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• W3170266525 abstract "•3D CLEM of cryo-FM and FIB-SEM datasets using fluorescently labeled lipid droplets•Cryo-FIB-SEM imaging of organelle-organelle interactions and nuclear organization Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells. Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells. Gaining a mechanistic understanding of biological processes often depends on developing the capability of visualizing cells and tissues in their native hydrated state at high resolution. Groundbreaking advances in volume electron microscopy and specimen preparation enable the 3D visualization of cells in unprecedented detail. These advances include adapting focused ion beam milling followed by scanning electron microscopy (FIB-SEM) to the life sciences (Heymann et al., 2006Heymann J.A. Hayles M. Gestmann I. Giannuzzi L.A. Lich B. Subramaniam S. Site-specific 3D imaging of cells and tissues with a dual beam microscope.J. Struct. Biol. 2006; https://doi.org/10.1016/j.jsb.2006.03.006Crossref Scopus (254) Google Scholar). FIB-SEM has since been used to gain previously inaccessible insights into both cells and tissues under physiological and pathological conditions (Heymann et al., 2006Heymann J.A. Hayles M. Gestmann I. Giannuzzi L.A. Lich B. Subramaniam S. Site-specific 3D imaging of cells and tissues with a dual beam microscope.J. Struct. Biol. 2006; https://doi.org/10.1016/j.jsb.2006.03.006Crossref Scopus (254) Google Scholar; Knott et al., 2008Knott G. Marchman H. Wall D. Lich B. Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling.J. Neurosci. 2008; https://doi.org/10.1523/JNEUROSCI.3189-07.2008Crossref PubMed Scopus (446) Google Scholar; Merchán-Pérez et al., 2009Merchán-Pérez A. Rodriguez J.R. AlonsoNanclares L. Schertel A. DeFelipe J. Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction.Front. Neuroanat. 2009; 3: 1-14https://doi.org/10.3389/neuro.05.018.2009Crossref PubMed Scopus (129) Google Scholar; Schneider et al., 2010Schneider P. Meier M. Wepf R. Müller R. Towards quantitative 3D imaging of the osteocyte lacuno-canalicular network.Bone. 2010; 47 (Elsevier Inc): 848-858https://doi.org/10.1016/j.bone.2010.07.026Crossref PubMed Scopus (119) Google Scholar, Schneider et al., 2011Schneider P. Meier M. Wepf R. Müller R. Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuno-canalicular network.Bone. 2011; 49 (Elsevier Inc): 304-311https://doi.org/10.1016/j.bone.2011.04.005Crossref PubMed Scopus (99) Google Scholar; Weiner et al., 2011Weiner A. Dahan-Pasternak N. Shimoni E. Shinder V. von Huth P. Elbaum M. Dzikowski R. 3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum.Cell Microbiol. 2011; 13: 967-977https://doi.org/10.1111/j.1462-5822.2011.01592.xCrossref PubMed Scopus (62) Google Scholar, Weiner et al., 2016Weiner A. Mellouk N. Lopez-Montero N. Chang Y.Y. Souque C. Schmitt C. Enninga J. Macropinosomes are key players in early shigella invasion and vacuolar escape in epithelial cells.PLoS Pathog. 2016; 12: 1-24https://doi.org/10.1371/journal.ppat.1005602Crossref Scopus (57) Google Scholar; Reznikov et al., 2013Reznikov N. Almany-Magal R. Shahar R. Weiner S. Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures.Bone. 2013; 52 (Elsevier Inc): 676-683https://doi.org/10.1016/j.bone.2012.10.034Crossref PubMed Scopus (100) Google Scholar; Revach et al., 2015Revach O.Y. Weiner A. Rechave K. Sabanay I. Livne A. Geiger B. Mechanical interplay between invadopodia and the nucleus in cultured cancer cells.Sci. Rep. 2015; 5: 1-13https://doi.org/10.1038/srep09466Crossref Scopus (49) Google Scholar). The principle of FIB-SEM is that a biological sample is exposed to a focused ion beam (usually consisting of Ga ions) capable of removing thin layers of material by milling in a highly precise manner (5–10 nm). Between each sample milling, a scanning electron beam is used to image the newly exposed surface. By repeating this process hundreds or even thousands of times, a large sample volume can be acquired with an isotropic voxel reaching 3 nm (Wei et al., 2012Wei D. Jacobs S. Modla S. Zhang S. Young C.L. Cirino R. Caplan J. Czymmek K. High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy.BioTechniques. 2012; 53: 41-48https://doi.org/10.2144/000113850Crossref PubMed Scopus (46) Google Scholar; Xu et al., 2020Xu C.S. Pang S. Shtengel G. Müller A. Ritter A.T. Hoffman H.K. Takemura S.Y. Lu Z. Pasolli H.A. Iyer N. Chung J. Isotropic 3D electron microscopy reference library of whole cells and tissues.bioRxiv. 2020; https://doi.org/10.1101/2020.11.13.382457Crossref Scopus (0) Google Scholar), in resin and at room temperature. With the recent development of automated acquisition procedures, even relatively large volumes (>1,000 μm3) can be acquired within a few days, providing large ultrastructural datasets (Peddie and Collinson, 2014Peddie C.J. Collinson L.M. Exploring the third dimension: volume electron microscopy comes of age.Micron. 2014; https://doi.org/10.1016/j.micron.2014.01.009Crossref PubMed Scopus (172) Google Scholar; Narayan and Subramaniam, 2015Narayan K. Subramaniam S. Focused ion beams in biology.Nat. Methods. 2015; 12 (Nature Publishing Group): 1021-1031https://doi.org/10.1038/nmeth.3623Crossref PubMed Scopus (107) Google Scholar). The ability to acquire large volumes with high and isometric resolution holds the potential to look at the cellular environment in a more holistic manner. One major drawback of conventional FIB-SEM imaging at room temperatures is that it requires dehydration and resin embedding, precluding the possibility to visualize cellular structures closer to their native hydrated state (Sviben et al., 2016Sviben S. Gal A. Hood M.A. Bertinetti L. Politi Y. Bennet M. Krishnamoorthy P. Schertel A. Wirth R. Sorrentino A. Pereiro E. A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga.Nat. Commun. 2016; 7: 1-9https://doi.org/10.1038/ncomms11228Crossref Scopus (84) Google Scholar; Vidavsky et al., 2016Vidavsky N. Addadi S. Schertel A. Ben-Ezra D. Shpigel M. Addadi L. Weiner S. Calcium transport into the cells of the sea urchin larva in relation to spicule formation.Proc. Natl. Acad. Sci. U S A. 2016; 113: 12637-12642https://doi.org/10.1073/pnas.1612017113Crossref PubMed Scopus (44) Google Scholar; Vidavsky et al., 2016Vidavsky N. Akiva A. Kaplan-Ashiri I. Rechav K. Addadi L. Weiner S. Schertel A. Cryo-FIB-SEM serial milling and block face imaging: large volume structural analysis of biological tissues preserved close to their native state.J. Struct. Biol. 2016; 196 (Elsevier Inc.): 487-495https://doi.org/10.1016/j.jsb.2016.09.016Crossref PubMed Scopus (36) Google Scholar; Kumar et al., 2020Kumar S. Rechav K. Kaplan-Ashiri I. Gal A. Imaging and quantifying homeostatic levels of intracellular silicon in diatoms.Sci. Adv. 2020; 6https://doi.org/10.1126/sciadv.aaz7554Crossref Scopus (8) Google Scholar). Recent studies have shown that cellular membranes are particularly visible using in-column secondary electron detection (InLens SE) in scanning electron microscopy (SEM) (Schertel et al., 2013Schertel A. Snaidero N. Han H.M. Ruhwedel T. Laue M. Grabenbauer M. Möbius W. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens.J. Struct. Biol. 2013; 184 (Elsevier Inc): 355-360https://doi.org/10.1016/j.jsb.2013.09.024Crossref PubMed Scopus (97) Google Scholar; Spehner et al., 2020Spehner D. Steyer A.M. Bertinetti L. Orlov I. Benoit L. Pernet-Gallay K. Schertel A. Schultz P. Cryo-FIB-SEM as a promising tool for localizing proteins in 3D.J. Struct. Biol. 2020; 211 (Elsevier): 107528https://doi.org/10.1016/j.jsb.2020.107528Crossref PubMed Scopus (8) Google Scholar). This finding was unexpected because without staining by heavy atoms to generate contrast based on back-scattered electrons from the flat surface, the sample should be electron transparent. It has since been suggested that contrast may be a product of low-energy type 1 secondary electrons, generated at the electron beam focal point at low voltage (<3 kV), which are sensitive to the local surface potential of different biological material (Schertel et al., 2013Schertel A. Snaidero N. Han H.M. Ruhwedel T. Laue M. Grabenbauer M. Möbius W. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens.J. Struct. Biol. 2013; 184 (Elsevier Inc): 355-360https://doi.org/10.1016/j.jsb.2013.09.024Crossref PubMed Scopus (97) Google Scholar). Nevertheless, the extent to which three dimensional (3D) organellar structure and organization can be studied at high resolution using cryo-FIB-SEM needs to be explored further. In parallel to the increasing popularity of FIB-SEM instruments, correlative light and electron microscopy (CLEM) workflows are becoming an important tool to study rare, dynamic, or undescribed cellular events by mapping information from fluorescence microscopy (FM) onto electron microscopy (EM) data of the exact same sample (Kukulski et al., 2011Kukulski W. Schorb M. Welsch S. Picco A. Kaksonen M. Briggs J.A. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.J. Cell Biol. 2011; 192: 111-119https://doi.org/10.1083/jcb.201009037Crossref PubMed Scopus (290) Google Scholar; Bykov et al., 2016Bykov Y.S. Cortese M. Briggs J.A. Bartenschlager R. ‘Correlative light and electron microscopy methods for the study of virus–cell interactions’.FEBS Lett. 2016; 590: 1877-1895https://doi.org/10.1002/1873-3468.12153Crossref PubMed Scopus (43) Google Scholar; Karreman et al., 2016Karreman M.A. Mercier L. Schieber N.L. Solecki G. Allio G. Winkler F. Ruthensteiner B. Goetz J.G. Schwab Y. Fast and precise targeting of single tumor cells in vivo by multimodal correlative microscopy.J. Cell Sci. 2016; 129: 444-456https://doi.org/10.1242/jcs.181842Crossref PubMed Scopus (55) Google Scholar; Khalifa et al., 2016Khalifa G.M. Kirchenbuechler D. Koifman N. Kleinerman O. Talmon Y. Elbaum M. Addadi L. Weiner S. Erez J. ‘Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence–SEM–EDS technique’.J. Struct. Biol. 2016; 196 (Elsevier Inc.): 155-163https://doi.org/10.1016/j.jsb.2016.01.015Crossref PubMed Scopus (24) Google Scholar; Weiner et al., 2016Weiner A. Mellouk N. Lopez-Montero N. Chang Y.Y. Souque C. Schmitt C. Enninga J. Macropinosomes are key players in early shigella invasion and vacuolar escape in epithelial cells.PLoS Pathog. 2016; 12: 1-24https://doi.org/10.1371/journal.ppat.1005602Crossref Scopus (57) Google Scholar; Weiner and Enninga, 2019Weiner A. Enninga J. The pathogen–host interface in three dimensions: correlative FIB/SEM applications.Trends Microbiol. 2019; 27 (Elsevier Ltd): 426-439https://doi.org/10.1016/j.tim.2018.11.011Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar; Scher and Avinoam, 2020Scher N. Avinoam O. 50 Shades of CLEM: how to choose the right approach for you.Methods Cell Biol. 2020; (Elsevier Inc)https://doi.org/10.1016/bs.mcb.2020.08.001Crossref PubMed Scopus (1) Google Scholar). For correlative microscopy, visible features in both imaging modalities must be identified. This can be achieved either by using intrinsic features of the sample, such as the shape of cells, organelles, and other landmarks, or by adding fiducial markers that are both fluorescent and electron opaque. The latter is typically challenging under cryogenic conditions, even for cryosections (Masich et al., 2006Masich S. Östberg T. Norlén L. Shupliakov O. Daneholt B. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography.J. Struct. Biol. 2006; 156: 461-468https://doi.org/10.1016/j.jsb.2006.05.010Crossref PubMed Scopus (44) Google Scholar), and even more so if a 3D correlation is needed, because the fiducials would have to be incorporated into the specimen volume. To overcome these challenges and develop a cryo-3D-CLEM approach, we examined whether organelles such as lipid droplets (LDs), which are relatively abundant in cells and well resolved by cryo-FIB-SEM imaging, can be used as internal fiducial markers to target the acquisition of large volumes in plunge-frozen cells grown on EM grids. Plunge freezing of cells grown on EM grids is a well-established method to vitrify samples for several downstream cryo-EM techniques (Medalia et al., 2002Medalia O. Weber I. Frangakis A.S. Nicastro D. Gerisch G. Baumeister W. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography.Science. 2002; 298: 1209-1213https://doi.org/10.1126/science.1076184Crossref PubMed Scopus (643) Google Scholar, Medalia et al., 2007Medalia O. Beck M. Ecke M. Weber I. Neujahr R. Baumeister W. Gerisch G. Organization of actin networks in intact Filopodia.Curr. Biol. 2007; 17: 79-84https://doi.org/10.1016/j.cub.2006.11.022Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar; Sartori et al., 2007Sartori A. Gatz R. Beck F. Rigort A. Baumeister W. Plitzko J.M. Correlative microscopy: Bridging the gap between fluorescence light microscopy and cryo-electron tomography.J. Struct. Biol. 2007; 160: 135-145https://doi.org/10.1016/j.jsb.2007.07.011Crossref PubMed Scopus (248) Google Scholar; Mahamid et al., 2016Mahamid J. Pfeffer S. Schaffer M. Villa E. Danev R. Cuellar L.K. Förster F. Hyman A.A. Plitzko J.M. Baumeister W. Visualizing the molecular sociology at the HeLa cell nuclear periphery.Science. 2016; 351: 969-972https://doi.org/10.1126/science.aad8857Crossref PubMed Scopus (248) Google Scholar; Spehner et al., 2020Spehner D. Steyer A.M. Bertinetti L. Orlov I. Benoit L. Pernet-Gallay K. Schertel A. Schultz P. Cryo-FIB-SEM as a promising tool for localizing proteins in 3D.J. Struct. Biol. 2020; 211 (Elsevier): 107528https://doi.org/10.1016/j.jsb.2020.107528Crossref PubMed Scopus (8) Google Scholar). In the present study, we grew mammalian cells on holey carbon-coated EM grids, cryo-immobilized them by plunge freezing, and imaged them using mixing of InLens SE and type 2 secondary electron (SE2) detection in cryo-FIB-SEM. Because the SE2 detector is less sensitive to charge accumulation on the cross section than the InLens SE detector, blending the two reduced the appearance of charging artifacts in the final image, resulting in a better signal to noise ratio. In all experiments (N = 5), cells appeared flat on the carbon film with the region close to the nucleus accounting for most of the volume (Figure 1A). The maximum volume acquired with an isometric voxel of 10 nm was ∼5,584μm3, which is approximately 45% of the volume of the cell. Much like in transmission electron microscopy (TEM) membranes and lipids generated a darker contrast compared with their surroundings, as previously shown (Schertel et al., 2013Schertel A. Snaidero N. Han H.M. Ruhwedel T. Laue M. Grabenbauer M. Möbius W. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens.J. Struct. Biol. 2013; 184 (Elsevier Inc): 355-360https://doi.org/10.1016/j.jsb.2013.09.024Crossref PubMed Scopus (97) Google Scholar; Spehner et al., 2020Spehner D. Steyer A.M. Bertinetti L. Orlov I. Benoit L. Pernet-Gallay K. Schertel A. Schultz P. Cryo-FIB-SEM as a promising tool for localizing proteins in 3D.J. Struct. Biol. 2020; 211 (Elsevier): 107528https://doi.org/10.1016/j.jsb.2020.107528Crossref PubMed Scopus (8) Google Scholar). Hence, membranous organelles such as the endoplasmic reticulum, Golgi apparatus, multi-vesicular bodies, and mitochondria could be readily observed and segmented (Figures 1B–1G) (Schertel et al., 2013Schertel A. Snaidero N. Han H.M. Ruhwedel T. Laue M. Grabenbauer M. Möbius W. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens.J. Struct. Biol. 2013; 184 (Elsevier Inc): 355-360https://doi.org/10.1016/j.jsb.2013.09.024Crossref PubMed Scopus (97) Google Scholar; Spehner et al., 2020Spehner D. Steyer A.M. Bertinetti L. Orlov I. Benoit L. Pernet-Gallay K. Schertel A. Schultz P. Cryo-FIB-SEM as a promising tool for localizing proteins in 3D.J. Struct. Biol. 2020; 211 (Elsevier): 107528https://doi.org/10.1016/j.jsb.2020.107528Crossref PubMed Scopus (8) Google Scholar). Re-localizing cells of interest on EM grids is relatively straight forward (Figures 2A–2C ). However, targeting specific regions of interest within the huge volume of the cell is still challenging. LDs have been recently used as fiducial markers post acquisition of 2D TEM data from cryo-lamella (Klein et al., 2021Klein S. Wimmer B.H. Winter S.L. Kolovou A. Laketa V. Chlanda P. Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies.Commun. Biol. 2021; 4 (Springer US): 1-12https://doi.org/10.1038/s42003-020-01567-zCrossref PubMed Scopus (6) Google Scholar). Here, we tested whether fluorescently labeled LDs can be used for 3D correlative cryo-FM and FIB-SEM imaging. LDs, also called lipid bodies, are organelles that store neutral lipids including triglycerides and cholesterol esters (Tauchi-Sato et al., 2002Tauchi-Sato K. Ozeki S. Houjou T. Taguchi R. Fujimoto T. The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition.J. Biol. Chem. 2002; 277 (© 2002 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology): 44507-44512https://doi.org/10.1074/jbc.M207712200Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar; Olzmann and Carvalho, 2019Olzmann J.A. Carvalho P. Dynamics and functions of lipid droplets.Nat. Rev. Mol. Cell Biol. 2019; 20: 137-155https://doi.org/10.1038/s41580-018-0085-zCrossref PubMed Scopus (422) Google Scholar). To use LDs as internal fiducials for correlation, we stained mammalian cells grown on grids with the fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) before plunge freezing and visualized the cells by cryo-FM (Schorb et al., 2017Schorb M. Gaechter L. Avinoam O. Sieckmann F. Clarke M. Bebeacua C. Bykov Y.S. Sonnen A.F.P. Lihl R. Briggs J.A. New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.J. Struct. Biol. 2017; 197 (The Authors): 83-93https://doi.org/10.1016/j.jsb.2016.06.020Crossref PubMed Scopus (52) Google Scholar). We observed that 100% of the cells displayed some BODIPY staining (Figure 2) and selected cells for acquisition based on their LDs distribution. Cells suitable for acquisition showed an average of 37 ± 22 LDs/cell (N = 63, median = 29 LDs/cell) (Figure 2D), which assured having at least 15 LDs for correlation within a sub-volume of the cell. Whole grid landmarks such as symbols or missing grid squares were used to relocate cells in the FIB-SEM (Figure 2A). One or two cells were acquired in one acquisition session, which lasted 36–60 h (Figure 2E). Correlation of both datasets was performed under the ICY software (De Chaumont et al., 2012De Chaumont F. Dallongeville S. Chenouard N. Hervé N. Pop S. Provoost T. Meas-Yedid V. Pankajakshan P. Lecomte T. Le Montagner Y. Lagache T. Icy: an open bioimage informatics platform for extended reproducible research.Nat. Methods. 2012; 9: 690-696https://doi.org/10.1038/nmeth.2075Crossref PubMed Scopus (721) Google Scholar). We initialized the correlation by manually marking the centers of each LD in the FIB-SEM stack. In parallel, the centroids of the LDs were automatically defined by wavelet spot detection in the FM stack. We subsequently used AutoFinder in the eC-CLEM image registration plugin (Paul-Gilloteaux et al., 2017Paul-Gilloteaux P. Heiligenstein X. Bell M. Domart M.C. Larijani B. Collinson L. Raposo G. Salamero J. EC-CLEM: Flexible multidimensional registration software for correlative microscopies.Nat. Methods. 2017; 14: 102-103https://doi.org/10.1038/nmeth.4170Crossref PubMed Scopus (103) Google Scholar) to automatically match the centers of LDs in FIB-SEM and the centroids of LDs in FM, which were independently identified. AutoFinder then computed and applied the rigid transformation to the image. Correlation precision was estimated using a leave-one-out approach (Kukulski et al., 2011Kukulski W. Schorb M. Welsch S. Picco A. Kaksonen M. Briggs J.A. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.J. Cell Biol. 2011; 192: 111-119https://doi.org/10.1083/jcb.201009037Crossref PubMed Scopus (290) Google Scholar) with 17 of 18 LDs, yielding an error estimation on the omitted LD of 3.5 μm. Use of a validated statistical approach (Paul-Gilloteaux et al., 2017Paul-Gilloteaux P. Heiligenstein X. Bell M. Domart M.C. Larijani B. Collinson L. Raposo G. Salamero J. EC-CLEM: Flexible multidimensional registration software for correlative microscopies.Nat. Methods. 2017; 14: 102-103https://doi.org/10.1038/nmeth.4170Crossref PubMed Scopus (103) Google Scholar; Potier et al., 2021Potier G. Lavancier F. Kunne S. Paul-Gilloteaux P. A Registration Error Estimation Framework for Correlative Imaging. arXiv preprint, 2021: 1-11Google Scholar) yielded an average expected error for every point in the volume of 2–4.7 μm (Figure S1A), for the rigid registration. Since the rigid registration did not give an accurate enough correlation (Figures S1 and S2A), we used an affine transformation on the rough registration from the AutoFinder using the same 17 LDs, yielding an error estimation on the omitted LD of 730 nm and an average expected error estimation of 370–880 nm (Figures 2F–2I and S2C). On analysis of the resulting image transformation (Figures 2F–2I), we observed image stretching in the yz direction of the FIB-SEM coordinate system (Figure 2H). This distortion can be explained by uncompensated drift during stack acquisition. We used fast scanning with line averaging to provide an acceptable compromise between the signal to noise ratio and charging artifacts. However, some charging of the surface occurs, which likely causes the slight image distortion in the xy plane. FIB-SEM data are also collected at a 90° with respect to the FM data, which means that the axis with the lowest resolution (z) is different in FM and FIB-SEM. However, the contribution of this factor to the overall correlation precision is accounted for in the error estimation calculation. Although in theory more fiducials should increase the correlation precision, this is not always the case as it also depends on their localization accuracy and on how they are distributed in the volume (Paul-Gilloteaux et al., 2017Paul-Gilloteaux P. Heiligenstein X. Bell M. Domart M.C. Larijani B. Collinson L. Raposo G. Salamero J. EC-CLEM: Flexible multidimensional registration software for correlative microscopies.Nat. Methods. 2017; 14: 102-103https://doi.org/10.1038/nmeth.4170Crossref PubMed Scopus (103) Google Scholar; Potier et al., 2021Potier G. Lavancier F. Kunne S. Paul-Gilloteaux P. A Registration Error Estimation Framework for Correlative Imaging. arXiv preprint, 2021: 1-11Google Scholar). To test the robustness of the correlation with less fiducials, we repeated the refinement process using only nine well-distributed LDs, some of which were from a cluster of LDs in the center of the cell. This approach reduced the average expected error to 270–600 nm (Figures S2C and S2D). Measuring the discrepancy between the assigned and transformed positions for all 18 or only the 9 left-out LDs yielded an error estimation of 220 ± 164 and 247 ± 203 nm, respectively. This improvement in accuracy is likely because local inaccuracies in picking LD centers within a tight cluster disproportionately increases the error (Figures S2C and S2D). These experiments show that LDs are suitable and attractive as internal fiducial markers for 3D correlation. From the volumes that we obtained in cryo-FIB-SEM, we segmented a large fraction of the mitochondrial network (Figure 3A , Video S1). We observed that interactions between adjacent mitochondria and the mitochondrial cristae could be readily resolved as well as small granules inside the mitochondria matrix (Figures 1C and 3B, Video S1). Since it is not possible to determine the elemental composition of such small objects in the cryo-FIB-SEM, we could only speculate based on their size distribution (Figures 3C and 3D) that they were calcium phosphate granules as observed by cryo-scanning transmission electron microscopy (Wolf et al., 2017Wolf S.G. Mutsafi Y. Dadosh T. Ilani T. Lansky Z. Horowitz B. Rubin S. Elbaum M. Fass D. 3D visualization of mitochondrial solid-phase calcium stores in whole cells.eLife. 2017; 6: 1-18https://doi.org/10.7554/eLife.29929Crossref Scopus (32) Google Scholar). The nuclear envelope (NE) and its nuclear pores were also clearly visible. The nucleus contrast was not uniform, and structures such as the heterochromatin in the periphery of the nucleus, the nucleoli, and nuclear speckles were also clearly visible (Figure 4A). Occasionally, we observed NE invaginations into the nucleus (Figure 4B). We segmented the NE invaginations and observed that they often followed a sinuous path (Figure 4B, Video S1). On one occasion the NE invagination formed a tube approximately 3.4 μm in length and ∼200 nm in diameter that crosses the nucleus (Figures 4C–4G, Video S1). We also observed small particles 42 ± 7 nm in diameter (N = 5) within the NE invagination (Figures 4B, 4E, and 4F). Together, these observations demonstrate that cryo-FIB-SEM is a particularly informative approach to study the 3D organization of membrane and membrane-less organelles in intact hydrated cells and highlights the need to combine cryo-FIB-SEM with fluorescence information.Figure 4Nuclear organization as visualized by cryo-FIB-SEMShow full caption(A) A representative slice from a cryo-FIB-SEM acquisition highlighting the nucleus. Differences in nuclear content is visible based on contrast change. N, nucleolus; Ai, heterochromatin; Aii, nuclear speckles. Scale bar: 1 μm.(B) Segmentation of nucleoli (yellow) and nuclear envelope invaginations (green). One of the observed invaginations passed through the whole nucleus and was less sinuous than others. Holey carbon support film (gray).(C) The nuclear invagination crossing the entire nucleus spanning approximately 3.4 μm in length and approximately 200 nm in diameter. Inset shows the particles inside the invagination (blue).(D–G) Representative slices through the nuclear envelope invagination showing the particles inside the spherical appendage (E and F). Inset shows a 1.5× magnification of the boxed area. The small particles measured are 42 ± 7 nm in diameter (N = 5). Scale bar: 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) A representative slice from a cryo-FIB-SEM acquisition highlighting the nucleus. Differences in nuclear content is visible based on contrast change. N, nucleolus; Ai, heterochromatin; Aii, nuclear speckles. Scale bar: 1 μm. (B) Segmentation of nucleoli (yellow) and nuclear envelope invaginations (green). One of the observed invaginations passed through the whole nucleus and was less sinuous than others. Holey carbon support film (gray). (C) The nuclear invagination crossing the entire nucleus spanning approximately 3.4 μm in length and approximately 200 nm in diameter. Inset shows the particles inside the invagination (blue). (D–G) Representative slices through the nuclear envelope invagination showing the particles inside the spherical appendage (E and F). Inset shows a 1.5× magnification of the boxed area. The small particles measured are 42 ± 7 nm in diameter (N = 5). Scale bar: 1 μm. https://www.cell.com/cms/asset/a821b28a-892a-41b4-8f4b-18ac569dc03c/mmc2.mp4Loading ... Download .mp4 (19.29 MB) Help with .mp4 files Video S1. Intra-cellular organization as visualized by cryo-FIB-SEM, related to Figures 3 and 4 The cryo-FM and FIB-SEM correlative imaging workflow presented here can be used to study any cell that has LDs dispersed around the region of interest. It is particularly useful for studying cellular structures that are not well preserved by conventional FIB-SEM. The resolution and correlation precision can be further improved by using cryo-confocal or super-resolution FM (Arnold et al., 2016Arnold J. Mahamid J. Lucic V. De Marco A. Fernandez J.J. Laugks T. Mayer T. Hyman A.A. Baumeister W. Plitzko J.M. Site-pecific cryo-focused ion beam sample preparation guided by 3D correlative microscopy.Biophys. J. 2016; 110: 860-869https://doi.org/10.1016/j.bpj.2015.10.053Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar; Wolff et al., 2016Wolff G. Hagen C. Grünewald K. Kaufmann R. Towards correlative super-resolution fluorescence and electron cryo-microscopy.Biol. Cell. 2016; 108: 245-258https://doi.org/10.1111/boc.201600008Crossref PubMed Scopus (59) Google Scholar; Hoffman et al., 2020Hoffman D.P. Shtengel G. Xu C.S. Campbell K.R. Freeman M. Wang L. Milkie D.E. Pasolli H.A. Iyer N. Bogovic J.A. Stabley D.R. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.Science. 2020; 367https://doi.org/10.1126/science.aaz5357Crossref PubMed Scopus (96) Google Scholar). Further improving the correlation precision will be important if specific regions need to be targeted for milling of thin lamella suitable for TEM. Much can also be done to enhance the success rate of data acquisition, alignment, image processing, and analysis." @default.
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• W3170266525 cites W1523109634 @default.
• W3170266525 cites W1965637518 @default.
• W3170266525 cites W2004277164 @default.
• W3170266525 cites W2005158297 @default.
• W3170266525 cites W2025042015 @default.
• W3170266525 cites W2034184519 @default.
• W3170266525 cites W2037947871 @default.
• W3170266525 cites W2055853285 @default.
• W3170266525 cites W2063752629 @default.
• W3170266525 cites W2072291193 @default.
• W3170266525 cites W2086485894 @default.
• W3170266525 cites W2087765858 @default.
• W3170266525 cites W2099934586 @default.
• W3170266525 cites W2106393979 @default.
• W3170266525 cites W2122804888 @default.
• W3170266525 cites W2125238092 @default.
• W3170266525 cites W2143368436 @default.
• W3170266525 cites W2145494795 @default.
• W3170266525 cites W2167279371 @default.
• W3170266525 cites W2213565211 @default.
• W3170266525 cites W2235248450 @default.
• W3170266525 cites W2279402243 @default.
• W3170266525 cites W2293502639 @default.
• W3170266525 cites W2303909136 @default.
• W3170266525 cites W2336589732 @default.
• W3170266525 cites W2400565571 @default.
• W3170266525 cites W2406638036 @default.
• W3170266525 cites W2418573148 @default.
• W3170266525 cites W2460061039 @default.
• W3170266525 cites W2523814097 @default.
• W3170266525 cites W2539070134 @default.
• W3170266525 cites W2583762284 @default.
• W3170266525 cites W2767435910 @default.
• W3170266525 cites W2802442955 @default.
• W3170266525 cites W2903107073 @default.
• W3170266525 cites W2907130154 @default.
• W3170266525 cites W2999528257 @default.
• W3170266525 cites W3023557184 @default.
• W3170266525 cites W3038883326 @default.
• W3170266525 cites W3093070307 @default.
• W3170266525 cites W3121535788 @default.
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