Transcript Samuel Roberts Noble Electron Microscopy Laboratory

Samuel Roberts Noble Electron
Microscopy Laboratory
770 Van Vleet Oval
University of Oklahoma
Norman, OK 73019-6131
Voice: 1-405-325-4391
FAX: 1-405-325-7619
URL: http://www.microscopy.ou.edu/
Faculty and Staff of the SRNEML
• Dr. Scott D. Russell, Ph.D., Director NML and Professor of
Botany & Microbiology, email: [email protected]
• Dr. Preston Larson, Ph.D., Research scientist, email:
[email protected]
• Greg Strout, M.S., TEM specialist, email: [email protected]
• All are at the SRNEML phone #: 325-4391
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)
What Can You See….
http://nobelprize.org/educational_games/physics/microscopes/powerline/index.html
Types of Microscopy
Electromagnetic
lenses
Glass lenses
Direct observation
Video imaging (CRT)
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
Image
– Formed by transmitted
light
– Charged electrons bend
due to magnetic field
Image
− 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
Transmission Electron Microscopy
ZEISS 10A conventional transmission
electron microscope (100,000 volts)
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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
Transmission Electron Microscopy
JEOL 2000-FX intermediate voltage (200,000 volt)
scanning transmission research electron microscope
(configured for both biological and physical sciences
specimens)
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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 -150°C)
$700,000 as currently configured at current prices
JEOL 2010-F intermediate voltage (200,000 volt) field
emission high resolution scanning transmission
research electron microscope
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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
Scanning Electron Microscopy
ZEISS DSM-960A scanning electron microscope – filament e- source
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magnification: X 10 to X
300,000)
30 Ångstrom resolution
(approximate)
OXFORD Link Pentafet Xray analyzer with IXRF
software imaging capability,
feature analysis and
quantitative software.
digital images are usually
acquired through a PC
interface
Scanning Electron Microscopy
JEOL JSM-880 high resolution SEM – LaB6 electron source
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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
Overview of a model TEM:
Zeiss 10A
The main components of a transmission electron
microscope are:
1. Vacuum System
2. Electron Optics Column
3. Control and Display Consoles
Vacuum System
Schematic of Zeiss 10A Vacuum System
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Low Vacuum Pumps
High Vacuum Pumps
Vacuum Gauges
Valves
Water Cooling
Pump Tube to Cathode Head
HV Pump Column
Column
Specimen Airlock Valve
Pump Tube to Double
Projector Lens
Pump Tube to Specimen
Chamber
Plate Valve
Plate Valve
By-Pass Valve
Manual Valve for Dessicator
Dessicator Valve
Pirani Gauge
Pre-Vacuum Manifold
Ventilation Valve
Rotary Pump 2 (LV System)
Pump Tube to Viewing
Chamber
Penning Gauge
Water Flow Operated Switch
Main Valve (V1)
Magnetic Water Valve
Baffle
Diffusion Pump
High Voltage
Cascade
Rotary Pump 1 (Backs DP)
Electron Optics Column
 Electron Beam Generation
− Produces electrons and accelerates them
toward specimen at HV
 Electromagnetic Lenses
− Condenser Lens (2)
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Condenses electrons into nearly parallel beam
(controls spot size, and brightness or intensity)
− Objective Lens
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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
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Allows higher mags, more compact, shorter
column, no distortion
− Projector Lens
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Magnifies a portion of the first image to form the
final image
− Stigmators
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Used to adjust the shape of the beam (circular)
Caused by lens imperfections, aperture
contamination, etc.
− Gun Alignment
− Deflector Coils
Electron Optics Column, cont.
 Apertures
− Spray or Fixed
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Provide contrast
− Movable
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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
Biological Specimen
Preparation
Emphasizing ultramicrotomy
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
Technology of Sectioning
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Ultramicrotome
Knife Selection
Specimen Preparation
Sectioning
Mounting Grids
Staining
A Few Sectioning Artifacts
Porter-Blum MT2B ultramicrotome
by Sorvall (ca. mid-1960s-1980)
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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
Reichert Ultracut Ultramicrotome
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All adjustments are on
viernier set screws
facilitating fine adj
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Lighting with above
and sub-stage lamps
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Mechanical advance
with thick sectioning
settings
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Water bath controls
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Fine control of speed
and duration of cut
and return cycle
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Future models had
innovations for serial
sectioning
RMC MT-6000 Ultramicrotome
RMC MT-6000 Ultramicrotome with FS-1000 Cryo-attachment
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
http://www.udel.edu/Biology/Wags/b617/micro/micro11.gif
Glass Knife Boat
Glass Knife Characteristics
Good for ultrathin sectioning
Good for thick sectioning
Glass spur
Edge defects
(not suitable for sectioning)
Wallner stress line
sharpness
durability
Caring for diamond knives:
http://www.emsdiasum.com/Diatome/diamond_knives/manual.htm
http://www.emsdiasum.com/Diatome/knife/images/
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
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
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
Technology of specimen preparation
• Coarse preparation of samples:
– Small objects (mounted on grids):
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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
Williams & Carter, 1996, Fig. 10-3
Grid selection
Specialized grids include:
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Bar grids
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Mixed bar grids
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Folding grids
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Slot grids
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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
90° Wedge specimen
The 90°-wedge specimen:
1. Prethin to create 2-mm square of the multilayers on a Si substrate.
2. Scribe Si through surface layers, turn over, and cleave.
3. Inspect to make sure the cleavage is clean, giving a sharp 90° edge, reject if not.
4. Mount 90° corner over edge of hole in Cu slot grid and insert in TEM.
5. Note two different orientations are available from single cleavage operation.
Williams & Carter, 1996, Fig. 10-17
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
Sandwiching techniques
Cross sectional preparation technique for layered specimens:
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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. 10-18
Window polishing
Procedures for performing window polishing of conductive sheet materials:
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A sheet of the metal1 cm2 is lacquered around the edges and made anode of an electrolytic cell.
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Initial perforation usually occurs at the top of the sheet.
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Lacquer is used to cover the initial perforation and sheet is rotated 180°.
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Thinning continues to ensure that final thinning occurs near the center of the sheet.
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If final edge is smooth rather than jagged it is probably too thick.
Williams & Carter, 1996, Fig. 10-2
Lithographic masking
Lithographic techniques applied to thinning a multi-layer specimen:
1. Unthinned sample is shown with a grid of Si3N4 barrier layers evident.
2. Etching between barrier layers produces undercutting down to the implanted layer, producing
uniform layer ~10 μm thick.
3. Further thinning with different solution produces large areas of uniformly thin material.
4. Si3N4 grid supports remaining unthinned regions.
Williams & Carter, 1996, Fig. 10-19
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”
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.
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
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
Electropolishing
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Electropolishing curve showing the increase in current between the anode and the cathode as
the applied voltage is increased.
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Polishing occurs on the plateau, etching at low voltages, and pitting at high voltages.
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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
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_localprobe.html
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
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.
Gatan Dual Ion Thinning Mill
Specimen port
Microscope viewer
Laser terminator
Argon tank
Mill controls
Ion mill selector
Vacuum meter
Elapsed time
Bias voltage
Gun current
Specimen current
Rotation
Epoxy mounting
Epoxy mounting of sectioned specimens prepared by thinning:
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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
Artifacts in Phy Sci specimens
Artifact/Problem
Consequence
Variable thickness
limited local area for chemical mapping (EP, IT, C, CD)
very limited area for EELS
somewhat limited area for absorption-free XEDS
omission of low density defects
distorted defect densities (EP, IT, TP)
Uniform thickness
limited diffraction information (UM)
limited microstructure information (UM)
handling difficulties (UM)
Surface films
bath 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 particle
C-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)
Artifacts in Phy Sci specimens
Artifact/Problem
Consequence
Differential thinning
different 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" defects
microstructure 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: extraction replication; IT: ion thinned; TP: tripod polish; C: cleavage (grinding, crushing).
Williams & Carter, 1996, Fig. 10-3
References
• Book resource:
– Williams DB, Carter CB (1996). Transmission Electron
Microscopy. I. Basics. Specimen preparation (Chapters 10)
Plenum Press, New York, pp 155-173.