021–1031 (2015). doi:10.1038/nmeth.3623;
pmid: 26513553
12. C. S. Xuet al., Enhanced FIB-SEM systems for large-volume 3D
imaging.eLife 6 , e25916 (2017). doi:10.7554/eLife.25916;
pmid: 28500755
13. K. J. Hayworth, N. Kasthuri, R. Schalek, J. W. Lichtman,
Automating the Collection of Ultrathin Serial Sections for Large
Volume TEM Reconstructions.Microsc. Microanal. 12 (S02),
86 – 87 (2006). doi:10.1017/S1431927606066268
14. D. D. Bocket al., Network anatomy andin vivophysiology of
visual cortical neurons.Nature 471 , 177–182 (2011).
doi:10.1038/nature09802; pmid: 21390124
15. W. Denk, H. Horstmann, Serial block-face scanning electron
microscopy to reconstruct three-dimensional tissue
nanostructure.PLOS Biol. 2 , e329 (2004). doi:10.1371/journal.
pbio.0020329; pmid: 15514700
16. P. de Boer, J. P. Hoogenboom, B. N. G. Giepmans, Correlated
light and electron microscopy: Ultrastructure lights up!Nat.
Methods 12 , 503–513 (2015). doi:10.1038/nmeth.3400;
pmid: 26020503
17. M. Hauseret al., Correlative Super-Resolution Microscopy: New
Dimensions and New Opportunities.Chem. Rev. 117 ,
7428 – 7456 (2017). doi:10.1021/acs.chemrev.6b00604;
pmid: 28045508
18. E. Wegelet al., Imaging cellular structures in super-resolution
with SIM, STED and Localisation Microscopy: A practical
comparison.Sci. Rep. 6 , 27290 (2016). doi:10.1038/
srep27290;pmid: 27264341
19. D. R. Keene, K. McDonald, The ultrastructure of the connective
tissue matrix of skin and cartilage after high-pressure freezing
and freeze-substitution.J. Histochem. Cytochem. 41 ,1141– 1153
(1993). doi:10.1177/41.8.8331280; pmid: 8331280
20. K. L. McDonald, Electron microscopy and EM
immunocytochemistry.Methods Cell Biol. 44 , 411–444 (1994).
doi:10.1038/nmeth.1855; pmid: 22290187
21. K. McDonald, M. Morphew, P. Verkade, T. Müller-Reichert,
Recent advances in high-pressure freezing: Equipment and
specimen loading methods.Methods Mol. Biol. 369 ,143– 173
(2007).doi:10.1038/nmeth.1855; pmid: 22290187
22. U. Schnell, F. Dijk, K. A. Sjollema, B. N. G. Giepmans,
Immunolabeling artifacts and the need for live-cell imaging.
Nat. Methods 9 , 152–158 (2012). doi:10.1038/nmeth.1855;
pmid: 22290187
23. D. Studer, B. M. Humbel, M. Chiquet, Electron microscopy of high
pressure frozen samples: Bridging the gap between cellular
ultrastructure and atomic resolution.Histochem. Cell Biol. 130 ,
877 – 889 (2008). doi:10.1007/s00418-008-0500-1;pmid:18795316
24. D. Studeret al., Capture of activity-induced ultrastructural
changes at synapses by high-pressure freezing of brain tissue.
Nat. Protoc. 9 , 1480–1495 (2014). doi:10.1038/
nprot.2014.099; pmid: 24874814
25. J. Z. Kiss, T. H. Giddings Jr., L. A. Staehelin, F. D. Sack,
Comparison of the ultrastructure of conventionally fixed and
high pressure frozen/freeze substituted root tips of Nicotiana
and Arabidopsis.Protoplasma 157 ,64–74 (1990).
doi:10.1007/BF01322639; pmid: 11538077
26. R. Dahl, L. A. Staehelin, High-pressure freezing for the
preservation of biological structure: Theory and practice.
J. Electron Microsc. Tech. 13 , 165–174 (1989). doi:10.1002/
jemt.1060130305; pmid: 2685196
27. W. Li, S. C. Stein, I. Gregor, J. Enderlein, Ultra-stable and
versatile widefield cryo-fluorescence microscope for single-
molecule localization with sub-nanometer accuracy.Opt.
Express 23 , 3770– 3783 (2015). doi:10.1364/OE.23.003770;
pmid: 25836229
28. M. A. Schwentker, thesis, Kirchhoff Institute for Physics
(2007). doi:10.11588/heidok.00007677
29. M. W. Tuijtel, A. J. Koster, S. Jakobs, F. G. A. Faas, T. H. Sharp,
Correlative cryo super-resolution light and electron microscopy
on mammalian cells using fluorescent proteins.Sci. Rep. 9 ,
1369 (2019). doi:10.1038/s41598-018-37728-8;
pmid: 30718653
30. Y.-W. Changet al., Correlated cryogenic photoactivated
localization microscopy and cryo-electron tomography.Nat.
Methods 11 , 737–739 (2014). doi:10.1038/nmeth.2961;
pmid: 24813625
31. R. Kaufmannet al., Super-resolution microscopy using
standard fluorescent proteins in intact cells under
cryo-conditions.Nano Lett. 14 , 4171–4175 (2014). doi:10.1021/
nl501870p; pmid: 24884378
32. P. D. Dahlberget al., Identification of PAmKate as a Red
Photoactivatable Fluorescent Protein for Cryogenic
Super-Resolution Imaging.J. Am. Chem. Soc. 140 , 12310– 12313
(2018). doi:10.1021/jacs.8b05960; pmid: 30222332
33. F. Moseret al., Cryo-SOFI enabling low-dose super-resolution
correlative light and electron cryo-microscopy.Proc. Natl.
Acad. Sci. U.S.A. 116 , 4804–4809 (2019). doi:10.1073/
pnas.1810690116; pmid: 30808803
34. L. Wanget al., Solid immersion microscopy images cells under
cryogenic conditions with 12 nm resolution.Commun. Biol. 2 ,
74 (2019). doi:10.1038/s42003-019-0317-6; pmid: 30820469
35. J. B. Grimmet al., A general method to fine-tune fluorophores
for live-cell andin vivoimaging.Nat. Methods 14 , 987– 994
(2017). doi:10.1038/nmeth.4403; pmid: 28869757
36.G. V. Loset al., HaloTag: A novel protein labeling technology
for cell imaging and protein analysis.ACS Chem. Biol. 3 ,
373 – 382 (2008). doi:10.1021/cb800025k; pmid: 18533659
37. A. Keppleret al., A general method for the covalent labeling of
fusion proteins with small molecules in vivo.Nat. Biotechnol.
21 ,86–89 (2003). doi:10.1038/nbt765; pmid: 12469133
38. A. Gautieret al., An engineered protein tag for multiprotein
labeling in living cells.Chem. Biol. 15 , 128–136 (2008).
doi:10.1016/j.chembiol.2008.01.007; pmid: 18291317
39. D. Kimet al., Correlative stochastic optical reconstruction
microscopy and electron microscopy.PLOS ONE 10 , e0124581
(2015). doi:10.1371/journal.pone.0124581; pmid: 25874453
40. C. J. Peddieet al., Correlative super-resolution fluorescence
and electron microscopy using conventional fluorescent
proteins in vacuo.J. Struct. Biol. 199 , 120–131 (2017).
doi:10.1016/j.jsb.2017.05.013; pmid: 28576556
41. M. G. Paez-Segalaet al., Fixation-resistant photoactivatable
fluorescent proteins for CLEM.Nat. Methods 12 , 215– 218
(2015). doi:10.1038/nmeth.3225; pmid: 25581799
42. E. Johnsonet al., Correlative in-resin super-resolution and
electron microscopy using standard fluorescent proteins.
Sci. Rep. 5 , 9583 (2015). doi:10.1038/srep09583;
pmid: 25823571
43. N. Matsko, M. Mueller, Epoxy resin as fixative during
freeze-substitution.J. Struct. Biol. 152 ,92–103 (2005).
doi:10.1016/j.jsb.2005.07.005; pmid: 16214372
44. J. A. Bogovic, P. Hanslovsky, A. Wong, S. Saalfeld, in2016 IEEE
13thInternational Symposium on Biomedical Imaging (ISBI)
(IEEE, 2016), pp. 1123–1126;http://ieeexplore.ieee.org/
document/7493463/.
45. A. Sugiura, G.-L. McLelland, E. A. Fon, H. M. McBride, A new
pathway for mitochondrial quality control: Mitochondrial-
derived vesicles.EMBO J. 33 ,2142–2156 (2014).
doi:10.15252/embj.201488104; pmid: 25107473
46. B. Burke, J. Ellenberg, Remodelling the walls of the nucleus.
Nat. Rev. Mol. Cell Biol. 3 , 487–497 (2002). doi:10.1038/
nrm860; pmid: 12094215
47. D. J. Anderson, M. W. Hetzer, Reshaping of the endoplasmic
reticulum limits the rate for nuclear envelope formation.
J. Cell Biol. 182 , 911–924 (2008). doi:10.1083/jcb.200805140;
pmid: 18779370
48. J. Ellenberget al., Nuclear membrane dynamics and
reassembly in living cells: Targeting of an inner nuclear
membrane protein in interphase and mitosis.J. Cell Biol. 138 ,
1193 – 1206 (1997). doi:10.1083/jcb.138.6.1193; pmid: 9298976
49. L. Lu, M. S. Ladinsky, T. Kirchhausen, Cisternal organization
of the endoplasmic reticulum during mitosis.Mol. Biol. Cell 20 ,
3471 – 3480 (2009). doi:10.1091/mbc.e09-04-0327;
pmid: 19494040
50. J. J. Smith, J. D. Aitchison, Peroxisomes take shape.Nat. Rev.
Mol. Cell Biol. 14 , 803–817 (2013). doi:10.1038/nrm3700;
pmid: 24263361
51. M. Grabenbauer, K. Sätzler, E. Baumgart, H. D. Fahimi,
Three-dimensional ultrastructural analysis of peroxisomes in
HepG2 cells.Cell Biochem. Biophys. 32 ,37–49 (2000).
doi:10.1385/CBB:32:1-3:37; pmid: 11330069
52. M. Veenhuis, S. E. Wendelaar Bonga, Cytochemical localization
of catalase and several hydrogen peroxide-producing oxidases
in the nucleoids and matrix of rat liver peroxisomes.
Histochem. J. 11 , 561–572 (1979). doi:10.1007/BF01012539;
pmid: 511592
53. S. Angermüller, H. D. Fahimi, Selective cytochemical
localization ofperoxidase, cytochrome oxidase and catalase in
rat liver with 3,3′-diaminobenzidine.Histochemistry 71 ,33– 44
(1981). doi:10.1007/BF00592568; pmid: 6262282
Hoffmanet al.,Science 367 , eaaz5357 (2020) 17 January 2020 11 of 12
RESEARCH | RESEARCH ARTICLE