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Practical
Scanning Electron Microscopy
Considerations in any microscopy:
Resolution
Magnification
Depth of field
Secondary information
Limits of Resolution (resolving power)
Unaided eye: 0.1mm
Light microscope: 0.2um
SEM:
1nm
TEM:
0.2nm
Evolution of Resolution
Depth of Field
Light Microscope vs Electron Microscope
General Diagram of the SEM System
Light Microscopy vs Electron Microscopy
Advantages of EM:
Resolution
Magnification
Depth of field
Disadvantages of EM:
Pricey
Better if conductive (SEM)
Maintenance
Vacuum
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Vacuum Systems
Why do we need a vacuum anyway?
Electrons are scattered by gas (or any other)
molecules
MFP at 1atm ~ 10cm
MFP at 10-5T ~ 4m
Some samples react with gases (O2)
Helps keep things clean!
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Vacuum
Systems
Terminology
Pressure
Units: atm, bar, mbar
Torr (mm of Hg)
Pa (N/m2)
1atm=1Bar=1000mBar=760Torr=105Pa
Pumping speed
l/min, l/sec
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Vacuum Systems
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Vacuum Systems
Quality of Vacuum
Low:
760-10-2 Torr
Medium:
10-2-10-5 Torr
High:
10-5-10-8 Torr
Ultrahigh: <~10-8 Torr
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Vacuum Systems
Measuring Vacuum in EM Systems
Thermocouple Gauge
Pirani Gauge
Cold cathode Gauge
Penning Gauge
Ion pump current
Very Broad Range of Vacuum to Measure
Grouped Ranges for Vacuum Gauges
Vacuum Gauge Choices and Working Ranges
Thermocouple/Pirani Gauges
Ionization Gauges
Ion Gauge Collection
Hot Cathode Ion Gauge
Penning gauge
Penning gauge
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Vacuum Systems
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Vacuum Systems
Types of Vacuum Pumps
1- Rotary (Fore, Rough, Aux, Mechanical)
2- Turbomolecular (Turbo)
3- Diffusion (Diff)
4- Ion (Sputter-ion)
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Vacuum Systems
Rotary Pump Basics
Always in the Foreline of the system
Exhausts pumped gases to atmosphere
Pumping rate decreases as vacuum increases
Usually has a low VP oil as a sealant to
facilitate pumping
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Vacuum Systems
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Vacuum Systems
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Vacuum Systems
Rotary Pump Problems
Cannot pump <10-2 Torr
Noisy
Backstreams
Vibration
Maintenance
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Vacuum Systems
Turbo Pump Basics
Direct drive electric motor-gas turbine
Rotor/stator assembly
Moves gas molecules through the assembly
by sweeping them from one to another
High rotational speed (>10,000 RPM)
Very clean final vacuum
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Vacuum Systems
Turbo Pump Problems
Needs a Foreline pump
Costly
Can fail abruptly
Whine
Needs to be protected from solid material
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Vacuum Systems
Diffusion Pump Basics
No moving parts
Heated oil bath and condensing chamber
Jet assembly to redirect condensing gas
Recycle of oil
Pressure gradient in condensing
chamber/Foreline pump removes from high
pressure side
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Vacuum Systems
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Vacuum Systems
Diffusion pump problems
Heat up/cool down time
Needs foreline pump
Can make a mess in vacuum
failures/overheating
Needs cooling water (usually)
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Vacuum Systems
Ion Pump Basics
High voltage creates electron flux
Ionizes gas molecules
Ions swept to titanium pole by magnetic field
Titanium erodes (sputters) as ions become
embedded
Getters collect Ti atoms and more gas ions
Current flow indicates gas pressure (vacuum)
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Vacuum Systems
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Vacuum Systems
Ion Pump Problems
Cannot work until pressure is <10-5 Torr
Low capacity storage-type pump
Needs periodic bake-out
Hard to startup (sometimes)
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Vacuum Systems
Summary
All electron microscopes require a vacuum system.
Usually consists of rotary-(turbo, diff)-(ion) pumps.
System should provide clean oil-free vacuum
at least 10-5 Torr or so.
Vacuum is usually measured with a combination of TC
and ion gauges.
Vacuum problems are some of the most challenging to
find and fix, and may even be caused by samples
outgassing
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Vacuum Systems
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Vacuum Systems
Typical TEM
Vacuum System
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Electron Sources and Lenses
Types of Electron Sources
Thermionic Sources
Tungsten filament
Lanthanum Hexaboride (LaB6) filament
CeB6
Field Emission sources
Cold
Schottky
Ideal Electron Source Characteristics
Low “work function” material so that it is easy to
remove electrons from the material
High melting point
Chemically and physically stable at high temps
Low vapor pressure
Rugged
Cheap
Thermionic Emission of Electrons
Filament material is heated with an electrical current
so that the “work function” of the material is exceeded
and the electrons are allowed to leave the outermost orbital.
Generates a fairly broad source of electrons (cloud)
Tungsten Hairpin Filaments
Most common of all filaments in electron guns
Low cost (~$20)
Lots of beam current
Not very intense illumination
Emission temperature ~2700K
Work function= 4.5ev
Can last about 100 hours
Tungsten Hairpin Filament Saturation
Tungsten Hairpin Filament
LaB6 (and CeB6) Filaments
Lower work function thermionic source (2.4ev)
Lots brighter (~50x) than W-hairpin
Relatively costly (~$700)
Can be direct replacement for W-hairpin
Heated to about 1700K
Can last hundreds of hours
LaB6 Emitter Problems
Need higher vacuum to reduce reactivity
More difficult to make
Heating/cooling must be slow (brittle material)
Heating is indirect through a graphite well
Thermionic Gun Layout
Optimization of Thermionic Emitter Lifetime
Keep vacuum system in good working order
Clean gun area
Do not oversaturate the filament
Minimize the number of heating/cooling cycles
Field Emission Electron Sources
Process proposed in 1954/Demonstrated in 1966
Usually a single crystal W-wire sharpened and shaped
Tip radius <1.0um
Usually includes a ZrO2 component to assist emission (if heated)
About 10,000 times brighter than W-hairpin
Small apparent source which helps obtain small probes
with high temporal coherence
Decreased energy spread in the beam
Can last many thousands of hours
Cold Field Emitters
Most intense (brightest) electron source
Tip radius very small (~0.1um)
Needs very high electric field intensity
Tips contaminate and need “flashing” to clean and/or anneal
Expensive (~$4000)
Requires ultrahigh vacuum in gun
Schottky Field Emitters
More stable than cold field emitters
Self annealling as ions impact tip
Lower work function than cold field emitters
Extraction field intensity can be lower
Vacuum requirements lower
Still expensive (~$4000)
Typical Schottky Field Emission Sources
Schottky Field Emitter Diagram
Schottky Field Emitter Parts
Suppressor Cap:
limits the electron emission to the desired area of the tip
actually blocks electrons from the heater and shaft
Heating Filament-tungsten hairpin:
heats the tungsten tip to enhance emission (1800K)
Emitter:
Single crystal W-needle w/ ZrO2 coating
Schottky Field Emitter Parts
Extractor Anode:
applies voltage to the filament to extract electrons
from the tip (1.8 - 7 keV)
Gun Lens:
Electrostatic lens which forms a crossover of the
electron source (acts similar to the C1 lens)
Optimizing Field Emission Emitter Lifetime
Keep vacuum system in good working order
Leave the emitter heated
Don’t over-extract
Don’t overheat
Electron Lenses
Electrostatic
Gun cap (Wehnelt cylinder)
Totally inside vacuum
Electromagnetic
All other lenses and stigmators
Partially outside of vacuum
Transmission Electron Microscope
Optical instrument in that it uses a lens to
form an image
Scanning Electron Microscope
Not an optical instrument (no image forming
lens) but uses electron optics.
Probe forming-Signal detecting device.
Electron Optics
Refraction, or bending,
of a beam of
illumination is caused
when the ray enters a
medium of a different
optical density.
Electron Optics
In light optics this is accomplished when a
wavelength of light moves from air into glass
In EM there is only a vacuum with an optical
density of 1.0 whereas glass is much higher
Electron Optics
In electron optics the beam cannot enter a
conventional lens of a different refractive index.
Instead a “force” must be applied that has the
same effect of causing the beam of illumination
to bend.
Classical optics: The refractive index changes
abruptly at a surface and is constant between the
surfaces. The refraction of light at surfaces separating
media of different refractive indices makes it possible
to construct imaging lenses. Glass surfaces can be
shaped.
Electron optics: Here, changes in the “refractive
index” are gradual so rays are continuous curves rather
than broken straight lines. Refraction of electrons
must be accomplished by fields in space around
charged electrodes or solenoids, and these fields can
assume only certain distributions consistent with
field theory.
Converging (positive) lens: bends rays toward the
axis. It has a positive focal length. Forms a real
inverted image of an object placed to the left of the
first focal point and an erect virtual image of an
object placed between the first focal point and the
lens.
Diverging (negative) lens: bends the light rays
away from the axis. It has a negative focal length.
An object placed anywhere to the left of a diverging
lens results in an erect virtual image. It is not
possible to construct a negative magnetic lens
although negative electrostatic lenses can be made
Electron Optics
Electrostatic lens
Must have very clean and high vacuum
environment to avoid arcing across plates
Electromagnetic Lens
Passing a current through a single coil of
wire will produce a strong magnetic field
in the center of the coil
Three Electromagnetic Lenses
Electromagnetic Lens
Pole Pieces of iron
concentrate lines of
magnetic force
Electromagnetic Lens
Electromagnetic Lens
Forces Acting on an Electron Beam as
it goes through an Electromagnetic Lens
...and the Result
The two force
vectors, one in the
direction of the
electron trajectory
and the other
perpendicular to
it, causes the
electrons to move
through the magnetic
field in a helical
manner.
The strength of the magnetic field is determined by the
number of wraps of the wire and the amount of current
passing through the wire. A value of zero current (weak
lens) would have an infinitely long focal length while a large
amount of current (strong lens) would have a short focal
length.
Condenser Lens: Weak and Strong Conditions
Lens Defects
Since the focal length f of a lens is dependent
on the strength of the lens, if follows that different
wavelengths will be focused to different positions.
Chromatic aberration of a lens is seen as fringes around
the image due to a “zone” of focus.
Lens Defects
In light optics wavelengths
of higher energy (blue) are
bent more strongly and have
a shorter focal length
In the electron microscope
the exact opposite is true in
that higher energy
wavelengths are less
effected and have a
longer focal length
Lens Defects
In light optics
chromatic
aberration can be
corrected by
combining a
converging lens
with a diverging
lens. This is known
as a “doublet” lens
The simplest way to correct for chromatic aberration is
to use illumination of a single wavelength! This is
accomplished in an EM by having a very stable
acceleration voltage. If the e velocity is stable the
illumination source is monochromatic.
Lens Defects
LEO Gemini Lens
A few
manufacturers
have combined
an
electromagnetic
(converging)
lens with an
electrostatic
(diverging) lens
to create an
achromatic lens
The effects of
chromatic aberration
are most profound at
the edges of the lens, so
by placing an aperture
immediately after the
specimen chromatic
aberration is reduced
along with increasing
contrast
Lens Defects
The fact that rays enter and leave the lens field at
different angles results in a defect known as spherical
aberration. The result is similar to that of chromatic
aberration in that rays are brought to different focal
points
Spherical aberrations
are worst at the
periphery of a lens,
so again a small
opening aperture that
cuts off the most
offensive part of the
lens is the best way
to reduce the effect.
Diffraction
Diffraction
occurs when a
wavefront
encounters an
edge of an
object. This
results in the
establishment
of new
wavefronts
Diffraction
When this occurs
at the edges of an
aperture the
diffracted waves
tend to spread
out the focus
rather
than concentrate them. This results in a
decrease in resolution, the effect becoming more
pronounced with ever smaller apertures.
Apertures
Advantages
Disadvantages
Increase contrast by blocking
scattered electrons
Decrease resolution due
to effects of diffraction
Decrease effects of chromatic
and spherical aberration by
cutting off edges of a lens
Decrease resolution by
reducing half angle of
illumination
Decrease illumination by
blocking scattered
electrons
Astigmatism
If a lens is not
completely
symmetrical
objects will be
focussed to
different focal
planes resulting in
an astigmatic
image
The result is a
distorted image.
This can best be
prevented by having
a near perfect lens,
but other defects
such as dirt on an
aperture etc. can
cause astigmatism
Astigmatism in light
optics is corrected by
making a lens with a
offsetting defect to
correct for the defect in
another lens.
In EM it is corrected using a stigmator which is a ring of
electromagnets positioned around the beam to “push”
and “pull” the beam to make it more circular in crosssection
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The SEM System
and
Electron Beam-Sample Interactions
The TEM system and components:
Vacuum Subsystem
Electron Gun Subsystem
Electron Lens Subsystem
Sample Stage
More Electron Lenses
Viewing Screen w/scintillator
Camera Chamber
The SEM System and Components:
Vacuum Subsystem
Electron Gun Subsystem
Electron Lens Subsystem
Scan Generator Subsystem
Scattered Signal Detectors
Observation CRT Display
Camera CRT/Digital Image Store
SEM Scan Generation System
Sets up beam sweep voltage ramp in
both X and Y directions (tells beam how far to move and
the number of increments)
Synchronized between beam on sample and beam on CRT display
Can be analog or digital in format
Includes interface to magnification module for changing
the beam sweep on the sample
Scan Generator Interface
Magnification control in the SEM
Beam sweep on sample is synchronized with beam sweep
on display CRT
CRT size never changes
Sweep distance on sample can vary (using magnification
module)
Small distance on sample--> large magnification to CRT
Large distance on sample--> small magnification to CRT
Mag=CRT size/Raster Size
Magnification Control in the SEM
Depth of Field in the SEM
The single most important thing in making SEM images
pleasing to look at and interpret
Range of distances above and below the optimal focus
of the final lens that produces acceptably focussed image
features
DOF in the SEM is a few hundred times that of the LM
at similar magnifications
DOF is inversely proportional to the aperture angle 
Depth of Field and Defocus
DOF in the SEM
DOF and Aperture Size
Table 1. Depth of Field at 10 mm working distance for SE images.
Magnification
100 m
aperture
200 m
aperture
300 m
aperture
(  = 0.005 rad)
(  = 0.01
(  = 0.015
30 1.9 mm
3000 10 m
30000 1 m
995 m
663 m
5 m
3 m
0.5 m
0.3 m
Table 2. Depth of Field at 25 mm working distance for SE images.
Magnification
100 m
aperture
200 m
aperture
300 m
aperture
(  = 0.002 rad)
(  = 0.004
rad)
(  = 0.006
rad)
30 4.9 mm
3000 25 m
2.5 mm
1.6 mm
12.5 m
8.3 m
Note the large depth of field which is possible with small probe semi-angle ( .
DOF and Sample Tilt
DOF and Working Distance
Spot Size
Resolution is a direct function of (and limited by) the
final spot size of the electron beam
This is a function of initial beam crossover size at the gun
and the final spot formed by the beam shaping
apertures and the condensing lenses
Shorter focal lengths produce smaller focussed spots
Short working distances have the smallest spots and
the best resolution
Smaller spots reduce the signals generated (S/N decreases)
Spot Size Control in the SEM
Signal Detectors for the SEM
Electron Beam-Specimen Interactions
First thing: electrons are scattered in a near-forward direction
Electron Beam-Sample Interaction
Electron Flight Simulator Demo
Smorgasbord of Electron Beam Sample Interactions
Elastic Scattering
Backscattered Electrons
Inelastic Scattering
Plasmon Excitation (coherent oscillations in free electron “plasma”)
Secondary Electrons from conduction band
Electron Shell Excitation (photons, characteristic x-rays and Auger electrons)
X-ray Continuum (braking radiation)
Phonon Excitation (thermal)
Electron Beam-Sample Interactions
Backscattered (Primary) Electrons
Backscatter Yield
n=-0.0254+0.016*A2-0.000186*A2*A2+0.00000083*A2*A2*A2
Backscatter Yield
0.6
0.5
Yield
0.4
0.3
0.2
0.1
0
0
20
40
60
Atomic #
80
100
Backscattered Electron Detectors
Backscattered Electron Image
Backscattered Electron Detector Placement
For either solid-state Si detectors or Robinson type
Secondary Electrons and Detectors
Secondary Electrons
Inelastic collision and ejection of weakly held conduction
band electrons (need only few eV to exceed work function
of the sample atoms)
Always low in energy (<50eV)
Can also be formed from backscattered electrons. Ratio is Z
dependent (SEBS/SEB increases with Z)
Usually a large fraction is produced within a region defined
by the primary beam
Some Secondary Electron Characteristics
Types of Secondary Electrons/Origins
Secondary Electrons: Edge Effects
Everhart-Thornley (ET)
Secondary Electron Detector
Photomultiplier Tube Electronics
Whole E-T Detector w/PMT Amplification
Secondary Electron Images
Auger Electron Generation
Auger Analytical Volume
Auger Electron Spectroscopy
Yielded inverse to BSE: lighter elements emit more
Electrons are VERY specific in energy...can indicate
type of bonding involved and oxidation state
MFP for typical Auger energies is about 0.1-2nm
Analytical volume is very small---> resolution is high
Signal is pretty weak
X-ray Photon Production
Bremsstrahlung (Braking) radiation
Characteristic X-rays
Bremsstrahlung Continuum X-rays
Formed by the release of energy from the primary electron
beam as it decelerates in the presence of the Coulombic
field of target (sample) atoms
Large energy spread (0-E0)
Not very useful
Forms a large portion of the x-ray spectral background
Characteristic X-rays
Formed when inner shell electrons are ejected by the
primary beam, followed by an outer shell electron
falling and filling the vacancy. Energy difference is
compensated by releasing a photon of “characteristic”
energy, defined by the energy level differences of the
orbitals, which is unique within a series of transitions.
Characteristic X-ray Production
Energy Dispersive X-ray Spectrometer
X-ray Spectrum from EDS Spectrometer
Wavelength Dispersive (crystal) Spectrometer
X-ray Spectra Comparison EDS vs WDS
Cathodoluminescence Signal Generation
Electron beam excitation of sample valence band electrons
into the conduction band (electron-hole pair production)
If allowed to recombine, the annihilation of the electron-hole pair
creates a photon (sometimes in the visible range)
A high efficiency collector (usually a parabolic mirror) and a
PMT are used to collect and amplify the signal
Absorbed Current or Specimen Current
Sample is detector
IB~= ISC+ IBS + (ISE + Iph +Ietc)
SC image looks like an inverted BSE image
Very useful and easy to obtain
Resolution not so good
Transmitted Electrons
In thin samples the beam may pass through the thickness
TED is located below the sample (like BSE detectors)
Sort of like TEM w/o the resolution
Relative Sizes of the Emission Zones (looking from above)
Image Collection, Recording
and Presentation
Rule-of-thumb microscope conditions
-best resolution
-best depth of field
-best sample preservation
Conventional Photographic Methods
Digital Methods
Presentation for:
Display
Publication
Image Collection
Proper subject identification
Proper subject orientation
Best selection of imaging conditions
-HV
-WD
-Spot size (aperture)
-Scan rate
Subject Identification/Orientation
Representative of the whole
Image background
Not too busy
Important image information is centered and prominent
Many times a slight tilt conveys more information
“Best” Imaging Conditions
High resolution
-short working distance
-small spot size
-high accel. Voltage
-high magnifications
Depth of field
-long working distance
-low magnifications
-larger spot size
Low magnification
-large spot
Selection of Scan Rate for Imaging
Sensitive samples
-may need to be fast
-low S/N
-maybe TV integration mode
Insulating (charging) samples
-decrease charging with small spot and
fast frame rate, maybe TV again
-focus/stigmate in an area adjacent to the area recorded
-use image shift function to quickly move small amounts
Normally conductive samples
-use slowest rate practical w/o degrading surface
Old Technology
Analog scan SEMs
2nd CRT for viewing the image as it scans
Film based camera focused on this CRT (low persistence)
Almost always a 4x5 inch Polaroid sheet film camera
Very slow scan for about a 2000 line image (~3 minutes)
P/N film or just an instant positive image
About $3/shot now
Generalized Photographic Processing
Needed for TEM image plates
(Can be used for SEM film images too)
Exposure of silver halide grains (latent image)
Development (reducing basic solution---> Ag0)
Rinse (water) or Stop (acid)
Fix (thiosulfates)
Rinse (water)
Dry
Scan or Print photographically
Good photographic processing results in the best images
and are still the images that are used to compare
other (newer) techniques
Newer Technology
Digital raster SEMs
Frame buffer storage of image info
Image processing
Digital image storage
-usually TIFF files so that header can contain
image and microscope specific data
Fully transportable formats
Easy incorporation of images into documents
LEO 982 Specific Digital Imaging
Detectors
-SEI (chamber)
-SEI (column)
-BSE
Signal mixer
-brightness
-ratio
Gamma correction
-corrects for desired brightness and contrast
Iout~=Iin
-power function deviation from 1:1
1.0 darkens and enhances lower greys
1.0 lightens and enhances higher greys
Gamma Corrections
<--- switch position 0
<--- switch position 1
Gamma corrections
<----- switch position 3
<----- switch position 4
<----- switch position 5
<----- switch position 6
LEO 982 Specific Digital Imaging
Slow scan rates 1-3 continuous scan
Slow scan rates 4-8 store one frame of data
-dump to disk as image file (TIFF)
Choose image pixel matrix density from 512x512 to
2048x2048 (lowest is usually OK)
Right mouse button will interrupt any scan and store
results in the buffer (incl. TV)
TV rate integration of frames can reduce random noise
in the final image at a fast scan rate
File path and naming convention
LEO 982 Specific Digital Imaging
Variable small raster
-used to increase scan rate for image adjustment
Can store multiple images in the same frame
-variable frame
-split screen
-kind of gimmicky.....don't use for important images
Stereo Pair Images (Anaglyphs)
By collecting two images offset by about 4-100 in tilt
Display them side by side and cross eyes to converge
Build a blue-red image composite and use stereo glasses
-In Photoimpact program:
convert images to RGB
adjust color balance (red-right, blue-left)
perform image calculation (difference operator and merge)
Special Scan Modes in the LEO 982
Line scan
-disable Y-axis scan to see grey-level variations
on a line
Y-modulation
-if very little Z-axis information this converts it
to Y-axis deflection (not very useful)
Spot scan
-mostly for x-ray data acquisition
Additional Scanning Features of the LEO 982
Dual magnification
-useful for “looking around”
-don't use for important images
Scan rotation
-electronically rotates the raster on the sample
-very useful for getting a good “presentation”
Dynamic focus
-use to compensate for the portions of the sample that
fall outside the depth of field distance. Sets up a
ramp on the focus current +- the center of the field
Tilt correction
-compensates for trapezoidal scan on highly tilted samples
Image Processing
Generally use “kernels” which are arrays of arithmetic
operators on a pixel
Standard kernels are used to blur, average, and sharpen
images. 3X3, 5x5, array of operators.
Photoshop and PhotoImpact have custom and standard
kernels
Kernel Operations for Sharpening an Image
Different Kernels
Effect of Kernel Size on
Operations
Contrast Enhancement
Original kernel
Average kernel
Sharpen kernel
Blur kernel
Pitfalls of Image Processing
Images can be distorted and data lost
Pixelization of images
Ethical behavior dictates a minimum of processing
Always better off collecting the best image and either
not processing or doing it only lightly
Image Manipulation
Erosion of edge pixels
-kernel operator to find edges
-erode or erase edge pixels one layer at a time
-break apart and separate touching features
Dilation of edge pixels
-kernel operator to find edges
-dilate or add edge pixels one layer at a time
-fuse separate features
Most useful in particle and other small repeating features
Presentation of Micrographs
Reports
-probably least critical
-must convey information concisely
Journal
-probably most critical
-size, grey-levels, resolution
-must be specific and representative of the narrative
Posters
-most variable in format
-otherwise like journal
-conducive to point and discuss
Web
-like journal
-can be interactive
Presentation Media
Photographic paper
Photo quality printer output
-dye sublimation
-ink jet....getting there!
-laser...maybe...
-consider viewing distance in choice
Include TIFF or JPEG files in reports using word processor
Powerpoint for talks
Micrographs as Art
Wonders of things small
Intricacies of natural samples
Subtle grey tones, like fine b/w photos
Can be psuedocolored to add interest
Comparisons to more familiar things
Explain phenomena in a “gee-whiz” way
Sample Preparation for Electron Microscopy
Electrically Conductive Samples
Electrically Insulating Samples
Biological Samples
“Odd” Samples
Why do samples need to be prepared???
Vacuum environment
Charged particle environment
Too big
Components migrate in response to the beam
Two General Samples Types
Bulk Samples
SEM only
Thin Samples
SEM and TEM
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Dehydration
Why?
Samples are incompatible with the vacuum
Surfaces will be disrupted while forced-drying
How?
Air dry
Critical Point Dry
HMDS Dry
What sample types?
Biologicals
Hydrated geologicals
Synthetics like polymers or solgels/aerogels
Air Drying
Can only be used on “rugged” samples
Biologicals like tough exoskeletons
Materials that won't change size/shape
Air Dried Sample
Critical Point Drying
Water is replaced with miscible 2nd fluid
Transitional fluid replaces 2nd fluid
Transitional fluid is driven past the “critical point”
by increasing pressure and temperature
Pressure is relieved as gas escapes
Samples are left water, 2nd fluid, and transitional fluid dry
CPD Sample
Critical Point Dryer
More CPD Dried
HMDS drying
Water is replaced with a 2nd fluid
2nd fluid is replaced with HMDS
HMDS is allowed to dry leaving surfaces intact
HMDS Dried
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Sample Coating
Why coat samples?
Electrical insulators need to be made conductive
Increase rigidity
Increase SE emission
Usual coatings
Metals like Au, Ag, Pt, Pd, Cr, Os or alloys
Carbon
Typical coating methods
Sputtering
Evaporation
Sample Coating
Things to watch out for:
Decoration artifacts
X-ray emission lines
Sample deformation during deposition
Sputter Coating Samples
Usually a simple DC sputtering system
Low vacuum
Argon backfill
inert and ionizable
relatively high mass
good pumping character
Relatively simple time vs current rate of deposition
Slower coating--->smaller islands--->smoother film
Usually +-5nm is sufficient for conductivity
Typical EM Lab Sputtering System
Cathode
Argon bleed
Vacuum chamber
Samples
Vacuum gauge
HV control
Current monitor
Timer
Sample Coating: Evaporation
Used when sputtering won't work well
Carbon
Making shadows
Line of sight deposition
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Revealing Interior Portions of Samples
Why?
Outside may be “weathered”
Inside may have different chemistry or morphology
Inside may have smaller pieces or details
Inside may be immature or undifferentiated
Inside may be source of problems or defects
Revealing Interior Portions of Samples
How?
Smash it! (don't make it any harder than necessary)
Cut it
Saw it
Grind it
Fracture it
Polish it (mechanical, electrochemical)
Etch it
Revealing Interior Portions of Samples
Tools
various types of knives and blades
Microtome
Polishing bench and wheels
Wet processing
Inside Structure
Microtomes and Microtomy
Tool with very sharp blade and a sample translation stage
Ultramicrotome for EM
Usually a glass or diamond knife
stationary cutting edge
moving sample
cut pieces float off on water surface held adjacent
to the blade edge
Can use thin sections in TEM or cleaned bulk surface
in the SEM
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Stabilization of loose parts
Why?
Loose stuff falls off
Loose stuff changes other surface details
How?
Use glues or tapes
Use clips
Make sandwiches
Embed in other materials
Sometimes a coating will do
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Sample Resizing
Why?
Too darned big for the system
How?
Similar to revealing interiors of samples
-smash, saw, cut, grind, polish, etc.
Concerns:
Part left over is representative of the whole
You don't lose the interesting part
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Methods to make similar measurements
with other techniques
Why?
Complementary data
Comparisons
How?
Use fiducial markings
Use sample holders with a grid of numbers/letters
Find a landmark
Use absolute or relative stage coordinates
Circle the area of interest
Processes Common to Many Samples
Dehydration
Coating
Methods to reveal interior details
Stabilization of loose parts
Sample resizing
Methods to make similar measurements with other techniques
Special imaging circumstances
Special imaging circumstances
Why?
Want sample in particular position
Need to see a certain area or side
Want proximity data to/from reference material
How?
Be creative
Mount samples so they protrude from stage
Make a multi-holder
Include a standard material on the stage
Spring clips/tape/wire
Sample Preparation Flowchart
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
How to Prepare Small Particles
Dispersion of single particles or groupings?
Mixture of sizes or monodisperse?
Potential to move around on stage?
Want compositional information? What about the substrate?
From a solid mass, dry powder, airborne, or liquidborne?
Reactive outside of their usual environment?
Small Particle Dispersion
Agglomeration is a problem
-camphor/napthalene method
-sticky dot method
-dust and remove method
-filter onto membranes (Nuclepore filters)
Drying ring dispersions
Mortar and pestle size modification
Small Particles
Most will stick electrostatically
Large ones may need some help to stay in place
-carbon coating
-metal coating
-sticky dots
Coatings often are not continuous
-special stages for evaporators and sputter coaters
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Cross Sections
Why?
-to see interior or sub-surface details
How?
-fracture
-cleaving
-microtome
-polishing
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Electrically Insulating Materials
Four Choices:
Try to view as-is w/low energy beam
-small aperture
-vary accelerating voltage
Try a faster scan rate to limit electron dose
Make it conductive w/o destroying the
surface topography
Use a variable pressure instrument (we don't have one)
Insulators
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Electrically Conductive Samples
The best sample
The most unusual sample
Simply attach to sample stub and “go”
Beware of contaminated surfaces
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Biological Materials
Generally require extensive preparation
Most important to remove water w/o
destroying the surfaces
May need to ruggedize (fix) tissues
May be possible to freeze and view directly
Given rise to “environmental” or “low vacuum”
systems to obviate need to dry samples
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Untouchable Samples
Historically significant samples
Forensic samples
Samples from litigations
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Preparing Samples for Automated SEM Scans
Usually a size/shape/compositional analysis
Usually requires a grey-level segmentation of the image
Usually needs some parameters to keep or discard data
-edges
-too small
-too big
Samples must be flat and relatively featureless except for your target
Examples:
gunshot residue analysis
asbestos analysis
bone implant analysis
small particle analysis (IPA, SPOT sampler)
Gunshot Residue Analysis
When a gun is fired, small particles are generated during the explosion of the primer,
and leave the gun via the smoke.
The particles are deposited on parts of the body.
These small particles are called gunshot residue (GSR).
Particles are very characteristic, therefore presence of these particles forms evidence of
firing a gun.
Particles normally consist of Pb (lead), Sb (antimony) and Ba (barium).
Gunshot Residue
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Sample Preparation of Semiconductors
Usually Silicon
Increasingly III-V or II-VI compounds
Do not need conductive coatings unless a thick oxide,
nitride or resist is present
p-type and n-type seem to image differently due to
variation in conductivity and dopant concentration
Some areas may be “floating” electrically and need
separate grounding
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Manipulated Samples
Stressed in tension or compression
Samples irradiated to simulate high dose -exposure
Electron beam induced current (EBIC)
Voltage contrast
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Temperature Controlled Viewing in the SEM
Some glasses have mobile components
-Na+
-Ag+
Cooling to <-140C seems to stabilize the electromigration
Some high VP or liquid samples can be frozen and viewed
w/o a coating
Watch the crystallization of materials from solution
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Low Vacuum SEM
ESEM (environmental SEM)
Differentially pumped gun/column and chamber
High vacuum in former; adjustable vacuum in latter
Many types of backfill gasses and vapors
Up to about 1 Torr in chamber
Dissipates surface charging
Eliminates the need to fully dry samples
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Hazardous Samples
Biohazards (DNA, Viruses, Bacteria, etc.)
Radioisotopes
Fine dust
Toxic materials (Be metal)
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Quick and Dirty Analyses
80% of what you'll ever know about something you learn
in the first dirty experiment
Stabilize sample
Make it fit mechanically
Protect the instrument
Try it!
Individual Processing of Samples for EM Observation
Small Particles
Cross-sections
Insulators
Conductors
Biologicals
“Untouchables”
For automated analyses
Semiconductor devices
Manipulated samples
High or Low temperature processing
Low vacuum observation
Hazardous materials
“Quick-and-dirty” analyses
Magnetic samples
Magnetic Sample Materials
Deflect the electron beam
High mag work very difficult
Low mag work approachable
X-ray analysis OK
Make sure pieces are stable on stage
Small particles need to be FIRMLY adhered
TEM Sample Prep for Materials
Thin Sample Prep for TEM or SEM
Dispersion of small particles
SEM: sticky dots, conductive tabs or glue
TEM: alcohol dispersion on thin film
Ultramicrotomy
Mechanical thinning
Chemical thinning
Ion thinning
Image Collection, Recording
and Presentation
Rule-of-thumb microscope conditions
-best resolution
-best depth of field
-best sample preservation
Conventional Photographic Methods
Digital Methods
Presentation for:
Display
Publication
Image Collection
Proper subject identification
Proper subject orientation
Best selection of imaging conditions
-HV
-WD
-Spot size (aperture)
-Scan rate
Subject Identification/Orientation
Representative of the whole
Image background
Not too busy
Important image information is centered and prominent
Many times a slight tilt conveys more information
“Best” Imaging Conditions
High resolution
-short working distance
-small spot size
-high accel. Voltage
-high magnifications
Depth of field
-long working distance
-low magnifications
-larger spot size
Low magnification
-large spot
Selection of Scan Rate for Imaging
Sensitive samples
-may need to be fast
-low S/N
-maybe TV integration mode
Insulating (charging) samples
-decrease charging with small spot and
fast frame rate, maybe TV again
-focus/stigmate in an area adjacent to the area recorded
-use image shift function to quickly move small amounts
Normally conductive samples
-use slowest rate practical w/o degrading surface
Old Technology
Analog scan SEMs
2nd CRT for viewing the image as it scans
Film based camera focused on this CRT (low persistence)
Almost always a 4x5 inch Polaroid sheet film camera
Very slow scan for about a 2000 line image (~3 minutes)
P/N film or just an instant positive image
About $3/shot now
Generalized Photographic Processing
Needed for TEM image plates
(Can be used for SEM film images too)
Exposure of silver halide grains (latent image)
Development (reducing basic solution---> Ag0)
Rinse (water) or Stop (acid)
Fix (thiosulfates)
Rinse (water)
Dry
Scan or Print photographically
Good photographic processing results in the best images
and are still the images that are used to compare
other (newer) techniques
Newer Technology
Digital raster SEMs
Frame buffer storage of image info
Image processing
Digital image storage
-usually TIFF files so that header can contain
image and microscope specific data
Fully transportable formats
Easy incorporation of images into documents
LEO 982 Specific Digital Imaging
Detectors
-SEI (chamber)
-SEI (column)
-BSE
Signal mixer
-brightness
-ratio
Gamma correction
-corrects for desired brightness and contrast
Iout~=Iin
-power function deviation from 1:1
1.0 darkens and enhances lower greys
1.0 lightens and enhances higher greys
Gamma Corrections
<--- switch position 0
<--- switch position 1
Gamma corrections
<----- switch position 3
<----- switch position 4
<----- switch position 5
<----- switch position 6
LEO 982 Specific Digital Imaging
Slow scan rates 1-3 continuous scan
Slow scan rates 4-8 store one frame of data
-dump to disk as image file (TIFF)
Choose image pixel matrix density from 512x512 to
2048x2048 (lowest is usually OK)
Right mouse button will interrupt any scan and store
results in the buffer (incl. TV)
TV rate integration of frames can reduce random noise
in the final image at a fast scan rate
File path and naming convention
LEO 982 Specific Digital Imaging
Variable small raster
-used to increase scan rate for image adjustment
Can store multiple images in the same frame
-variable frame
-split screen
-kind of gimmicky.....don't use for important images
Stereo Pair Images (Anaglyphs)
By collecting two images offset by about 4-100 in tilt
Display them side by side and cross eyes to converge
Build a blue-red image composite and use stereo glasses
-In Photoimpact program:
convert images to RGB
adjust color balance (red-right, blue-left)
perform image calculation (difference operator and merge)
Special Scan Modes in the LEO 982
Line scan
-disable Y-axis scan to see grey-level variations
on a line
Y-modulation
-if very little Z-axis information this converts it
to Y-axis deflection (not very useful)
Spot scan
-mostly for x-ray data acquisition
Additional Scanning Features of the LEO 982
Dual magnification
-useful for “looking around”
-don't use for important images
Scan rotation
-electronically rotates the raster on the sample
-very useful for getting a good “presentation”
Dynamic focus
-use to compensate for the portions of the sample that
fall outside the depth of field distance. Sets up a
ramp on the focus current +- the center of the field
Tilt correction
-compensates for trapezoidal scan on highly tilted samples
Image Processing
Generally use “kernels” which are arrays of arithmetic
operators on a pixel
Standard kernels are used to blur, average, and sharpen
images. 3X3, 5x5, array of operators.
Photoshop and PhotoImpact have custom and standard
kernels
Kernel Operations for Sharpening an Image
Different Kernels
Effect of Kernel Size on Operations
Contrast Enhancement
Original
kernel
Sharpen kernel
Average
Blur kernel
Pitfalls of Image Processing
Images can be distorted and data lost
Pixelation of images
Ethical behavior dictates a minimum of processing
Always better off collecting the best image and either
not processing or doing it only lightly
Image Manipulation
Erosion of edge pixels
-kernel operator to find edges
-erode or erase edge pixels one layer at a time
-break apart and separate touching features
Dilation of edge pixels
-kernel operator to find edges
-dilate or add edge pixels one layer at a time
-fuse separate features
Most useful in particle and other small repeating features
Presentation of Micrographs
Reports
-probably least critical
-must convey information concisely
Journal
-probably most critical
-size, grey-levels, resolution
-must be specific and representative of the narrative
Posters
-most variable in format
-otherwise like journal
-conducive to point and discuss
Web
-like journal
-can be interactive
Presentation Media
Photographic paper
Photo quality printer output
-dye sublimation
-ink jet....getting there!
-laser...maybe...
-consider viewing distance in choice
Include TIFF or JPEG files in reports using word processor
Powerpoint for talks
Micrographs as Art
Wonders of things small
Intricacies of natural samples
Subtle grey tones, like fine b/w photos
Can be psuedocolored to add interest
Comparisons to more familiar things
Explain phenomena in a “gee-whiz” way
Introduction to X-ray Microanalysis
Review of Physics of X-ray Generation
Hardware
-EDS
-WDS
-electron microprobe vs. SEM/EDS
Software
-Spectral acquisition
-Spectral match
-Qualitative analysis
-Quantitative analysis
-X-ray images (maps)
-Spectral mapping
-simulation of electron scattering/x-ray emission
X-ray Generation
Hardware for X-ray Microanalysis
WDS
-Roland circle based Bragg-diffracting crystals and
detector arrangement
-either horizontal or vertical design
EDS
-cooled solid state detector
-integrated FET and preamplifier
Computer accumulator/conditioner of signals
MCA output for energy vs intensity
Some hardware facility for control of the electron beam
position for mapping and DBC
WDS System
Rowland Circle in WDS Spectrometer
Typical EMPA
EDS Topics (from Notes)
Spatial Resolution
Directionality of Signals
Rough Surfaces
Hardware/Signal Processing
-dead time and time constants
Microscope Parameters
-overvoltage
-TOA
-WD (EA)
EDS Spectral Interpretation
Background Continuum
Characteristic x-rays
Excitation and absorption
Detector efficiency
Artifacts
Peak ID function (qualitative analysis)
Spectral matching
Structure of a Si(Li) Detector for X-rays
Nomogram of
E-beam Penetration
Beam Diameter vs
Beam Current
Quantitative EDS Analysis
Clean spectrum
Standards vs. no-standards
K-ratio
Corrections
-atomic # (Z)
-absorption (A)
-fluorescence (F)
Advanced X-ray Techniques
X-ray image maps
Spectral Mapping
Particle and Phase Analysis
X-ray Image Maps
Edax Imaging and Mapping program
Process
-take a look at your sample with eds
-look for elements of interest
-setup ROI (region of interest) on the peaks
-start mapping function
-DBC on
-dwell time
-pixel density for map
-maps show up line by line in different colors
for each ROI (element)
-color intensity is related to # of x-rays detected
-can collect SE image simultaneously
Qualitative x-y spatial distribution of elements
Not very high resolution
Spectral Mapping
Sort of like previous x-ray maps
Collect full spectra at each pixel
Store data in a raw form so that it can be massaged later
Take “phases” and additively process the spectra of all the
pixels that determine that phase
-leads to pretty good quantitative analysis
-averages small inhomogeneities in the phase
Huge file sizes (stores greylevel and data for each pixel)
->30Mbytes
Particle and Phase Analysis
Similar to mapping
Additional sizing information (area, feret diameters, calc. Volume...)
Mixes qualitative spectral matching info and morphological info
to come up with a particle or phase ID
Steers the beam on the sample to collect the data for binarized
“white” areas (as determined by threshold setup)
Good for collecting statistically significant amount of data on
feature groups
Imaging Artifacts
What is an “artifact”
Sources of Artifacts
sample preparation
vacuum compatibility
electron beam “issues”
too low/too high KV (not really an artifact)
vibrations
stray magnetic fields
acoustic noise
Micrograph Critique Session