Advances in EM Sample Preparation Advances in EM Sample Preparation 1. Automated Grid Plunging 2. cryoCLEM 3. Broad Ion Beam preparation 2 1. Plunge Freezing 3 Types of biological specimens Suspended samples • Single particle analysis Purifed macromolecular complexes Viruses Liposomes Small (non-adherent) cells Icosahedral reconstruction Internal structure of small cells by cryo-ET Cell monolayers grown on grids 4 Leica Workflow CLEM Workflow Solution for cryo TEM Grid Plunging Adapter for Cryo TEM Transfer Image Analysis Cryo-Transfer Cryo-Electron Microscopy 5 Leica EM GP Grid plunger 21 March 2014 Page 6 The Dewar and the secondary cryogen filling Liquefier in place over ethane container in Dewar 7 Enviromental Chamber 8 9 Enviromental Chamber 10 Software Interface 11 Plunge Freezing After freezing the grid remains in or above the ethane ready for transfer to the grid box 12 EM GP Adapter for Cryo TEM Transfer 21 March 2014 Page 13 EM GP Adapter 1 2 5 3 21 March 2014 4 Page 14 Results 15 16 Results Virus-Like Particles of a Fish Nodavirus Mag 78000x Courtesy of Dr. G. Resch CSF Electron Microscopy Facility, Austria 17 Results Yeast RNA polymerase II Mag 78000x Courtesy of Dr. G. Resch CSF Electron Microscopy Facility, Austria 18 Results Microtubules Mag 40000x Courtesy of Dr. Formanek, Leibniz-Institut für Polymerforschung,, Germany 19 Results Liposomes Mag 31000x Pure DPPC liposomes CS coated DPPC liposomes Courtesy of Dr. Julia C. Schwarz, Univ. Of Vienna, Austria 20 Results Nanocarriers for dermal drug delivery Mag 31000x Nano-structured lipid carriers (NLC) Solid lipid nanoparticles (SLN) 21 2. Cryo CLEM - Correlative Workflow Solution Introduction Application Correlative Light and Electron Microscopy (CLEM) combines fluorescence microscopy and electron microscopy imaging of the same sample. A method which allows light microscopy rapid screening of large areas and fast determination of regions of interest (ROI) in the electron microscope. W. Baumeister, 2008 Leica Cryo CLEM Workflow Solution Or Grid Plunging Cryo-Transfer System with Loading Station Leica Cryo Light Microscopy High Pressure Freezing and Cryo-Ultramicrotomy CLEM Software (Mark and Find Information) Image Analysis Cryo Loading Station Cryo-Electron Microscopy 24 CLEM Solution for Cryo Microscopy The cryo-stage with transfer system is the "missing link" to combine cryo LM with the cryo EM . • Safe (sample safety), easy and controlled procedure to maintain cryo conditions from sample preparation to cryo EM • Cryo objective with low working distance (<0,5 mm) for higher resolution, speeds up location of target structure in EM. • Software enables user to find LM-marked structures during EM analysis Cryo transfer system docked to cryo stage Leica Cryo Objectives (50x and 20x magnification) Cryo Stage with lid Cryo Light Microscopy with Leica Microscope, Camera, Cryo Stage and Cryo Objectives Alignment of LM software with EM software => mark and find function 25 Resch et al., in press Beijing, August 11, 2010 26/127 Resch et al., in press Beijing, August 11, 2010 27/127 Cryo CLEM system, available 2014 28 3. Broad Ion Beam Preparation Fußzeile 29 21.03.2014 Slope cutting with the EM TIC 3X ….. …biological possibilities? Ion beam slope cut of the Leica EM TIC 3X mask prepared area 31 Leica EM TIC 3X “Triple ion beam” slope cutting Principle Three ion beams hitting the sample from different directions (reduction of curtaining) Fixed sample (better heat transfer) sample slope prepared area Features Cutting depth >1000µm Cutting width > 4000µm Cutting speed >150µm/h 32 mask ion beams Model of the ion beam slope cut process: Collision cascade Primary ion can remove an atom if: • Ione energy > bonding energy of the atom • Transferred impulse on the lattice atom is directed to the surface of the sample Ion beam Sample area of interest mask Application Examples Application Examples SiC abrasive paper Veneer Ultramicrotomed section on a grid EM TIC 3X Cooling Stage LN2 flow design with external dewar and pump Temperature range 30°C … -150°C Cooled mask First Animal Experiment – The Gummi Bear Gummi bear brain TIC 020 ~80°C Cryo (TIC 3X) -150°C Cross section of Gummi Bear's brain Marshmallow cooling stage -150°C; 5kV Heat-sensitive polymer fibers with water-soluble portion Without cooling (~80°C) Leica EM TIC 3X (cooling stage -120°C) Comparison heat sensitive coaxial polymer fibers with water-soluble portion FC7 -140°C TIC 3X -120°C Cooling stage result Heat-sensitive coaxial polymer fibers with water-soluble portion Coming soon EM VCT 100 EM TIC 3X EM VCT100 - connectivity for environmental control VCT100 Cryo Loading Station Samples at RT cooled in TIC TIC 3X / VCT connectivity VCT transfer >-150°C sample holder with mask several holder available ofor EM Pact holders ofor HPM holders oindustrial samples •max. 10x7x4mm Retrofit for existing TIC 3X oFSE (first time installation) Fußzeile 48 21.03.2014 Preparation for ion beam slope cutting the samples Instruments: Cryo-loading station with cryo-saw to perform sample preparation for the TIC 3X under LN2 conditions to fulfil max. protruded length of the sample above the mask edge VCT 100 shuttle to transfer the sample EM TIC 3X with VCT docking station to perform cryo-slope cut (cross section) Fußzeile 49 21.03.2014 Preparation Workflow Transfer frozen sample to cryo-loading station filled with LN2 Insert in the VCT holder and pre-prepare under LN2 using the cryo-saw Attach VCT 100 shuttle onto loading station for transfer to TIC3X Ion beam mill then transfer to cryoSEM Fußzeile 50 21.03.2014 TIC 3X / VCT connectivity Application Fußzeile 51 21.03.2014 Samples and Possibilities Environmentally sensitive samples eg: 1. Oil shale 2. Batteries 3. Polymers 4. Cryo-transfer needed, min. temperature -150°C Biological Applications…….? 1. Ion beam milling pre-FIB to expose large area 2. Soft biological materials with hard inclusions 3. Stents 4. Artificial joints 5. Cell scaffolds 6. ? Fußzeile 52 21.03.2014 Thank you 53
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