Center for Microscopy and Image Analysis Introduction to Electron Microscopy Andres Kaech Instrumentation The types of electron microscopes Transmission electron microscope (TEM) Scanning electron microscope (SEM) Hela Cells 1
The types of electron microscopes Transmission electron microscope (TEM) Electron beam Scanning electron microscope (SEM) Electron beam Specimen ~100 nm Specimen Projection Surface Examples Mouse intestine Actin filaments Junction Glycocalix Microvilli 1 µm Elektronenmikroskopie ETH Zurich Specimens courtesy of Bärbel Stecher, Institute of Microbiology, ETH Zurich 2
Examples Mouse intestine Membrane (lipid bilayer) Actin filaments 500 nm Elektronenmikroskopie ETH Zürich Examples Immunolabelling: Localization of proteins Correlation of structure with function 500nm Subcellular localization of H /K -ATPase in cimetidine-treated resting gastric parietal cells (rabbit). (A) Immunolabeling on the Golgi apparatus. (B) Tubulovesicles. Inset depicts the labeling on an encapsulated microvesicle (arrow). (C,D) Note the labeling on a diverging tubulovesicle (arrow in C) and a concentric multi-laminar structure (D). Bars 100 nm. Akira Sawaguchi, Kent L. McDonald, and John G. Forte, Volume 52(1): 77 86, 2004, Journal of Histochemistry & Cytochemistry 3
Examples Rat intestine Light microscopy Electron microscopy Microvilli 1 µm Adherence junction: ß-catenin visualized by immunolabelling using immunogold 10 µm Green F-actin (fluorescein isothiocyanate) Yellow ß-catenin (Cy3) Orange Nuclei (propidium iodide) Schwarz & Humbel 2007: Methods in Molecular Biology, vol. 369, Electron Microscopy: Methods and Protocols, Second Edition Edited by: J. Kuo Humana Press Inc., Totowa, NJ From photons to electrons e - Similarities to photons: Wave-particle duality of electrons Optical properties (Diffraction, chromatic abberation, spherical abberation, astigmatism etc.) Resolution depends on aperture and wavelength (Diffraction limited resolution) Abbe s equation d = 0.61 λ/na NA = n sinα 4
Resolution of electron microscopes The higher the energy of the electrons, the lower the wavelength, the higher the resolution Acceleration voltages of electrons: Transmission electron microscopes (TEM): 40 300 kv (1200 kv) Scanning electron microscopes (SEM): 1 30 kv Effective instrument resolution TEM: 0.1 nm Effective instrument resolution SEM: 1 nm However: Resolution of biological objects is limited by specimen preparation: Practical resolution: > 1 nm Resolution of electron microscopes Resolution Limit Wavelength/Size Object MRI, CT 1 mm Radio Human eye 100 μm Cells Infrared 10 μm Red blood cells Visible 1 μm Bacteria Light microscope Ultraviolet 100 nm Mycoplasma Viruses 10 nm Proteins x,γ-rays 1 nm Amino acids Electron microscope 0.1 nm Atoms Ultrastructure 5
Transmission electron microscope vs. Widefield light microscope Transmission electron microscope Widefield light microscope Electron gun Illumination Lamp Electromagnetic lens Condenser lens Glass lens TEM grid Specimen Slide Electromagnetic lens Objective lens Glass lens Electromagnetic lens Projector lens Glass lens Phosphorescent screen CCD camera Final image Eye CCD camera Scanning electron microscope vs. Confocal laser scanning microscope Scanning electron microscope Confocal laser scanning microscope Electron gun Photomultiplier (Detector) Illumination Detector Photomultiplier Laser Electromagnetic lenses Lens system condenser Glass lenses Electromagnetic lens Electromagnetic/ electrostatic lens Beam scanner Objective Mirror Glass lenses X-ray, photomultiplier Specimen 6
Electron source (Electron gun) (e.g. tungsten) High voltage (acceleration voltage of e - ) Electron microscopes are high vacuum systems Transmission electron microscope (TEM) Scanning electron microscope (SEM) High vacuum Without vacuum: Electrons would collide with gas molecules Electron source (tungsten) would blow 7
Electromagnetic lenses Electromagnetic lens of a transmission electron microscope Electromagnetic lenses Magnetic field depends on current and number of windings e - e - Advantage of a electromagnetic lenses: I The focal length can be changed by changing the current: No movement or exchange of the lens is required for focusing or changing magnification! Image is inverted and rotated Image rotation is corrected in microscopes 8
Electromagnetic lenses Chromatic aberration Due to energy difference of electrons (wavelength) Spherical aberrations e - (98 kv) e - (100 kv) e - (102 kv) Curvature and distortion of field Electromagnetic lenses Axial astigmatism confusion of the image Most relevant aberration in biological electron microscopy Cellulose filter paper imaged in SEM With astigmatism Under focused image elliptic deformation Focus circle of least confusion Over focused image elliptic deformation Withouth astigmatism Focus, corrected astigmatism circle of confusion minimized 9
Electromagnetic lenses Axial astigmatism of electromagnetic lenses confusion of the image Objective lens x Corrector coils True image of object Optical axis Object: Source of e - (off-axis) y z Reasons: Inhomegenities of the lens Contamination of lenses and apertures Charging of specimen Focus Specimen holders and stages Specimen on a TEM grid 3 mm Specimen holder 10
Specimen holders and stages Transmission electron microscope Goniometer: x, y, z, r Specimen size: 3 mm in diameter! Ca. 100 nm in thickness (electron transparent) Specimen holders and stages Scanning electron microscope Viewing chamber = Specimen chamber Gun Objective lens Specimen stub Stage Stub holder Specimen stage (x, y, z, r, tilt) Specimen size: 100 mm in diameter 2 cm in z-direction (not electron transparent) 11
Electron specimen interactions Backscattered electrons (E=E0) Primary electrons (E 0 ) Elastic (higher angle, E=E 0 ) Inelastic (low angle, E=E 0 - E) Unscattered (E=E 0 ) Electron specimen interactions Inelastic scattering: Primary electrons hit electrons of the specimen atom Energy is transferred from the primary electron to the specimen Emission of electrons and radiation K L M N 2 1 12
Electron specimen interactions Inelastic scattering: Inner-shell ionisation Electron hole is filled by an electron of an outer shell: Surplus energy is either emitted as characteristic x-ray or transferred to another electron, which is emitted (Auger electron) Bremsstrahlung (continuum x-rays) Deceleration of electrons in the Coulomb field of the nucleus Emission of X-ray carrying the surplus energy E Uncharacteristic X-rays Secondary electrons (SE) Loosely bound electrons (e.g., in the conduction band) can easily be ejected low energy (< 50 ev) Phonons Lattice vibrations (heat) ( beam damage) Plasmons Oscillations of loosely bound electrons in metals Cathode luminescence Electron specimen interactions Primary electrons Backscattered electrons X-rays Auger electrons Specimen Secondary electrons Cathode luminescense Heat Interaction volume SEM analysis Elastically scattered electrons Inelastically scattered electrons TEM analysis Unscattered electrons 13
Imaging in the transmission electron microscope Contrast formation in TEM Absorption of electrons Scattering of electrons / phase contrast NOTE: Mechanisms occur at the same time (superposition) Question: Which mechanism is most relevant for biological specimens? Imaging in the transmission electron microscope Contrast formation in TEM Biological specimen consist of light elements: Absorption contrast weak Scattering/phase contrast weak LOW CONTRAST Contrast enhancement required: Treatment with heavy metals (Ur, Pb, Os)! Heavy metals attach differently to different components 14
Imaging in the transmission electron microscope Main contrast formation in plastic embedded specimens Scattering of electrons through heavy metals Primary electron beam Specimen phospholipids ribosome Heavy metal ions Objective aperture (back focal plane) Brightness Image plane Imaging in the transmission electron microscope Thin section of alga stained with heavy metals (Ur, Pb) 15
Imaging in the transmission electron microscope Thin section of alga without heavy metal staining 1 µm Imaging in the transmission electron microscope Contrast enhancement by underfocusing Thin section of a frozen-hydrated apple leaf ( unstained ) 1 µm Electron microscopy ETH Zurich Phase contrast only (H 2 O vs. biological material) 16
Imaging in the scanning electron microscope Scanning and signal detection Primary electron beam secondary electrons, backscattered electrons, x-rays Imaging in the scanning electron microscope Signal and detection Primary electrons Backscattered electrons X-rays Auger electrons Specimen Secondary electrons Cathode luminescense Heat Interaction volume SEM analysis Elastically scattered electrons Inelastically scattered electrons TEM analysis Unscattered electrons 17
Imaging in the scanning electron microscope Secondary electron detector Primary electrons +200-500V Collector voltage +7-12kV HV Photomultiplier SE Electrons Photons Electrons Imaging in the scanning electron microscope Contrast formation in SEM using SE Different number of electrons from different spots of the specimen Dependent on location of the detector topography of the specimen acceleration voltage of primary electrons composition of the specimen 18
Imaging in the scanning electron microscope Contrast based on SE Virtual light source Leg of an ant, coated with ca. 10 nm Platinum Electron microscopy ETH Zurich Imaging in the scanning electron microscope Contrast formation in SEM Biological material (light elements): Only few electrons escape from specimen Almost no contrast, similar contrast everywhere on specimen Unsharp image (electrons from large volume) Contrast enhancement important & needed: Localization of the signal to the surface Coating of biological specimen with thin heavy metal layer (a few nm) Reducing acceleration voltage 19
Imaging in the scanning electron microscope Contrast formation Uncoated Coated with 4 nm platinum Primary electron beam Primary electron beam Platinum Imaging in the scanning electron microscope Contrast based on SE: Non-coating vs. coating with heavy metals Uncoated Coated with 4 nm platinum 500 nm Freeze-fractured yeast Electron microscopy ETH Zurich 20