Correlative Microscopy - From living cells to 3D electron microscopy A comprehensive workflow approach
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1 Correlative Microscopy - From living cells to 3D electron microscopy A comprehensive workflow approach Maria Marosvoelgyi, Liesbeth Hekking, Ben Lich, Matthias Geisbauer and Alex de Marco (FEI) Maja Guenthert and Roger Wepf (ETH Zuerich)
2 Overview Introduction of FEI Company Correlative Microscopy: From LM to EM FEI s CorrSight concept Correlative workflows SEM / FIB solutions Summary
3 Life Sciences applications FEI Company portfolio Life Science Correlative Light Microscopy SEM / SDB (3D SEM) Maps software CorrSight icorr Inspect Quanta NovaNano Teneo Verios Helios Scios TEM (2D - 3D) Tecnai Talos Titan Halo Titan Krios 3
4 Overview Introduction of FEI Company Correlative Microscopy: From LM to EM FEI s CorrSight concept Correlative workflows SEM / FIB solutions Summary
5 Publications on Correlative microscopy Combination of Light fluorescence microscopy and electron microscopy in 1 experiment
6 Organism (~10 mm) Tissue (~1 mm) Glomerulus (~100 μm) Cell (~10 μm) Synapse (~500nm) Vesicle (~30nm) Lipid bi-layer (4.3nm) Z-Resolution Resolution limit resin embedded specimen 5 STEM Tomo TEM Tomo 10 DualBeam FIB tomography Serial Section SEM Serial Section SEM (ATUM) Serial Section TEM 500 Light Microscopy Structure size (nm)
7 4D imaging: 3D over time in living cells GFP-tagged end-binding proteins (EB3-GFP) in retina pigment epithelial cells. Max. intensity projection, 84x84 µm, 488 nm excitation Image courtesy of Dr. T. Schwarz and Dr. J. Hehl (ETHZ) Cells are happy!!!!
8 Two approaches for CLEM 1. Integrated approach: 1 step sample preparation workflow LM/EM sample prep LM imaging EM imaging Easy imaging and navigation Maximum sample protection Special sample preparation (need for compromises) 2. Combined, sequential approach : 2 steps sample preparation workflow LM imaging EM sample prep EM imaging Flexibility in LM imaging conditions Flexibility in sample carrier and size Easy sample preparation
9 Overview Introduction of the FEI company Correlative Microscopy: from LM to EM FEI s CorrSight concept Correlative workflows SEM / FIB solutions Summary
10 Electron microscopy sample preparations living biological specimen chemical fixation cryoprotection Cryo-fixation preembedding labelling Rehydration Dehydration Freezing Freeze-substitution Freeze-drying Cryo-sectioning Critical-point-drying CEMOVIS Resin embedding resin embedding Freeze-fracture Resin resin section Thawed section Resin section whole mount replica Frozen hydrated Adapted from slide of Bruno Humbel, UNIL Lausanne
11 Requirements for CLEM instrumentation Solutions for CLEM must be flexible = adapt to different workflows efficient = help to increase throughput and reproducibility user-friendly = be easy-to-use, quick to learn
12 Inside is a high-end fluorescence microscope High-end fluorescence microscope Wide field fluorescence Spinning disk confocal Structured illumination The sample is completely immobile (x,y and z) The whole microscope system is mounted on a large xy-stage
13 CorrSight: a microscope, sample prep and navigation tool Unique modular concept: - Easy exchange of sample environments for different experiments Screen Live Cryo
14 Live Cell Imaging platform CorrSight Dedicated Correlative Light Microscope Screen Cryo Live
15 Live Cell Imaging platform CorrSight Dedicated Correlative Light Microscope Screen Cryo Live
16 Prescreening of samples to find the needle in the haystack
17 Prescreening of samples to find the needle in the haystack Automated scanning of multiple samples to identify promising areas Sample holders directly compatible to LM and EM imaging Fiducials for automated correlation
18 CorrSight: Fiducials for automated correlation
19 CorrSight: Fiducials for automated correlation Automatic correlation with 2 clicks only
20 Example in plants: Root nodule sample with bacteria High Pressure Frozen / Freeze-Substitute root nodule sample with bacteria (OS/Uac) and fluorescence dyes: Phalloidin-Alexa 660 and SytoxG. Fluorescence and SEM images Images and samples from Roger Wepf (ETH Zürich) Overview with Fluorescence section Fluorescence section SEM image Overlay SEM/LM Overlay SEM/LM
21 Example in plants: Root nodule sample with bacteria High Pressure Frozen / Freeze-Substitute root nodule sample with bacteria (OS/Uac) and fluorescence dyes: Phalloidin-Alexa 660 and SytoxG. Fluorescence and SEM images Images and samples from Roger Wepf (ETH Zürich) Find the right place for imaging using fluorescence High resolution SEM image at the same place
22 Example in plants: Root nodule sample with bacteria High Pressure Frozen / Freeze-Substitute root nodule sample with bacteria (OS/Uac) and fluorescence dyes: Phalloidin-Alexa 660 and SytoxG. Fluorescence and SEM images Images and samples from Roger Wepf (ETH Zürich) SEM image
23 Example in plants: Root nodule sample with bacteria High Pressure Frozen / Freeze-Substitute root nodule sample with bacteria (OS/Uac) and fluorescence dyes: Phalloidin-Alexa 660 and SytoxG. Fluorescence and SEM images Images and samples from Roger Wepf (ETH Zürich) SEM image (b&w inverted)
24 Live Cell Imaging platform CorrSight Dedicated Correlative Light Microscope Screen Cryo (cooling with liquid nitrogen) Live
25 Live Cell Imaging platform CorrSight Dedicated Correlative Light Microscope Screen Cryo Live
26 The following slides are confidential, please contact us at if you wish to receive the complete slide set Images in Electron microscopy mode
27 New generation of DualBeam systems from FEI: Scios & Helios
28 FEI Small Dual Beam systems How does a DualBeam work? Ga LMIS 52º SEM Beams Coincidence Point FIB SEM FIB Ions 35
29 Summary FEI offers a complete portfolio for CLEM workflows: CorrSight supporting several workflows in Light Microscopy Variety of EM solutions for data collection to image ultrastructure in nm-resolution In 2D and 3D MAPS software Runs both LM and EM experiments Enables large areas of data acquisition Correlates, manages and presents all collected data
30 Thank you for your interest and please come & visit our booth Maria
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