Cryo Electron Microscopy John Mitchels Thanks to Miloš Hovorka and Alex Rigort Define cryo… The word cryo or cryos (κρύο) is Greek and means "icy cold" (from crystallos) Typically temperatures lower than -100 Centigrade, but hardware generally allows temperatures lower than room temperature. • (N) Nitrogen -196 • (He) Helium -269 • (C6H6) Ethane -88 2 3 4 Transmission Electron Microscopes (TEM) Scanning Electron Microscopes (SEM) Dual Beams (FIB/SEM) FEI Product Line - Technology Leadership Optical solutions, data processing, etc. Bio samples (or other soft water) • Biological samples are full of water. This has the implications for the sample preparation and observation (body water content around 60%, brain 73%, cell 70%). • Most/less abundant elements: H, C, N, O/Na, Mg, P, S. • Biological sample = poor scattering = poor contrast in electron microscope. • In EM context low sample conductivity = charging. – coating by metal layer, low dose, low kV, scanning strategy – HiVac x LoVac/ESEM 5 Specimen preparation 9 • Fixation = to stop the biological activity and to preserve the tissue structure for subsequent treatments. • “The objective is the process tissues and cells without significant change in size, shape, positional relationship of the cellular components and to preserve as much of the biological activity and chemical nature of cellular components…” • Physical fixation – Heat/Microwaves (+ chemical fixation) – Cryofixation (rapid freezing of cells or tissues to the very low temperature) • Chemical fixation – traditional technique using chemical or mixture of chemicals Francis Bacon (1561-1626) First scientific publication about preservation with ice. He stuffed a chicken with snow to see what would happen, he caught pneumonia that day, then he died. The problem with Fixation 10 Normal Strawberry Freeze Dried Good for general morphology at tissue level; at macro level lots of distortion, very easy method and easy to image in SEM. CPD Better preservation of the macro structure; loss of components via solvent extraction; very easy and quick. Chemical Fixation Good macroscopic control; ultra structure can be well preserved but soft and squishy; needs support for sectioning; a lot of extraction; quick easy. Heat Preservation Well you can see it does not work for strawberries unlike eggs. Cryo Preservation Good macro and micro, and ultra structure and tissue support when frozen. Bad when thawed! 11 Adapted,imagebyAndresKaech,UniversityofZürich. Samplepreparationflowchart (selectedtechniques) + staining FIB/SEM Samplepreparationflowchart (selectedtechniques) Tissue preparation (using chemicals) Sample preparation flow. • chemical fixation – e.g. glutaraldehyde, formaldehyde, osmium tetroxide, … – result is influenced by sample size, a way how to fix, concentration of solution, speed of penetration of fixative, temperature, time etc. • dehydration = to remove water  to enable infiltration (ethanol/aceton replaces water) • infiltration/embedding into suitable medium (resin - EM, paraffin - LM) – examples of resins: acrylic (Lowicryl, LR White), epoxy (Epon (1956), Araldites, Spurr, Durcupan, etc.) – ideally well soluble in dehydration agents, low viscosity, minimal shrinkage, stability under e-beam, minimal granularity • block observation / cutting into sections  post staining 13 • Why, advantages – The best method of preservation. Rapid freezing in milliseconds = near perfect preservation (minimal chemical and physical changes if done well). – Offers a SnapShot at a particular time, very important when studying function. • Sample vitrification – Cool the specimen so rapidly that there is not time for ICE (crystalline water) to form!  ICE is what does the damage as it rips structures apart. – Increase cooling speed by the reducing size of the specimen. • Methods – High pressure freezing (HPF, up to 200 µm), plunge freezing, slam (metal-mirror) freezing, spray freezing, double jet propane freezing (all up to units or tens of µm) 14 Cryofixation 15 Adapted,imagebyAdresKaech,UniversityofZurich. Samplepreparationflowchart (selectedtechniques) + staining FIB/SEM No dehydration!Cryofixation High Pressure Freezing 17 Adopted from www.leica.com; Adres Kaech, University of Zurich; Kanno H, et al. Science 189: 880–881 (1975) • freezing of aqueous specimens up to approx. 200 µm thickness • high pressure (2100 bars) + rapid cooling (>104 K/s) = vitrified specimen • Water increases its volume upon freezing  great pressure applied during cooling makes ice formation difficult. Plunge Freezing 18 • freezing of aqueous specimens: cell suspensions or thin slices (< 10 µm) • plunging blotted specimens into melting e.g. ethane at high velocity • high freezing velocity + coolant with high thermal conductivity needed Adopted from presentation of Adres Kaech, University of Zurich. Why ethane at -88? 19 Methods of cooling 20 Direct Liquid/Gas Cooling Methods of Cooling 21 Passive and Closed Loop Flow cooling Peltier? -90 Methods of cryo pumping 22 Cryogen storage and safety 23 Problems with contamination 24 Elucidating the assembled structure of amphiphiles in solution via cryogenic transmission electron microscopy Honggang Cui,a Travis K. Hodgdon,b Eric W. Kaler,b Ludmila Abezgauz,c Dganit Danino,c Maya Lubovsky,d Yeshayahu Talmond and Darrin J. Pochan*a Show Affiliations Soft Matter, 2007,3, 945-955 DOI: 10.1039/B704194B Received 20 Mar 2007, Accepted 31 May 2007 First published online 28 Jun 2007 https://www.emsdiasum.com Cryo SEM 25 Features 26 Images Bio - Macro 27 Images 28 Mounting Methods for Cryo SEM 29 Freeze Fracture 30 Sublimation Etching 31 From LeicaFrom ZTH, Zurich Metal Coating in Cryo 32 Cryo SEM Fun 33 TEM 34 Structure of the thermally stable Zika virus Nature (2016) doi:10.1038/nature17994 Single Particle Analysis 35 http://cns.fas.harvard.edu/CryoEM www.pnas.orgcgidoi10.1073pnas.0711623105 Spa workflow 36 Crystallography 37 Three-dimensional electron crystallography of protein microcrystals Dan Shi,1,† Brent L Nannenga,1,† Matthew G Iadanza,1,† and Tamir Gonen1,* Data – use zika and ebola 38 Structure of the thermally stable Zika virus Nature (2016) doi:10.1038/nature17994 39 Adapted,imagebyAdresKaech,UniversityofZurich. Samplepreparationflowchart (selectedtechniques) + staining FIB/SEM No dehydration!Cryofixation 40 • To replace all water in the sample with liquid resin. • To form the tissue into a hard block to allow the sectioning of thin (< 100 nm) sections. Resins • Epoxy, cured with heat at 60-70o C. • Acrylic, can be cured at 50o C or at low temps of -20 to -30o C with UV. Infiltration and embedding 41 Normal sectioning of resin blocks Cryo Ultra Microtomy 42 Cryo-FIB sample preparation and observation “A great challenge for cell biology is to study the complex interplay of molecular assemblies in cellular systems in situ and at different scales of resolution (e.g. from ‘cells to molecules’).”… “Especially for ultrastructural imaging in cell biology this (FIB) preparation route is becoming increasingly important, similar to the impact micro- and nanomachining techniques had on the progress in materials science. A key instrument in this context is the FIB system.”… (Rigort A., Plitzko J. M., ABB 581 (2015), 122-130.) • basic idea = to prepare cellular samples for TEM investigations directly on the EM grid • hardware and protocols adapted to cryogenic conditions (first successful cryo-FIB experiment on bio sample in 2003) • frozen samples (plunge freezing, HPF) in situ thinned • native status/context  image is generated by the density of the biological material itself, no artifacts generated by sample preparation 43 Cryo TEM prep workflow 44 Vitrobot Sample Plunge Freezing. High quality sample preparation. KriosCorrSight Scios Amira & Maps Correlative Light and Confocal Imaging of Cryo Samples. Correlative Electron/Ion imaging and FIB milling of Lamella, Slice and View. Correlative TEM imaging, tomography, diffraction and automation. Extraction of the data from the Krios and the correlation with the light and electron. FEI is the only manufacturer to offer a complete workflow solution. The Scios FIB transfer system 45 In situ cryo lamella milling 46 A. Rigort, J.M. Plitzko; Archives of Biochemistry and Biophysics 581 (2015) 122-130. Multiple in-situ cryo-FIB lamellae can be prepared before transfer into TEM. 47 Cryo FIB process Protective Coating Preventing Beam Erosion 48 Cryo-focused-ion-beam applications in structural biology. Rigort A, Plitzko JM. Arch Biochem Biophys. 2015 Sep 1;581:122-30. Schaffer et al. http://www.bio-protocol.org/e1575 Electron tomography 49 Magnetotactic bacteria (Courtesy: Dr. Kobayashi, National Institute of Advanced Industrial Science and Technology, Osaka, Japan.) sample tilt image reconstruction 3D image 50 A. Rigort, F. J. B. Bäuerlein, et al. J.M. Plitzko; Proc. Natl. Acad.Sci. U.S.A. 109 (12) (2012) 4449-4454.. 500 nm 500 nm TEM projection 3D reconstruction Cryo lamella inspection Cryo-Electron Tomography FIB Lamella 51 VOLUME RENDERING Remarks 52 FIB /SEM tomography vs. vitrified samples Protective coating on milling. A. Rigort, J.M. Plitzko; Archives of Biochemistry and Biophysics 581 (2015) 122-130. Schertel A., Journal of Structural Biology 184 (2013) 355–360. Correlative microscopy 53 K.A. Jahna, et al., Correlative microscopy…, Micron 43, p.5 (2012). 54 Correlative microscopy Adapted from R. Wepf (ETH Zürich) presentation. Cryo FIB Traditional 55 http://www.ged.rwth- aachen.de/files/pictures/original/picture_1783.jpeg https://youtu.be/XfmFmeLU0Sg Bulk Specimens: Cryo-Lift Out 56 Mahamid et al. Journal of Structural Biology 192 (2015) 262–269 • Create a thick lamella at stage position S1 • Pick up the lamella with a cold needle • Transfer the lamella from stage position S1 to stage position S2 • Thin the lamella • STEM image So why/why not Cryo? Pros • Rapid • No chemicals • Easy (…ish) • Versatile • Allows in situ etching • Light correlation possible 57 Cons • Requires Cryogens/specialised equipment • Artefact if incorrect • Some materials don’t freeze well • Contamination • Contrast?! Which method? • Basically required magnification/resolution • >10mm – uCT under or Light Microscopy (can be used for higher magnification also) • 10-0.2mm – Standard SEM cryo but artifact at high magnification • 200um-20nm – Cryo TEM prep (vitrification) • 5um-0.5A – TEM (SPA, crystallography, tomography and/or lamella) 58 Thank you for your attention! Questions? Or email john.mitchels@fei.com