Presentation on theme: "Samuel Roberts Noble Electron Microscopy Laboratory"— Presentation transcript:
1 Samuel Roberts Noble Electron Microscopy Laboratory 770 Van Vleet Oval University of Oklahoma Norman, OK Voice: FAX:URL:
2 Faculty and Staff of the SRNEML Dr. Scott D. Russell, Ph.D., Director NML and Professor of Botany & Microbiology,Dr. Preston Larson, Ph.D., Research scientist,Greg Strout, M.S., TEM specialist,All are at the SRNEML phone #:Major Equipment AvailableTransmission Electron Microscopes (3 mm grid)JEOL 2010 (Pending) – FEG (field emission) – molecular resolutionJEOL 2000 – LaB6 – 200 KV for physical & biological samplesZeiss 10 – Tungsten filament – 100 KV for biological samplesScanning Electron MicroscopesJEOL JSM 880 High Resolution – small samples (1 x 1 x 3 mm)Zeiss 960 Digital SEM – larger samples (a few cm3)
5 Comparison of LM and TEM Light SourceElectron SourceGlass LensesEM LensesLight has different speeds in different mediums (refraction)Light bends due to refractionCharged electrons bend due to magnetic fieldImageImageFormed by transmitted lightFormed by transmitted electrons impinging on phosphor coated screenBoth glass and EM lenses subject to same distortions and aberrationsGlass lenses have fixed focal length, change objective lens to chang mag., move objective lens closer to or farther away from specimen to focusEM lenses to specimen distance fixed, focal length varied by varying current through lensLight wavefront moves in a straight line while electrons move in helical orbits, EM lenses change trajectory but no huge change in electron velocity
6 Transmission Electron Microscopy ZEISS 10A conventional transmissionelectron microscope (100,000 volts)Configured for conventional imaging in the biological sciences and other simple specimensRobust and simple to operate (in comparison)Monostable switch controls hysteresisMeasured stability of magnification ±1%Magnification range X100 to 200,0003.4 Ångstrom resolution (point to point)Microscope used for student instructionConventional 100 KV instruments are now ~$200,000
7 Transmission Electron Microscopy JEOL 2000-FX intermediate voltage (200,000 volt) scanning transmission research electron microscope (configured for both biological and physical sciences specimens)magnification: X 50 to X 1,000,0001.4 Ångstom resolution (LaB6 source)backscattered and secondary electron detectorsGatan Digi-PEELS Electron Energy Loss Spectrometer, software and off axis imaging cameraKevex Quantum 10 mm2 X-ray detector (detects elements down to boron), with spatial resolution to as little as 20 nanometers (on thin sections)IXRF X-ray analyzer with digital imaging capability, X-ray mapping, feature analysis and quantitative software.Gatan Be double-tilt analytical holder for quantitative X-ray workGatan cryo-TEM specimen holder (to -150°C)$700,000 as currently configured at current prices
8 JEOL 2010-F intermediate voltage (200,000 volt) field emission high resolution scanning transmission research electron microscopeMagnification: X 50 to X 1,000,000High resolution field emission gun (FEG) source producing coherent electron beamPlanned Gatan GIF and Electron Energy Loss Spectrometer (EELS)Planned X-ray detector (detects elements to boron), spatial resolution to as little as 20 nm (on thin sections)Specified res: ~1.2 ÅOther cool stuffPlanned acceptance date: Fall 2007
11 Scanning Electron Microscopy ZEISS DSM-960A scanning electron microscope – filament e- sourcemagnification: X 10 to X 300,000)30 Ångstrom resolution (approximate)OXFORD Link Pentafet X-ray analyzer with IXRF software imaging capability, feature analysis and quantitative software.digital images are usually acquired through a PC interface
12 Scanning Electron Microscopy JEOL JSM-880 high resolution SEM – LaB6 electron sourcemagnification: X 10 to X 300,000)15 Ångstrom resolution (LaB6 source)backscattered electron detector, transmitted electron detector, electron channelling imagingDouble-tilt analytical holder with picoammeter for quantitative X-ray workKevex X-ray analyzer with IXRF software and digital imaging capability availableEquipped for x-ray feature analysis, mapping and quantitative analysisFilm support using sheet film or Polaroid is available, but most users opt for digital imagesCDs and sleeves are provided per each session$300,000 current value
14 Overview of a model TEM: Zeiss 10A The main components of a transmission electron microscope are:Vacuum SystemElectron Optics ColumnControl and Display Consoles
15 Vacuum System Schematic of Zeiss 10A Vacuum System Low Vacuum Pumps High Vacuum PumpsVacuum GaugesValvesWater CoolingColumnSpecimen Airlock ValvePlate ValveBy-Pass ValveManual Valve for DessicatorDessicator ValvePirani GaugePre-Vacuum ManifoldVentilation ValveRotary Pump 2 (LV System)Rotary Pump 1 (Backs DP)Pump Tube to Cathode HeadHV Pump ColumnPump Tube to Double Projector LensPump Tube to Specimen ChamberMain Valve (V1)BaffleDiffusion PumpHigh Voltage CascadePenning GaugePump Tube to Viewing ChamberMagnetic Water ValveWater Flow Operated Switch
16 Electron Optics Column Electron Beam GenerationProduces electrons and accelerates them toward specimen at HVElectromagnetic LensesCondenser Lens (2)Condenses electrons into nearly parallel beam (controls spot size, and brightness or intensity)Objective LensFocuses beam that has passed through specimen (primary and scattered) and forms a magnified intermediate image. Focusing accomplished by varying current through lensIntermediate LensAllows higher mags, more compact, shorter column, no distortionProjector LensMagnifies a portion of the first image to form the final imageStigmatorsUsed to adjust the shape of the beam (circular)Caused by lens imperfections, aperture contamination, etc.Gun AlignmentDeflector Coils
17 Electron Optics Column, cont. AperturesSpray or FixedProvide contrastMovableDepending on the aperture, can control brightness, resolution (balance diffraction versus spherical aberration), contrast, depth of fieldSpecimen Holder/AirlockViewing AreaFluorescent ScreenBinocularsColumn should be vibrationally isolated
19 Overview of Biological Specimen Preparation Killing & Fixation- Death; Molecular stabilizationDehydration- Chemical removal of H2OInfiltration- Replace liquid phase with resinEmbedding & Polymerization- Make solid, sectionable blockSectioning- Ultramicrotome, mount, stain
20 Technology of Sectioning UltramicrotomeKnife SelectionSpecimen PreparationSectioningMounting GridsStainingA Few Sectioning Artifacts
21 Porter-Blum MT2B ultramicrotome by Sorvall (ca. mid-1960s-1980) Simple belt device drives the microtome arm in MT2MT2B has adjustable duration and speed in the return stroke (much more complex)Limited movement possible in the fluorescent bulbHighly adjustable stage and specimen chuck, but all with spring locks rather than verniers making fine adj hardLocks on microscope used rather than screws (also awkward)Mechanical advance system
22 Reichert Ultracut Ultramicrotome All adjustments are on viernier set screws facilitating fine adjLighting with above and sub-stage lampsMechanical advance with thick sectioning settingsWater bath controlsFine control of speed and duration of cut and return cycleFuture models had innovations for serial sectioning
24 RMC MT-6000 Ultramicrotome with FS-1000 Cryo-attachment
25 Knives Razor blades did not last long Glass knives Diamond knives Took hours of honing to achieve translucenceEdge gone after one sectionGlass knivesMore durable and can be made easilyInexpensiveEdge may last over 60 sectionsDiamond knivesExpensive and fragileRequires highly skilled user (no room for error)Edge may last for years depending on user & cleanliness
31 Estimating ThicknessInterference reflection angle from Sjöstrand (1967)Sections of varying thicknesses as indicated by Sorvall interference colors (right). Image (left) is from
32 Physical Sciences Specimen Preparation - general techniques formaterials sciencesDirect lattice resolution in polydiacetylene single crystal showing (010)lattice planes spaced at 1.2 nm.
33 Physical Sciences Specimen Preparation - general techniques formaterials sciencesDirect lattice resolution in polydiacetylene single crystal showing (010)lattice planes spaced at 1.2 nm.
34 Technology of specimen preparation Coarse preparation of samples:Small objects (mounted on grids):StrewSprayCleaveCrushDisc cutter (optionally mounted on grids)Grinding deviceIntermediate preparation:Dimple grinderFine preparation:Chemical polisherElectropolisherIon thinning millPIMS: precision milling (using SEM on very small areas (1 X 1 μm2)PIPS: precision ion polishing (at 4° angle) removes surface roughness with minimum surface damageBeam blockers may be needed to mask epoxy or easily etched areasEach technique has its own disadvantages and potential artifacts
36 Grid selection Specialized grids include: Bar grids Mixed bar grids Folding gridsSlot gridsHexagonal gridsMesh is designated in divisions per inch (50 – 1000)Materials vary from copper and nickel to esoteric selections (Ti, Pt, Au, Ag etc.) based on various demandsThese are available from routine TEM suppliers – coated or not.Williams & Carter, 1996, Fig. 10-2
37 90° Wedge specimen The 90°-wedge specimen: Prethin to create 2-mm square of the multilayers on a Si substrate.Scribe Si through surface layers, turn over, and cleave.Inspect to make sure the cleavage is clean, giving a sharp 90° edge, reject if not.Mount 90° corner over edge of hole in Cu slot grid and insert in TEM.Note two different orientations are available from single cleavage operation.Williams & Carter, 1996, Fig
38 Cross sectional viewsCross sectional views of reasonably thin sliceable materials:Sheet sample is cut into slices and stacked with spacers placed to the outsideSandwiched materials are mounted in slot and glued together for supportMaterial is observed in TEMWilliams & Carter, 1996, Fig
39 Sandwiching techniques Cross sectional preparation technique for layered specimens:Etching of a multilayer sample.Etch away most of the sample, leaving a small etched plateau.Mask a region < 50 nm across.Etch away the majority of the surrounding plateau.If this thin region is turned 90° and mounted in a specimen holder.Interface is viewed parallel to electron beam.Williams & Carter, 1996, Fig
40 Window polishingProcedures for performing window polishing of conductive sheet materials:A sheet of the metal1 cm2 is lacquered around the edges and made anode of an electrolytic cell.Initial perforation usually occurs at the top of the sheet.Lacquer is used to cover the initial perforation and sheet is rotated 180°.Thinning continues to ensure that final thinning occurs near the center of the sheet.If final edge is smooth rather than jagged it is probably too thick.Williams & Carter, 1996, Fig. 10-2
41 Lithographic maskingLithographic techniques applied to thinning a multi-layer specimen:Unthinned sample is shown with a grid of Si3N4 barrier layers evident.Etching between barrier layers produces undercutting down to the implanted layer, producing uniform layer ~10 μm thick.Further thinning with different solution produces large areas of uniformly thin material.Si3N4 grid supports remaining unthinned regions.Williams & Carter, 1996, Fig
42 Disc punch / drillDisc of 3 mm diameter is cut from raw “bulk” specimenHeating plate is provided for gluing specimensRough polishing proceeds to a thickness of ~100 μm or soRim provides a gripping area imparting structural rigidity to the specimenPressure meter provides a guide to how cutting proceedsSamples from this step are often differentially ground in the center in a “dimple grinder”
43 Dimple grinderGrinding wheel provides thin center and durable rimPressure, speed, and depth of grinding can be selected by controlsStop at several μm thicknessDimple grinding of 3 mm discs is usually preparative to another more precise method of thinning, such as ion milling, chemical or electropolishing.
44 Chemical polishing Chemical polishing procedure: This device is gravity fed.Punched 3 mm specimen is suspended in meniscus of etchant.Etchant flow is started.Progress in etching specimen is monitored by illuminating glass tube.Light in glass tube and etchant acts as a fiber optic sourceSpecimen transparency is viewed in mirror.Unidirectional polishing in this designDesign could, if needed, be redesigned for bidirectional etching.Williams & Carter, 1996, Fig. 10-5
45 Gravity-fed & twin-jet electropolishing Gravity-fed one surface electropolisher (left), which uses reservoir as cathode.Twin-jet electropolisher uses specimen as conductor (above).Williams & Carter, 1996, Fig. 10-7
46 ElectropolishingElectropolishing curve showing the increase in current between the anode and the cathode as the applied voltage is increased.Polishing occurs on the plateau, etching at low voltages, and pitting at high voltages.Ideal conditions for obtaining a polished surface require the formation of a viscous film between the electrolyte and the specimen surface.Williams & Carter, 1996, Fig. 10-6
47 TEM sample preparation using the method of electrochemical polishing TEM sample preparation using the method of electrochemical polishing. Best results were obtained using 30% HNO3 in CH3OH at temperature of -200 C and a voltage of V. This method was used because of the larger amounts of transparent area compared with ion beam milling.
48 Ion mill schematic Schematic diagram of an ion-beam thinning device: Ar gas bleeds into the partial vacuum of ionization chamber6 keV potential creates beam of Ar ions on rotating specimenEither one or both guns may be selectedRotation speed and angle may be alteredProgress in thinning is viewed using a monocular microscope & back lighting.Specimen may be cooled to LN2 temperatures.Perforation is detected by penetration of ions through specimen.Williams & Carter, 1996, Fig. 10-8
49 Gatan Dual Ion Thinning Mill General ion milling procedure:Sample bombarded by an argon or iodine plasma.Bombardment dislodges atoms from specimen surface.Preparation is terminated when specimen is thin enough to see through or perforated.Layers of 1 to several atoms of thickness are observed in TEM.Can be adapted for en face thinning and for cross sectional views.Milling speed is controlled by: (1) specimen current, (2) plasma density (partial vacuum & gas concentration), (3) type of plasma (argon or iodine gas), (4) specimen angle, (5) milling temperature (LN2 dewars can be used), (6) ion guns (one or both) activated and (7) time.Intervention often needed: to adjust specimen current as specimen thinning proceeds.Laser cut-off device: is provided to terminate milling once a selected intensity of light passage is reached.
51 Epoxy mountingEpoxy mounting of sectioned specimens prepared by thinning:Sequence of steps for thinning particles and fibers.Materials are first embedding them in epoxy3 mm outside diameter brass tube is filled with epoxy prior to curingTube and epoxy are sectioned into disks with diamond sawSpecimens are then dimple ground and ion milled to transparencyWilliams & Carter, 1996, Fig
52 Artifacts in Phy Sci specimens Artifact/ProblemConsequenceVariable thicknesslimited local area for chemical mapping (EP, IT, C, CD)very limited area for EELSsomewhat limited area for absorption-free XEDSomission of low density defectsdistorted defect densities (EP, IT, TP)Uniform thicknesslimited diffraction information (UM)limited microstructure information (UM)handling difficulties (UM)Surface filmsbath residue, spec. dissolution and/or redeposition (EP)enhanced surface oxide (EP)extremely irregular topographies (IT)faster contamination buildup under beam (EP, R)retention of matrix on extracted particleC-redeposition (UM—embedded, UM, C, R—support films)Cu2O formation from Cu grids upon heating (R, UM, C)ion amorphization, diffusion-pump oil, redeposition (IT)
53 Artifacts in Phy Sci specimens Artifact/ProblemConsequenceDifferential thinningdifferent phases thin at different rates (EP, IT)different orientations thin at different rates (IT)grain/phase boundary grooving (EP, IT)anodic attack of matrix/particle (UM)"Selectivity"perforation influenced by local defect structure (EP, IT)very limited or no microstructure information (C, R)weak local regions debond and fall out (all)"False" defectsmicrostructure obscured by high defect density (UM, CD)deformation-induced defects (EP, TP)ion-induced loops, voids (IT)heat-altered defects (EP, IT)EP: electropolished; UM: ultramicrotomed; CD: controlled dimpling; R: extrac-tion replication; IT: ion thinned; TP: tripod polish; C: cleavage (grinding, crush-ing).
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