The world leader in serving scienceProprietary & Confidential Ondrej L. Shanel 7th of May, 2020 Transmission electron microscopy 2 Proprietary & Confidential Content • Contrast transfer function • Specimens • Specimen preparation • Aplications • Mass contrast • Phase contrast • Lorentz microscopy • Enviromental Transmission Electron Microscopy • Diffraction • Holography • Tomography • Cryo EM – SPA • CryoEM - MED • Scanning Transmission Electron Microscopy (STEM) • Bright field STEM • Dark field STEM • High Angular Angle Dark Field STEM • Electron Energy Lost Spectroscopy and Imaging • EDAX mapping 3 Proprietary & Confidential HR-TEM imaging II. Ψ(q)= FTΨ(r)*FTO(r) Ψ*(q)=A(q) konv. Ψ(q) Ψim(R)=M.FT-1 T(q).Ψ*(q) T(q)=D(α, ε,q)exp(iχ (q)) χ (q)= Δ λq2+0.5Csλq4 I(R)= │Ψim(R) │2 3 4 Proprietary & Confidential Contrast Transfer Function I. • Determines the capability of microscope to transfer the spatial frequencies from sample to visualizing device: where K(q) is envelope amplitude function L(q) is envelope frequence function • Where Cs is spherical abberation, q is spatial frequency, λ is wavelenght, Δf is defocus. 5 Proprietary & Confidential Contrast Transfer Function II. Tecnai T20 SuperTwin Tecnai TF20 SuperTwin Point-to-point resolution Point-to-point resolution Information limit Information limit Scherzer defocus – optimal for point-to-point resolution: Spatial abberation envelope Chromatic abberation envelope 6 Proprietary & Confidential Specimens • Thin foils, lamelas or edges of material. • Thin foils – biologic and metalurgic application. • Lamelas – semicondutor industry, universities, ... • Edges – material research, universities, ... • All of this specimens are worn on support grid. 6 7 Proprietary & Confidential Specimen preparation • Biological specimen – epoxy fixing or deep freezing plus microtom slicing, heavy elements marking. • Metalurgical specimen – rolling and argon etching. • Semiconductor specimen – acid and argon etching, SDB. • Other – replicas, ... • Not easy process, handy experience and knowledge required. 7 8 Proprietary & Confidential Bio samples preparation8 9 Proprietary & Confidential Lamelas preparation9 10 Proprietary & Confidential Material science – standard method10 11 Proprietary & Confidential HR-TEM imaging I. • Image is created as interaction of electron beam with specimen and microscope optic including its abberation and phase shifts. • To calculate exact image wave function is used. • Interaction of electron with specimen is multiscattered act. 11 12 Proprietary & Confidential TEM – mass contrast. • Based on material absorbtion and reflection of electrons. • Cannot distinguish between material differences and thickness. • Main role to mag. 100kx. • Using amplitude envelope of CTF. • Most usage in biology. 13 Proprietary & Confidential TEM bright Field – Mass contrast13 Aluminium 7075 14 Proprietary & Confidential TEM bright Field - BioSpecimen14 Bakterie syfilisu – (2017) O.L.Sháněl 15 Proprietary & Confidential TEM – dark field. • Exploiting only deflected electrons to enhance the image contrast for different techniques (phase, mass contrast). • Using tilted illumination and objective apperture. • Use in defect imaging, HR-TEM. 16 Proprietary & Confidential TEM Dark Field – Mass contrast16 Aluminium 7075 17 Proprietary & Confidential TEM Bright vs Dark Field17 Bright Field - Aluminium 7075 - Dark Field 18 Proprietary & Confidential TEM – phase contrast I. • Based on electron interference – sample is pattern. • Using phase part of CFT. • Main role above magnification 300kx. • Non-trully atomic resolution – vacancy atoms are not clear visible – only decreasing of intensity is detected. • This contrast is used in HR-TEM imaging. sample screen 19 Proprietary & Confidential TEM – phase contrast II. • Interpretation of image is not easy. • Importance to know what it should be seen – theoretical calculation. 20 Proprietary & Confidential TEM – phase contrast III. • Gold particles on thin carbon film imaged by 200kV SuperTWIN FEG under different defocus. 0 nm -67 nm -102 nm -128 nm 21 Proprietary & Confidential Low Contrast specimen – Phase contrast21 Adenovirus, Phoebe Stewart,Vanderbilt 22 Proprietary & Confidential Phase Plate22 Danev, R. and Baumeister, W. (2016) eLife, 5, e13046Danev, R. et al. (2014) PNAS, 111, 15635 23 Proprietary & Confidential Lorentz microscopy • To image magnetic or electrostatic specimen. • Specimen inserted out of magnetic lens field. • Resolution down to 0.34nm. 23 24 Proprietary & Confidential Enviromental TEM • Specimen is inserted in special capsule where gases can be introduced. • Maximal pressure varies from 100Pa to 600Pa. • Resolution 0.344 nm. • Usage for lifetime experiment observation. • http://www.youtube.com/watch?v=sHtKG-Z-AVI 24 25 Proprietary & Confidential Diffraction • Focal plane is imaged instead of image itself. • Two types - SAED – Illumination with plane wave. - CBED – Illumination with focused beam. 25 SAED – YMnO3 (Figure 1: Sets of electron diffraction patterns of YMn0.75Ti0.25O3 CBED - Fe0.7Pb1.3Sb207 (pyrochlore-type) along [111]. 26 Proprietary & Confidential TEM – potential contrast. • Using holography. • Added phase shift to original wave. Inverse Transform Original Image (called ‘the hologram’) Fourier transform Extract sideband and put origin at centre wrapped phase vacuum glue 100 nm gives amplitude and phase SrTiO3 SrRuO3 27 Proprietary & Confidential Tomography • Specimen is imaged under different angles and then is constructed with mathematical module to 3D image. • Resolution down to 0.5 nm (limited by specimen stage shift and tilt accuracy). 27 28 Proprietary & Confidential MicroED • Small crystals • Needs few crystals • Low dose imaging (1.5 - 3 el/Å2) • Crystals imaged under cryo-conditions • Provides high resolution Vitrification of small crystals TEM low-dose screening TEM diffraction screening Diffraction tilt series data collection Reconstruction 29 Proprietary & Confidential MicroED 30 Proprietary & Confidential Single Particle CryoEM Workflow Animace se svolením Max Planck Institutu Biochemie, Martinsried, Germany 31 Proprietary & Confidential Correlative EM-light microscopy • Imaging with light microscopy first (fluorescence or reflection mode) – up to 1µm details. • EM imaging for more details up to 1nm. 31 32 Proprietary & Confidential Ziegler A., Graafsma H., Zhang X. F., Frenken J. W. M.: In-situ Materials Characterization: Across Spatial and Temporal Scales, (2014) The need of ultrafast Time resolution TEM: miliseconds (camera frame rate limit) UEM: femtoseconds (pulse length limit) 33 Proprietary & Confidential Operation modes Ultrafast TEM (UTEM) Stroboscopic mode (laser on sample) Dynamic TEM (DTEM) Single-shot mode Movie mode Operation modes Stroboscopic mode laser RF cavity 34 Proprietary & Confidential Schematic overview of UEM setup with laser induced/assisted emission Arbouret A.,Caruso G. M., Houdellier F.: Advances in Imaging and Electron Physics, 207, (2018) 35 Proprietary & Confidential • RF-cavity beam deflection: pulses are created by deflection of the continuous beam over a slit aperture using RF-cavity Realization of femtosecond electron pulses Verhoeven W. et al: In-situ High quality ultrafast transmission electron microscopy using resonant microwave cavities, (2018) 36 Proprietary & Confidential Stroboscopic mode • also called Ultrafast TEM (UTEM) • millions of electron pulses are detected to create a single image • time delay between laser hitting the sample and electron pulse arrival is kept unchanged during one image capture • pulse repetition rate – order of MHz • pulse duration – order of 100 fs (laser pulse duration limit) • no significant change in spatial resolution Operation modes Arbouret A.,Caruso G. M., Houdellier F.: Advances in Imaging and Electron Physics, 207, (2003) 37 Proprietary & Confidential ONLY REVERSIBLE PROCESSES • nanostructures melting and recrystallization • dynamics of laser beam induced phase transitions and atomic structural expansion • debeye-waller factor measurement UTEM - applications Barwick B. et al:4D Imaging of Transient Structures and Morphologies in Ultrafast Electron Microscopy, 322, (2008) 38 Proprietary & Confidential Single-shot mode • also called Dynamic TEM (DTEM) • irreversible processes • millions (typically 108) electrons in a single pulse • spacecharge effect (Boersch effect) results in limited spatial resolution (typically 10-200 nm) and temporal resolution (typically 10-50 ns) which is no longer determined by laser pulse length Operation modes Arbouret A.,Caruso G. M., Houdellier F.: Advances in Imaging and Electron Physics, 207, (2003) 39 Proprietary & Confidential Movie mode • also called Movie DTEM • electron pulse train is created and individual pulses are then deflected to different sections of camera • sample is still illuminated by a single pulse • same limitations as for the case of the single-shot mode Operation modes Arbouret A.,Caruso G. M., Houdellier F.: Advances in Imaging and Electron Physics, 207, (2018) 40 Proprietary & Confidential IRREVERSIBLE PROCESSES • reactive multilayer foils – reaction wavefront propagation • various irreversible chemical reactions • study of biological reactions: utilizing also in-situ liquid microscopy or cryo-electron microscopy DTEM and movie DTEM - applications Evans J. E.,Browning N. D.: Enabling direct nanoscale observations of biological reactions with dynamic TEM, 62, (2013) Kim J. S., et al: Imaging of Transient Structures Using Nanosecond in Situ TEM, 321, (2008) 41 Proprietary & Confidential STEM - principle • Scaning with small probe through sample – collection of signal gone through the sample on the detector below imaging system. • STEM and SEM is not the same technique. STEM is of course much better! • Almost true atomic resolution – easier interpretation regarding to HR- TEM. • Many detection techniques – BF, DF, SED, BSED, EDX. Interaction volume SEM specimen thickness TEM specimen thickness BF DF CCD HAADF SED BSED EDX Specimen 42 Proprietary & Confidential STEM – image creation. • STEM image is influenced by three main factors: 1.) Sample-electron interaction. 2.) Detector type (BF, DF, HAADF). 3.) Diffraction camera lenght. • Image contrast is defined by: 1.) Sample thickness – BF (transmitted primary). 2.) Elements composition – DF (diffraction on cryst. structure). 3.) Elements weight – HAADF (Z-contrast). 43 Proprietary & Confidential STEM – electron sample interaction. • Electron sample interaction change the energy of primary beam differently based on atom weight and crystal structure. HAADFBF DF Incoherent scattering by a single atom or amorphous materials (proportional to Z at high q) Bragg diffraction by crystals q Relative intensity Incoherent scattering (not on scale) Total sum of signals 44 Proprietary & Confidential STEM detectors • Three main detectors collect different spectrum of deflected beam: • BF – transmitted primary beam. • DF – deflected beam on crystall structures. • HAADF – high angle delflected beam – Z-contrast. High-Angle Annular Dark Field BF/DF Dark field detector Bright field detector 45 Proprietary & Confidential STEM – camera lenght • The diffraction camera lenght significantly influences the detected signal. • With camera lenght changing is possible to obtain a different contrast using the same detector. camera lenght 100 mm camera lenght 300 mm 46 Proprietary & Confidential HR-STEM II. High Resolution ADF STEM image of a triple-junction in Au poly-crystal. Numerous voids occur at defects at the interface. Sample courtesy of Dr. T. Radetic, U.Dahmen & C. Kisielowski, NCEM Berkeley, USA 47 Proprietary & Confidential HR-STEM II. 5 nm • Ag concentration in individual atomic columns of Al 48 Proprietary & Confidential HR-TEM vs. HR-STEM I. • HREM and HR-STEM images from the same SrTiO3 bi-crystal boundary 49 Proprietary & Confidential HR-TEM vs. HR-STEM II. Visualization of dislocation in silicon using TEM and STEM techniques BF 300 nm WBDF STEM-DF 50 Proprietary & Confidential Electron Energy Loss Spectroscopy I • Electron energy filtering for spectroscopy or imaging. • Resolution to 0.004 eV. • Possible to distuingish element type, plasmon energy, type of atomic band. 50 51 Proprietary & Confidential Electron Energy Loss Spectroscopy II • Using magnetic Biprism. • Two HW solution – in-column (Zeiss, Jeol). - under column (Gatan – FEI, Jeol). 51 52 Proprietary & Confidential Energy Filtering TEM • Used same filtering technique as EELS. 52 53 Proprietary & Confidential EDAX mapping • Using STEM technique and EDAX detector. • Resolution to 0.2 nm. • Energy resolution down to 100 eV. 53 54 Proprietary & Confidential Big data54 55 Proprietary & Confidential Conclusion • TEM and STEM offer extremly powerful scientific tool. • There are many possibilities how to create image under different contrast conditions – enable to distinguish many types of physical properities of sample: 1.) Atomic structure. 2.) Electric and magnetic potential distribution. 3.) Bound type and its energy. 4.) Thickness. 5.) Elements distribution over sample.