Opt 307/407 Practical Scanning Electron Microscopy

Opt 307/407 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

Opt 307/407 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!

Opt 307/407 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

Opt 307/407 Vacuum Systems

Opt 307/407 Vacuum Systems Quality of Vacuum Low: 760-10-2 Torr
Medium: Torr High: Torr Ultrahigh: <~10-8 Torr

Measuring Vacuum in EM Systems
Opt 307/407 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

Opt 307/407 Vacuum Systems Types of Vacuum Pumps
1- Rotary (Fore, Rough, Aux, Mechanical) 2- Turbomolecular (Turbo) 3- Diffusion (Diff) 4- Ion (Sputter-ion)

Opt 307/407 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

Opt 307/407 Vacuum Systems Rotary Pump Problems
Cannot pump <10-2 Torr Noisy Backstreams Vibration Maintenance

Direct drive electric motor-gas turbine Rotor/stator assembly
Opt 307/407 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

Needs to be protected from solid material
Opt 307/407 Vacuum Systems Turbo Pump Problems Needs a Foreline pump Costly Can fail abruptly Whine Needs to be protected from solid material

Opt 307/407 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

Opt 307/407 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)

Opt 307/407 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)

Opt 307/407 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)

Opt 307/407 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

Opt 307/407 Vacuum Systems Typical TEM Vacuum System

Electron Sources and Lenses
Opt307/407 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 ( 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

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 LEO Gemini 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 Disadvantages Advantages
Decrease resolution due to effects of diffraction Decrease resolution by reducing half angle of illumination Decrease illumination by blocking scattered electrons Advantages Increase contrast by blocking scattered electrons Decrease effects of chromatic and spherical aberration by cutting off edges of a lens

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
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 cross-section

Electron Beam-Sample Interactions
Opt 307/407 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

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= *A *A2*A *A2*A2*A2

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

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

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 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 -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

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 Vacuum chamber Samples Vacuum gauge HV control Current monitor Timer Argon bleed

Sample Coating: Evaporation
Used when sputtering won't work well Carbon Making shadows Line of sight deposition

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

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

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

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

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

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

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

-to see interior or sub-surface details
Cross Sections Why? -to see interior or sub-surface details How? -fracture -cleaving -microtome -polishing

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

Electrically Conductive Samples
The best sample The most unusual sample Simply attach to sample stub and “go” Beware of contaminated surfaces

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

Untouchable Samples Historically significant samples Forensic samples
Samples from litigations

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

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

Manipulated Samples Stressed in tension or compression
Samples irradiated to simulate high dose -exposure Electron beam induced current (EBIC) Voltage contrast

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

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

Hazardous Samples Biohazards (DNA, Viruses, Bacteria, etc.)
Radioisotopes Fine dust Toxic materials (Be metal)

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!

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