Cryo-electron tomography of large biological specimens vitrified by plunge freezing

Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to voluminous biological specimens such as tissues or particularly large mammalian cells, which usually require high-pressure freezing for vitrification. Here we show that adult mouse cardiomyocytes and dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of complex native specimens. Our data provides unanticipated insights into the molecular architecture of samples hitherto unexplored by cryo-ET.

[1]  Danielle A. Grotjahn,et al.  Quantifying organellar ultrastructure in cryo-electron tomography using a surface morphometrics pipeline , 2023, The Journal of cell biology.

[2]  T. Friede,et al.  Direct proteomic and high-resolution microscopy biopsy analysis identifies distinct ventricular fates in severe aortic stenosis. , 2022, Journal of molecular and cellular cardiology.

[3]  R. Danev,et al.  Parallel cryo electron tomography on in situ lamellae , 2022, bioRxiv.

[4]  B. Carragher,et al.  Waffle Method: A general and flexible approach for improving throughput in FIB-milling , 2022, Nature Communications.

[5]  S. Lehnart,et al.  The role of junctophilin proteins in cellular function , 2022, Physiological reviews.

[6]  M. Gautel,et al.  The molecular basis for sarcomere organization in vertebrate skeletal muscle , 2021, Cell.

[7]  Automated electron microscope , 2020, Metal Powder Report.

[8]  W. Baumeister,et al.  Molecular-scale visualization of sarcomere contraction within native cardiomyocytes , 2020, Nature Communications.

[9]  Esther Pueyo,et al.  Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images , 2020, Biomolecules.

[10]  R. Robergs,et al.  Glycerol , 2020, Reactions Weekly.

[11]  Wolfgang Baumeister,et al.  Reliable estimation of membrane curvature for cryo-electron tomography , 2020, PLoS Comput. Biol..

[12]  M. Marko,et al.  Cryo‐FIB preparation of whole cells and tissue for cryo‐TEM: use of high‐pressure frozen specimens in tubes and planchets , 2020, Journal of microscopy.

[13]  J. Heuser,et al.  Introducing a mammalian nerve-muscle preparation ideal for physiology and microscopy, the transverse auricular muscle in the ear of the mouse , 2020, Neuroscience.

[14]  Jaime Fern'andez del R'io,et al.  Array programming with NumPy , 2020, Nature.

[15]  D. Agard,et al.  Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae. , 2019, Journal of structural biology.

[16]  Sjors H.W. Scheres,et al.  Faculty Opinions recommendation of Real-time cryo-electron microscopy data preprocessing with Warp. , 2019, Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature.

[17]  W. Baumeister,et al.  A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue , 2019, Nature Methods.

[18]  W. Baumeister,et al.  A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue , 2019, Nature Methods.

[19]  D. Agard,et al.  Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae , 2019, bioRxiv.

[20]  Z. Su,et al.  Unraveling the Regulation of Hepatic Gluconeogenesis , 2019, Front. Endocrinol..

[21]  W. Baumeister,et al.  In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment , 2018, Cell.

[22]  M. Alves-Bezerra,et al.  Triglyceride Metabolism in the Liver. , 2017, Comprehensive Physiology.

[23]  Wolfgang Baumeister,et al.  In Situ Architecture and Cellular Interactions of PolyQ Inclusions , 2017, Cell.

[24]  Rubén Fernández-Busnadiego,et al.  Cryo‐electron tomography—the cell biology that came in from the cold , 2017, FEBS letters.

[25]  David Bilder,et al.  Taking Stock of the Drosophila Research Ecosystem , 2017, Genetics.

[26]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[27]  Jianping Wu,et al.  Structural basis for the gating mechanism of the type 2 ryanodine receptor RyR2 , 2016, Science.

[28]  R. Harding,et al.  Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections , 2016, Scientific Reports.

[29]  Hugo J. Bellen,et al.  COLLECTION : TRANSLATIONAL IMPACT OF DROSOPHILA Drosophila tools and assays for the study of human diseases , 2016 .

[30]  A. Hyman,et al.  Visualizing the molecular sociology at the HeLa cell nuclear periphery , 2016, Science.

[31]  A. Hyman,et al.  Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. , 2016, Biophysical journal.

[32]  José-Jesús Fernández,et al.  Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms. , 2016, Biophysical journal.

[33]  A. Hyman,et al.  A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms. , 2015, Journal of structural biology.

[34]  V. Lučić,et al.  Electron cryotomography of vitrified cells with a Volta phase plate. , 2015, Journal of structural biology.

[35]  W. Baumeister,et al.  Volta potential phase plate for in-focus phase contrast transmission electron microscopy , 2014, Proceedings of the National Academy of Sciences.

[36]  S. Lehnart,et al.  Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles , 2014, Journal of visualized experiments : JoVE.

[37]  Shoh Asano,et al.  Robust membrane detection based on tensor voting for electron tomography. , 2014, Journal of structural biology.

[38]  J. Gray,et al.  Thinning of large mammalian cells for cryo‐TEM characterization by cryo‐FIB milling , 2012, Journal of microscopy.

[39]  George S. B. Williams,et al.  Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction , 2012, Circulation research.

[40]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[41]  B. Murray,et al.  Freezing injury: the special case of the sperm cell. , 2012, Cryobiology.

[42]  Felix J. B. Bäuerlein,et al.  Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography , 2012, Proceedings of the National Academy of Sciences.

[43]  L. W. Liu,et al.  Chronic constipation: current treatment options. , 2011, Canadian journal of gastroenterology = Journal canadien de gastroenterologie.

[44]  P. Bishop,et al.  Skin ice nucleators and glycerol in the freezing-tolerant frog Litoria ewingii , 2011, Journal of Comparative Physiology B.

[45]  B. Humbel,et al.  The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. , 2010, Journal of structural biology.

[46]  Matthias Chiquet,et al.  Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution , 2008, Histochemistry and Cell Biology.

[47]  John D. Hunter,et al.  Matplotlib: A 2D Graphics Environment , 2007, Computing in Science & Engineering.

[48]  R. Schalek,et al.  Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy , 2007, Nature Methods.

[49]  C. Xiao,et al.  Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: Glycerol replacement of NaCl protects CNS neurons , 2006, Journal of Neuroscience Methods.

[50]  David N Mastronarde,et al.  Automated electron microscope tomography using robust prediction of specimen movements. , 2005, Journal of structural biology.

[51]  R. Sterzi,et al.  Glycerol for Acute Stroke , 2005, The Cochrane database of systematic reviews.

[52]  J. Dubochet,et al.  Cryo‐electron microscopy of vitreous sections , 2004, The EMBO journal.

[53]  R. Robergs,et al.  Glycerol. Biochemistry, pharmacokinetics and clinical and practical applications. , 1998, Sports medicine.

[54]  R. Sousa.  Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization. , 1995, Acta crystallographica. Section D, Biological crystallography.

[55]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[56]  K. E. Zachariassen.  Physiology of cold tolerance in insects. , 1985, Physiological reviews.

[57]  M. Blackman,et al.  The cubic and other structural forms of ice at low temperature and pressure , 1957, Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences.

[58]  Gáspár Jékely,et al.  Content-aware image restoration for electron microscopy. , 2019, Methods in cell biology.

[59]  Florian Beck,et al.  In Situ Structure of Neuronal C 9 orf 72 Poly-GA Aggregates Reveals Proteasome Recruitment Graphical , 2018 .

[60]  W. Baumeister,et al.  Cryo-Electron Tomography: Can it Reveal the Molecular Sociology of Cells in Atomic Detail? , 2016, Trends in cell biology.

[61]  M. Marko,et al.  Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography. , 2014, Journal of structural biology.

[62]  P. Chlanda,et al.  Cryo-electron microscopy of vitreous sections. , 2014, Methods in molecular biology.

[63]  Daniel Baum,et al.  Automated segmentation of electron tomograms for a quantitative description of actin filament networks. , 2012, Journal of structural biology.

[64]  J. Dubochet,et al.  The cell in absence of aggregation artifacts. , 2001, Micron.

[65]  J R Kremer,et al.  Computer visualization of three-dimensional image data using IMOD. , 1996, Journal of structural biology.