Samuel Roberts Noble Electron Microscopy Laboratory

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Samuel Roberts Noble Electron Microscopy
Laboratory

• 770 Van Vleet OvalUniversity of OklahomaNorman,
  OK 73019-6131Voice 1-405-325-4391FAX
  1-405-325-7619

URL http//www.microscopy.ou.edu/
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Faculty and Staff of the SRNEML

• Dr. Scott D. Russell, Ph.D., Director NML and
  Professor of Botany Microbiology, email
  srussell_at_ou.edu
• Dr. Preston Larson, Ph.D., Research scientist,
  email plarson_at_ou.edu
• Greg Strout, M.S., TEM specialist, email
  gstrout_at_ou.edu
• All are at the SRNEML phone [phone]

Major Equipment Available

• Transmission Electron Microscopes (3 mm grid)
• JEOL 2010 (Pending) FEG (field emission)
  molecular resolution
• JEOL 2000 LaB6 200 KV for physical
  biological samples
• Zeiss 10 Tungsten filament 100 KV for
  biological samples
• Scanning Electron Microscopes
• JEOL JSM 880 High Resolution small samples (1 x
  1 x 3 mm)
• Zeiss 960 Digital SEM larger samples (a few cm3)

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What Can You See.
http//nobelprize.org/educational_games/physics/mi
croscopes/powerline/index.html
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Types of Microscopy
Electromagnetic lenses
Glass lenses
Direct observation
Video imaging (CRT)
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Comparison of LM and TEM
Light Source
Electron Source
Glass Lenses
EM Lenses

• Light has different speeds in different mediums
  (refraction)
• Light bends due to refraction

• Charged electrons bend due to magnetic field

Image
Image

• Formed by transmitted light

• Formed by transmitted electrons impinging on
  phosphor coated screen

• Both glass and EM lenses subject to same
  distortions and aberrations
• Glass lenses have fixed focal length, change
  objective lens to chang mag., move objective lens
  closer to or farther away from specimen to focus
• EM lenses to specimen distance fixed, focal
  length varied by varying current through lens
• Light wavefront moves in a straight line while
  electrons move in helical orbits, EM lenses
  change trajectory but no huge change in electron
  velocity

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Transmission Electron Microscopy
ZEISS 10A conventional transmission electron
microscope (100,000 volts)

• Configured for conventional imaging in the
  biological sciences and other simple specimens
• Robust and simple to operate (in comparison)
• Monostable switch controls hysteresis
• Measured stability of magnification 1
• Magnification range X100 to 200,000
• 3.4 Ångstrom resolution (point to point)
• Microscope used for student instruction
• Conventional 100 KV instruments are now 200,000

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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 1,000,000
• 1.4 Ångstom resolution (LaB6 source)
• backscattered and secondary electron detectors
• Gatan Digi-PEELS Electron Energy Loss
  Spectrometer, software and off axis imaging
  camera
• Kevex 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 work
• Gatan cryo-TEM specimen holder (to -150C)
• 700,000 as currently configured at current prices

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JEOL 2010-F intermediate voltage (200,000 volt)
field emission high resolution scanning
transmission research electron microscope

• Magnification X 50 to X 1,000,000
• High resolution field emission gun (FEG) source
  producing coherent electron beam
• Planned 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 Å
• Other cool stuff
• Planned acceptance date Fall 2007

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Scanning Electron Microscopy
ZEISS DSM-960A scanning electron microscope
filament e- source

• magnification 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

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Scanning Electron Microscopy
JEOL JSM-880 high resolution SEM LaB6 electron
source

• magnification X 10 to X 300,000)
• 15 Ångstrom resolution (LaB6 source)
• backscattered electron detector, transmitted
  electron detector, electron channelling imaging
• Double-tilt analytical holder with picoammeter
  for quantitative X-ray work
• Kevex X-ray analyzer with IXRF software and
  digital imaging capability available
• Equipped for x-ray feature analysis, mapping and
  quantitative analysis
• Film support using sheet film or Polaroid is
  available, but most users opt for digital images
• CDs and sleeves are provided per each session
• 300,000 current value

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Overview of a model TEMZeiss 10A
The main components of a transmission electron
microscope are

• Vacuum System
• Electron Optics Column
• Control and Display Consoles

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Vacuum System
Schematic of Zeiss 10A Vacuum System

• Low Vacuum Pumps
• High Vacuum Pumps
• Vacuum Gauges
• Valves
• Water Cooling

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Electron Optics Column

• Electron Beam Generation
• Produces electrons and accelerates them toward
  specimen at HV
• Electromagnetic Lenses
• Condenser Lens (2)
• Condenses electrons into nearly parallel beam
  (controls spot size, and brightness or intensity)
• Objective Lens
• Focuses beam that has passed through specimen
  (primary and scattered) and forms a magnified
  intermediate image. Focusing accomplished by
  varying current through lens
• Intermediate Lens
• Allows higher mags, more compact, shorter column,
  no distortion
• Projector Lens
• Magnifies a portion of the first image to form
  the final image
• Stigmators
• Used to adjust the shape of the beam (circular)
• Caused by lens imperfections, aperture
  contamination, etc.
• Gun Alignment
• Deflector Coils

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Electron Optics Column, cont.

• Apertures
• Spray or Fixed
• Provide contrast
• Movable
• Depending on the aperture, can control
  brightness, resolution (balance diffraction
  versus spherical aberration), contrast, depth of
  field
• Specimen Holder/Airlock
• Viewing Area
• Fluorescent Screen
• Binoculars
• Column should be vibrationally isolated

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Biological Specimen Preparation

• Emphasizing ultramicrotomy

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Overview of Biological Specimen Preparation
Killing Fixation
- Death Molecular stabilization
Dehydration
- Chemical removal of H2O
Infiltration
- Replace liquid phase with resin
Embedding Polymerization
- Make solid, sectionable block
Sectioning
- Ultramicrotome, mount, stain
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Technology of Sectioning

• Ultramicrotome
• Knife Selection
• Specimen Preparation
• Sectioning
• Mounting Grids
• Staining
• A Few Sectioning Artifacts

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• Porter-Blum MT2B ultramicrotome by Sorvall (ca.
  mid-1960s-1980)
• Simple belt device drives the microtome arm in
  MT2
• MT2B has adjustable duration and speed in the
  return stroke (much more complex)
• Limited movement possible in the fluorescent bulb
• Highly adjustable stage and specimen chuck, but
  all with spring locks rather than verniers making
  fine adj hard
• Locks on microscope used rather than screws (also
  awkward)
• Mechanical advance system

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Reichert Ultracut Ultramicrotome

• All adjustments are on viernier set screws
  facilitating fine adj
• Lighting with above and sub-stage lamps
• Mechanical advance with thick sectioning settings
• Water bath controls
• Fine control of speed and duration of cut and
  return cycle
• Future models had innovations for serial
  sectioning

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RMC MT-6000 Ultramicrotome
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RMC MT-6000 Ultramicrotome with FS-1000
Cryo-attachment
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Knives

• Razor blades did not last long
• Took hours of honing to achieve translucence
• Edge gone after one section
• Glass knives
• More durable and can be made easily
• Inexpensive
• Edge may last over 60 sections
• Diamond knives
• Expensive and fragile
• Requires highly skilled user (no room for error)
• Edge may last for years depending on user
  cleanliness

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http//www.udel.edu/Biology/Wags/b617/micro/micro1
1.gif
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Glass Knife Boat
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Glass Knife Characteristics
Good for ultrathin sectioning
Good for thick sectioning
Glass spur
Edge defects(not suitable for sectioning)
Wallner stress line
sharpness
durability
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Caring for diamond knives http//www.emsdiasum.co
m/Diatome/diamond_knives/manual.htm
http//www.emsdiasum.com/Diatome/knife/images/
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Estimating Thickness
Interference reflection angle from Sjöstrand
(1967)
Sections of varying thicknesses as indicated by
Sorvall interference colors (right). Image
(left) is from http//www.jasonhostetter.com/pics/
gallery/emu/bigpics/ultramicrotome.jpg
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Physical Sciences Specimen Preparation

• - general techniques for
• materials sciences

Direct lattice resolution in polydiacetylene
single crystal showing (010)lattice planes spaced
at 1.2 nm.
http//www.ph.qmw.ac.uk/images/molwires.jpg
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Physical Sciences Specimen Preparation

• - general techniques for
• materials sciences

Direct lattice resolution in polydiacetylene
single crystal showing (010)lattice planes spaced
at 1.2 nm.
http//www.ph.qmw.ac.uk/images/molwires.jpg
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Technology of specimen preparation

• Coarse preparation of samples
• Small objects (mounted on grids)
• Strew
• Spray
• Cleave
• Crush
• Disc cutter (optionally mounted on grids)
• Grinding device
• Intermediate preparation
• Dimple grinder
• Fine preparation
• Chemical polisher
• Electropolisher
• Ion thinning mill
• PIMS 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
  damage
• Beam blockers may be needed to mask epoxy or
  easily etched areas
• Each technique has its own disadvantages and
  potential artifacts

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Williams Carter, 1996, Fig. 10-3
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Grid selection

• Specialized grids include
• Bar grids
• Mixed bar grids
• Folding grids
• Slot grids
• Hexagonal grids
• Mesh 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
  demands

These are available from routine TEM suppliers
coated or not.
Williams Carter, 1996, Fig. 10-2
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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. 10-17
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Cross sectional views

• Cross sectional views of reasonably thin
  sliceable materials
• Sheet sample is cut into slices and stacked with
  spacers placed to the outside
• Sandwiched materials are mounted in slot and
  glued together for support
• Material is observed in TEM

Williams Carter, 1996, Fig. 10-12
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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 lt 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. 10-18
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Window polishing

• Procedures 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
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Lithographic masking

• Lithographic 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. 10-19
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Disc punch / drill
Disc of 3 mm diameter is cut from raw bulk
specimen Heating plate is provided for gluing
specimens Rough polishing proceeds to a thickness
of 100 µm or so Rim provides a gripping area
imparting structural rigidity to the
specimen Pressure meter provides a guide to how
cutting proceeds Samples from this step are often
differentially ground in the center in a dimple
grinder
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Dimple grinder
Grinding wheel provides thin center and durable
rim Pressure, speed, and depth of grinding can be
selected by controls Stop at several µm thickness
Dimple grinding of 3 mm discs is usually
preparative to another more precise method of
thinning, such as ion milling, chemical or
electropolishing.
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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 source
• Specimen transparency is viewed in mirror.
• Unidirectional polishing in this design
• Design could, if needed, be redesigned for
  bidirectional etching.

Williams Carter, 1996, Fig. 10-5
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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
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Electropolishing

• Electropolishing 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
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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 15-20 V. This method
was used because of the larger amounts of
transparent area compared with ion beam milling.
http//www.phys.rug.nl/mk/research/98/hrtem_localp
robe.html
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Ion mill schematic

• Schematic diagram of an ion-beam thinning device
  
• Ar gas bleeds into the partial vacuum of
  ionization chamber
• 6 keV potential creates beam of Ar ions on
  rotating specimen
• Either one or both guns may be selected
• Rotation speed and angle may be altered
• Progress 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
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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.
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Gatan Dual Ion Thinning Mill
Microscope viewer
Specimen port
Laser terminator
Argon tank
Mill controls
Ion mill selector
Vacuum meter
Elapsed time
Rotation
Gun current
Bias voltage
Specimen current
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Epoxy mounting

• Epoxy mounting of sectioned specimens prepared by
  thinning
• Sequence of steps for thinning particles and
  fibers.
• Materials are first embedding them in epoxy
• 3 mm outside diameter brass tube is filled with
  epoxy prior to curing
• Tube and epoxy are sectioned into disks with
  diamond saw
• Specimens are then dimple ground and ion milled
  to transparency

Williams Carter, 1996, Fig. 10-10
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Artifacts in Phy Sci specimens
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Artifacts in Phy Sci specimens
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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Williams Carter, 1996, Fig. 10-3
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References

• Book resource
• Williams DB, Carter CB (1996). Transmission
  Electron Microscopy. I. Basics. Specimen
  preparation (Chapters 10) Plenum Press, New York,
  pp 155-173.