1

Lecture Date: March 11

th
, 2008
Microscopy and Surface Anal ysi s 1
Reading Assignments for Microscopy and
Surface Anal ysi s
Skoog, Holler and Nieman, Chapter 21, Surface
Characterization by Spectroscopy and Microscopy
Hand-out Review Article: R. J . Hamers, Scanned Probe
Microscopies in Chemistry, J. Phys. Chem., 1996, 100,
13103-13120.
2
Microscopy and Surface Anal ysi s
Microscopic and imaging techniques:
Optical microscopy
Confocal microscopy
Electron microscopy (SEM and TEM, related methods)
Scanning probe microscopy (STM and AFM, related methods)
Surface spectrometric techniques:
X-ray fluorescence (from electron microscopy)
Auger electron spectrometry
X-ray photoelectron spectrometry (XPS/UPS/ESCA)
Other techniques:
Secondary-ion mass spectrometry (SIMS)
Ion-scattering spectrometry (ISS)
IR/Raman methods
Why Study Surfaces?
Surface the interface between two of matters common
phases:
Solid-gas (we will primarily focus on this)
Solid-liquid
Solid-solid
Liquid-gas
Liquid-liquid
The majority of present studies are applied to this type of
system, and the techniques available are extremely
powerful
The properties of surfaces often control chemical
reactions
3
Microscopy
Why is microscopy useful? What can it tell the analytical
chemist?
Sample topography
Structural stress/strain
Electromagnetic properties
Chemical composition
Plus - a range of spectroscopic techniques, from IR to X-
ray wavelengths/energies, have been combined with
microscopy to create some of the most powerful analytical
tools available
Imaging Resolution and Magnificati on
Some typical values for microscopic methods:
Method Resol uti on
Magni ficati on
(x)
Human Eye 0.1-0.2 mm -
Optical
Microscopy
0.1-0.2 um ~1200
Electron
Microscopy
30-50 10-75,000
Probe
Microscopy
<1 >500,000
4
Optical Microscopy - History
An ancient technique the lens has been around for
thousands of years. Chinese tapestries dating from 1000
B.C. depict eyeglasses.
In 1000 A.D., an Arabian mathematician (Al Hasan) made
the first theoretical study of the lens.
Copernicus (1542 A.D.) made the first definitive use of a
telescope.
As glass polishing skills developed, microscopes became
possible. J ohn and Zaccharias J annsen (Holland) made
the first commercial and first compound microscopes.
Then came lens grinding, Galileo, the biologists, and
many great discoveries.
Modern Optical Microscopy in Chemistry
As optical microscopy
developed, the compound
microscope was applied to
the study of chemical
crystals.
The polarizing microscope
(1880): can see boundaries
between materials with
different refractive indices,
while also detecting
isotropic and anisotropic
materials.
http://www.microscopyu.com/articles/polarized/polarizedintro.html
5
Optical Microscope Design
Objective lenses are
characterized NA
(numerical aperature)
The numerical aperture of a
microscope objective is a
measure of its ability to
gather light and resolve fine
specimen detail at a fixed
object distance
Large NA =finer detail =
better light gathering
http://www.microscopyu.com/articles/polarized/polarizedintro.html
DiagramfromWikipedia(publicdomain)
Microscope design has
not changed much in
300 years
But the lenses are more
perfect free of
abberations
The Diffraction Limit
The image of an infinitely
small point of light is not a
point it is an Airy disk
with concentric bright/dark
rings
http://www.cambridgeincolour.com/tutorials/diffraction-photography.htm,http://www.olympusmicro.com/primer/java/mtf/airydisksize/
SeeY Garini, CurrentOpinioninBiotechnology2005,16:312
min airy
d
NA
r
61 . 0
sin n NA
The minimum distance between resolved point objects of equal
intensity is the Airy disk radius (r
airy
), since resolution of a
conventional optical microscope is limited by Fraunhofer diffraction
at the entrance aperture of the objective lens
Airy disk
Resolved Not resolved
6
The Diffraction Limit
Traditional optical microscopy is known as far-field
microscopy. Its lateral resolution is limited to ~200 nm.
The need for the light-gathering objective lens and its aperture in
a conventional microscope leads to a diffraction limit
Newer techniques make use of near-field methods to
overcome the diffraction limit. A fiber tip with an aperture <100
nm is scanned over a sample (forming the basis of techniques
like NSOM/SNOM the scanning near-field optical
microscope).
NSOM is now being using in conjunction with AFM to study
nano-phototonics
Resolution now down to ~30 nm
Confocal Scanning Microscopy
Confocal imaging (or confocal scanning microscopy,
CSM) was first proposed by Marvin Minsky in 1957.
Confocal imaging: A technique in which a single axial
point is illuminated and focused at a time. The light
reflected (or produced e.g. by fluorescence) is detected
for just that point. Light from out-of-focus areas is
suppressed. A complete image is formed by scanning.
Advantages over conventional optical microscopy:
Greater depth of field from images
Images are free from out-of-focus blur
Greater signal-to-noise ratio (for a spot but images take time!)
Better effective resolution (diffraction limit)
M. Minsky, Memoir oninventingtheconfocal scanningmicroscope. Scanning, 1988, 10, 128-138.
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Confocal Scanning Microscopy: Imaging Types
One type of imaging mode is stage or object scanning:
A more modern mode is laser
scanning:
Nipkow disks can be used for studying moving samples
disks with staggered holes block all but a certain lateral portion of
the sample beam
Laser Confocal Scanning Microscopy
Laser confocal scanning
(LSCM) is the most
common type of CSM
Applications:
Biochemistry (including
fluorescence probes)
Materials science
Can be used with a
fluorescent dye to stain
biological samples
Diagramfromhttp://www.cs.ubc.ca/spider/ladic/images/system.gif
8
Laser Confocal Scanning Microscopy
A complete LCSM system:
Diagramfromhttp://www.cs.ubc.ca/spider/ladic/images/system.gif
Laser Confocal Scanning Microscopy
LCSM is often combined with fluorometry or with Raman
For fluorometry, there are numerous LCSM fluorophores:
Diagramfromhttp://www.cs.ubc.ca/spider/ladic/images/system.gif
9
IR Microscopy and Spectroscopy
Most FTIR microscopes image using array detectors
IR spectra from a region are acquired at once, better S/N
However, this is at the expense of resolution (limited to ca. 10 um),
in contrast with scanning techniques. Resolution in FTIR imaging is
of course limited by the diffration limit, which is even worse for IR
wavelengths.
FigurefromJ. L. Koenig, S. Q. Wang, andR. Bhargava, Anal. Chem., 73, 361A-369A (2001).
IR Microscopy: Image Anal ysis
Extraction of data from FTIR micrographs is done by
color-coding peaks based on their IR frequency (a)
Suitable IR frequencies can be
chosen via a scatter plot (c) of
every point in the image vs. two
(or more) frequencies, followed
by location of the center-of-gravity
and possible statistical analysis
False color images can then be
constructed (b)
FigurefromJ. L. Koenig, S. Q. Wang, andR. Bhargava, Anal. Chem., 73, 361A-369A (2001).
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IR Microscopy: Polymer Chemistry Applications
FTIR microscopy can analyze compositional differences in
material science, chemical and biochemical applications
Example the study of time-dependent processes like
dissolution of a polymer by a solvent
FigurefromJ. L. Koenig, S. Q. Wang, andR. Bhargava, Anal. Chem., 73, 361A-369A (2001).
IR Microscopy: Polymer Chemistry Applications
A complex, solvent-dependent dissolution, diffusion and
molecular motion process is observed for polymers (e.g.
polymethylstyrene) above their entanglement mwt:
FigurefromJ. L. Koenig, S. Q. Wang, andR. Bhargava, Anal. Chem., 73, 361A-369A (2001).
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Raman Microscopy
Raman microscopy better
inherent resolution than IR
(uses lasers at shorter
optical wavelengths)
Not capable of imaging
(must still scan the sample)
this does have its
advantages though
Often integrated with LCSM
systems for combined 3D
visualization and
spectroscopy
Raman Microscopy: Forensic Applications
Raman microscopy has many obvious applications
one that is not so obvious is for forensic analysis of
colored fibers.
The Raman spectra obtained from fibers acts as a
fingerprint, and the complex spectra obtained from dye
mixtures can be used to determine if two fibers are
from the same origin
The individual dyes used in fabics are varied, and their ratios
are especially varied (even from batch to batch!)
Competing techniques are generally destructive e.g.
LC or ESI MS on dye-containing extracts from fibers
For moreabout forensicRamanmicroscopy, see: T. A. Brettell, N. RudinandR. Saferstein, Anal. Chem., 75, 2877-2890(2003).
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Electron Microscopy (EM)
Scanning electron
microscopy (SEM) an
electron beam is scanned
in a raster pattern and
reflected effects are
monitored.
Transmission electron
microscopy (TEM)
transmitted electrons are
monitored. Most TEM are
actually scanning STEM!
Contrast is created in a totally
different manner in EM
Bottomphoto - http://www.mos.org/sln/sem/velcro.html
Topphoto - http://emu.arsusda.gov/snowsite/default.html
Velcro (x35) Ice crystals
optical SEM
Electron Microscopy: Basic Design
Basic layout of an electron microscope:
Electron
gun
(1-30 keV)
Magnetic
lenses and
scanning
coils
Sample
Detectors
Detectors
electrons
photons
electrons
Computer
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Electron Microscopy: Resolution
Why can an electron microscope resolve things that
are impossible to discern with optical microscopy?
Example calculate the wavelength of electrons
accelerated by a 10 kV potential:
nm 0.0123 m 10 23 . 1
) V C)(10 10 60 . 1 )( kg 10 2(9.11
s J 10 63 . 6
2 2
2
11
4 19 - 31 -
34
2
2
1

meV
h
eV
m
m
h
m
eV
v
eV mv
EM can see >10000x more detail than visible light!
Note: Resolution
is limited by lens
aberations!
Electron Microscopy: Resolution
What about relativistic corrections? The electrons in an
EM can in some cases be moving pretty close to the
speed of light.
Example what is the wavelength for a 100 kV potential?
nm 10 7 . 3
) 1 )( V C)(10 10 60 . 1 )( kg 10 2(9.11
s J 10 63 . 6
) 1 ( 2 2
3
) / 10 3 ( kg) 10 9.11 ( 2
) V C)(10 10 60 . 1 ( 4 19 - 31 -
34
2
2 8 -31
4 19 -
2

s m
mc
eV
meV
h
eV
m
m
h
At high potentials, EM can see atomic dimensions
Using the
relativistically
corrected form of the
previous equation:
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Electron Microscopy: Sample-Beam Interacti ons
Sample-beam interactions
control how both SEM and
TEM (i.e. STEM) operate:
Formation of images
Spectroscopic/diffractometric
analysis
There are lots (actually
eight) types of sample-beam
interactions (which can be
confusing and hard to
remember!)
It helps to classify these 8 types into two classes of
sample-beam interactions:
bulk specimen interactions (bounce off sample reflected)
thin specimen interactions (travel through sample- transmitted)
SEM: Sample-Beam Interactions
Backscattered Electrons (~30 keV)
Caused by an incident electron colliding with an
atom in the specimen which is almost normal to
the incident electrons path. The electron is then
scattered "backward" 180 degrees.
Backscattered electron intensity varies directly
with the specimen's atomic number. This
differing production rates causes higher atomic
number elements to appear brighter than lower
atomic number elements. This creates contrast
in the image of the specimen based on different
average atomic numbers.
Backscattered electrons can come from a wide
area around the beam impact point (see pg. 552
of Skoog) this also limits the resolution of a
SEM (along with abberations in the EM lenses)
15
SEM: Sample-Beam Interactions
Secondary Electrons (~5 eV)
Caused by an incident electron passing "near"
an atom in the specimen, close enough to
impart some of its energy to a lower energy
electron (usually in the K-shell). This causes a
slight energy loss, a change in the path of the
incident electron and ionization of the electron in
the specimen atom. The ionized electron then
leaves the atom with a very small kinetic energy
(~5 eV). One incident electron can produce
several secondary electrons.
Production of secondary electrons is closely
linked to sample topography. Their low energy
(~5 eV) means that only electrons very near to
the surface (<10 nm) are detected. They also
dont suffer from the backscattered electron
lateral resolution problem depicted in Fig. 21-16
of Skoog. Changes in topography in the sample
that are larger than this sampling depth can
change the yield of secondary electrons via
indirect effects (called collection efficiencies).
Electron Microscope: Image Formation
FromJEOL ApplicationNote, V. E. Robertson, ca. 1985.
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SEM: Sample-Beam Interactions
Auger Electrons (10 eV 2 keV)
Caused by relaxation of an ionized atom after a
secondary electron is produced. The lower
(usually K-shell) electron that was emitted from
the atom during the secondary electron process
has left a vacancy. A higher energy electron from
the same atom can drop to a lower energy, filling
the vacancy. This leaves extra energy in the atom
which can be corrected by emitting a weakly-
bound outer electron; an Auger electron.
Auger electrons have a characteristic energy,
which is unique and depends on the emitting
element. Auger electrons have relatively low
energy and are only emitted from the bulk
specimen from a depth of several angstroms.
SEM: Sample-Beam Interactions
X-ray Emission
Caused by relaxation of an ionized atom after a
secondary electron is produced. Since a lower
(usually K-shell) electron was emitted from the
atom during the secondary electron process an
inner (lower energy) shell now has a vacancy. A
higher energy electron can "fall" into the lower
energy shell, filling the vacancy. As the electron
"falls" it emits energy in the form of X-rays to
balance the total energy of the atom.
X-rays emitted from the atom will have a
characteristic energy which is unique to the
element from which it originated.
X-ray (elemental) mapping of sample surfaces is
a common applications and a very powerful
analytical approach.
17
SEM: Sample-Beam Interactions X-rays
SEM: Sample-Beam Interactions
Cathodoluminescence (CL)
Caused by electron hole pairs, which are created
by the electron beam in certain kinds of materials.
When the pairs recombine, cathodoluminescence
(CL) can result. CL is the emission of UV-Visible-
IR light by the recombination effect. CL is usually
very weak and covers a wide range of
wavelengths, and requires high beam currents,
lowering resolution and challenging detector
systems!
CL signals typically result from small impurities in
an otherwise homogeneous material, or lattice
defects in a crystal.
CL can be used effectively for some analytical
problems. Some random examples:
Differentiation of anatase and rutile
Studying ferroelectric domains in sodium niobate
Location of subsurface crazing in ceramics
Forensic analysis of glasses
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TEM: Sample-Beam Interactions (Thin Sample)
Unscattered Electrons
Incident electrons which are transmitted
through the thin specimen without any
interaction occurring inside the specimen.
Used to image - the transmission of
unscattered electrons is inversely proportional
to the specimen thickness. Areas of the
specimen that are thicker will have fewer
transmitted unscattered electrons and so will
appear darker, conversely the thinner areas
will have more transmitted and thus will
appear lighter.
TEM: Sample-Beam Interactions (Thin Sample)
Elastically-Scattered Electrons
Incident electrons that are scattered (deflected
from their original path) by atoms in the
specimen in an elastic fashion (without loss of
energy). These scattered electrons are then
transmitted through the remaining portions of
the specimen.
Electrons follow Bragg's Law and are
diffracted. All incident electrons have the
same energy (and wavelength) and enter the
specimen normal to its surface. So all incident
electrons that are scattered by the same
atomic spacing will be scattered by the same
angle. These "similar angle" scattered
electrons can be collated using magnetic
lenses to form a pattern of spots; each spot
corresponding to a specific atomic spacing,
This pattern can then yield information about
the orientation, atomic arrangements and
phases present in the area being examined.
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TEM: Sample-Beam Interactions (Thin Sample)
Inelastically-Scattered Electrons
Incident electrons that interact with sample atoms
inelastically (losing energy during the interaction).
These scattered electrons are then transmitted
through the rest of the sample.
Inelastically scattered electrons have two uses:
1. Electron Energy Loss Spectroscopy (EELS): The
amount of inelastic loss of energy by the incident
electrons can be used to study the sample.
These energy losses are unique to the bonding
state of each element and can be used to extract
both compositional and bonding (i.e. oxidation
state) information on the sample region being
examined.
2. Kakuchi bands: Bands of alternating light and dark
lines caused by inelastic scattering, which are
related to interatomic spacing in the sample.
These bands can be either measured (their width
is inversely proportional to atomic spacing) or
used to help study the elasticity-scattered electron
pattern
Electron Optics: Electron Source (Gun)
Positive electrical potential applied to the anode
The filament (cathode) is heated until a stream of
electrons is produced
The electrons are then accelerated by the positive
potential down the column (can be up to 30 kV)
A negative electrical potential (~500 V) is applied
to the Wehnelt cap
Electrons are forced toward the column axis by
the Wehnelt cap
Electrons collect in the space between the
filament tip and Wehnelt cap (a space charge or
pool)
Those electrons at the bottom of the space
charge (nearest to the anode) can exit the gun
area through the small (<1 mm) hole in the
Wehnelt cap
These electrons then move down the column
towards the EM lens and scanning systems
Figurefromhttp://www.unl.edu/CMRAcfem/interact.htm
Thermionic electron gun how it works:
The results:
- Electrons are emitted from
a nearly perfect point
source (the space
charge)
- The electrons all have
similar energies
(monchromatic)
- The electrons will travel
parallel to the column
axis
20
Electron Optics: Focusing and Scanning
Electron optics consist of several components:
Apertures usually made of platinum foil, with circular holes of 2 to
100 um.
Magnetic lenses: Circular electro-magnets capable of projecting a
precise circular magnetic field in a specified region. The field acts like
an optical lens, having the same attributes (focal length, angle of
divergence...etc.) and errors (spherical aberration, chromatic
aberration....etc.). They are used to focus and steer electrons in an
EM (SEM and STEM).
Goal a focused, monochromatic (I.e. same energy/wavelength)
electron beam!
Electron Microscopy: Electron Detectors
Electron detectors dont get them confused with other topics we will
discuss these are not for energy analysis! (We will discuss energy
analyzers/detectors with Auger and photoelectron spectroscopy.)
Only for detecting the presence of electrons to form images
The actual detector is usually a scintillator (doped glass, etc) that
generates a light burst detected by a photomultiplier tube.
Semiconductor transducers are now becoming more common, since
they can be placed closer to the sample.
The Everhart-Thornley detector is used to alternately detect
secondary and backscattered electrons based on their energy (see
previous slide)
Used as a screen, or basically a poor mans energy analyzer
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Electron Microscopy: Overall Design
Overall layout of a scanning
electron microscope (SEM):
Seepg. 551 of Skooget al. andthereferences at theendof thislecture for detailsabout SEM design.
TEM design is similar
however, nowdays, TEM
systems usually include a
cryo-stage for keeping
samples extremely cold
during analysis
Transmission Electron Microscopy: Applications
Morphology
The size, shape and arrangement of the particles which make up
the specimen as well as their relationship to each other on the
scale of atomic diameters.
Crystallographic Information
The arrangement of atoms in the specimen and their degree of
order, detection of atomic-scale defects in areas a few
nanometers in diameter
We will discuss this topic further during the crystallography
lecture
Compositional Information
The elements and compounds the sample is composed of and
their relative ratios, in areas a few nanometers in diameter
22
Scanning Probe Microscopy
SPM, also known as profilimetry
The first form, scanning tunneling microscopy (STM), was
invented by G. Binning and H. Roher (IBM) in 1982
Probe microscopies can achieve surface resolutions in the
x and y directions (parallel to the surface) of 1-20 A.
Also can achieve excellent z-resolution
STM involves scanning an atomic-scale tip across a
sample, recording an image based on the movement of
the tip and its associated cantilever
R. J. Hamers, Scanned ProbeMicroscopiesinChemistry, J. Phys. Chem., 1996, 100, 13103-13120.
Scanning Tunneling Microscopy (STM)
Besocke-beetle style STM head
Rastering
control
electronics
computer
DC
bias
Piezo actuators
tunnel
current
amp
display
X Y
Z
Constant current imaging:
A feedback loop adjusts the separation
between tip and sample to maintain a
constant current. The voltages applied to
the piezo are translated into an image.
Image represents a convolution of
topography and electronic structure
1/8 in
Slidecourtesyof B. MantoothandtheWeissGroupat PennState
23
Scanning Tunnelling Microscopy
Cd
t
Ve I

Tunnelling current is caused by

quantum mechanical phenomena
(confinement of an electron to a
box with finite walls)
The tunnelling current I
t
is given by:
Tips are prepared by cutting and
electrochemical etching atomic
scale can be achieved because the
tunnelling current falls off
exponentially with increasing gap.
Where:
V is thebias voltage
C is aconstant based on the
conductingmaterials
d is thespacingbetween theatom
at thetip and thesampleatom
R. J. Hamers, Scanned ProbeMicroscopiesinChemistry, J. Phys. Chem., 1996, 100, 13103-13120.
Atomic Force Microscopy
STM requires conducting samples. AFM scans a similar
cantilever across the surface, but instead of holding the
tunnelling current constant (and watching the piezo
voltages), the deflection of the tip is observed by a
sensitive apparatus.
In AFM the piezos just move the tip in x and y the
deflection in z is detected by a laser focused on the
cantilever and a photodiode array.
Individual atoms can be moved (pushed) by the AFM tip.
For sensitive samples, tapping-mode AFM (with a
tapping frequency of ~100 kHz) can be used to take less
intrusive images.
24
SPM Applicati ons
Numerous chemical and biochemical
applications where atomic-scale
magnification is useful
Example: an AFM image of DNA
replication
R. J. Hamers, Scanned ProbeMicroscopiesinChemistry, J. Phys. Chem., 1996, 100, 13103-13120.
Further Reading
Electron Microscopy and Electron Microprobe/X-ray Emission Analysis
1. J . I. Goldstein et al., Scanning Electron Microscopy and X-ray Microanalysis, 3
rd
Ed., Kluwer Academic,
2003.
2. J . J . Bozzola et al., Electron Microscopy: Principles and Techniques for Biologists, 2
nd
Ed., J ones and
Bartlett, 1998.
3. J . W. Edington, N. V Philips, Practical Electron Microscopy in Materials Science, Eindhoven, 1976.
Electron Microscopy and Electron Diffraction/Electron Energy Loss Spectroscopy
4. A. Engel and C. Colliex, Application of scanning transmission electron microscopy to the study of
biological structure, Current Biology 4, 403-411 (1993). (STEM and EELS)
5. W. Chiu and M. F. Schmid, Electron crystallography of macromolecules, Current Biology 4, 397-402
(1993). (ED and LEED)
6. W. Chiu, What does electron cryomicroscopy provide that X-ray crystallography and NMR cannot?,
Annu. Rev. Biophys. Biomol. Struct., 22, 233-255 (1993). (Electron Cryomicroscopy/Imaging)
7. L. Tang and J . E. J ohnson, Structural biology of viruses by the combination of electron cryomicroscopy
and X-ray crystallography, 41, 11517-11524 (2002). (Electron Cryomicroscopy/Imaging)
Optical Microscopy
8. R. H. Webb, "Confocal optical microscopy, Rep. Prog. Phys. 59, 427-471 (1996).
Force Microscopy:
9. R. J . Hamers, Scanned probe microscopies in chemistry, J. Phys. Chem., 100, 13103-13120 (1996).