Transcript Microscopy and Surface Analysis 1

Microscopy and Surface Analysis 1
Lecture Date: March 17th, 2008
Microscopy and Surface Analysis
 Microscopic and imaging techniques:
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Optical microscopy
Confocal microscopy
Electron microscopy (SEM and TEM, related methods)
Scanning probe microscopy (STM and AFM, related methods)
 Surface spectrometric techniques:
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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 matter’s common
phases:
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–
–
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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
Microscopy
 Why is microscopy useful?
What can it tell the analytical
chemist?
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Sample topography
Structural stress/strain
Electromagnetic properties
Chemical composition
 Plus - a range of spectroscopic techniques, from IR to Xray wavelengths/energies, have been combined with
microscopy to create some of the most powerful analytical
tools available…
Imaging Resolution and Magnification
 Some typical values for microscopic methods:
Method
Resolution
Magnification
(x)
Human Eye
0.1-0.2 mm
-
Optical
Microscopy
0.1-0.2 um
~1200
Electron
Microscopy
30-50 Å
10-75,000
Probe
Microscopy
<1 Å
> 500,000
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. John and Zaccharias Jannsen (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
Optical Microscope Design

Microscope design has
not changed much in
300 years
– But the lenses are more
perfect, i.e. free of
aberrations
 Objective lenses are
characterized by numerical
aperature (NA)
– 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
Diagram from Wikipedia (public domain)
http://www.microscopyu.com/articles/polarized/polarizedintro.html
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
Airy disk
0.61
rairy 
 d min
NA
NA  n sin 
The minimum distance between resolved point objects of equal
intensity is the “Airy” disk radius (rairy), since resolution of a
conventional optical microscope is limited by Fraunhofer diffraction
at the entrance aperture of the objective lens
Resolved
Not resolved
http://www.cambridgeincolour.com/tutorials/diffraction-photography.htm, http://www.olympusmicro.com/primer/java/mtf/airydisksize/
See Y Garini, Current Opinion in Biotechnology 2005, 16:3–12
Getting Around the Diffraction Limit

Traditional optical microscopy is known as “far-field”
microscopy. Lateral resolution is limited to ~200 nm at short
wavelengths.
– 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:
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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 on inventing the confocal scanning microscope”. Scanning, 1988, 10, 128-138.
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
Diagram from http://www.cs.ubc.ca/spider/ladic/images/system.gif
Laser Confocal Scanning Microscopy
 A complete LCSM system:
Diagram from http://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:
Diagram from http://www.cs.ubc.ca/spider/ladic/images/system.gif
IR Microscopy and Spectroscopy
 Most FTIR microscopes image using array detectors
 IR spectra from a region are acquired at once for 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
limited by the diffraction limit, which is even worse for IR
wavelengths.
Figure from J. L. Koenig, S. Q. Wang, and R. Bhargava, Anal. Chem., 73, 361A-369A (2001).
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, fluorescence
microscopy, and Raman
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 more about forensic Raman microscopy, see: T. A. Brettell, N. Rudin and R. Saferstein, Anal. Chem., 75, 2877-2890 (2003).
Electron Microscopy (EM)
 Scanning electron
microscopy (SEM) – an
electron beam is scanned
in a raster pattern and
“reflected” effects are
monitored.
Velcro (x35)
Ice crystals
optical
SEM
 Transmission electron

microscopy (TEM) –
“transmitted” electrons are
monitored. Most TEMs are
actually scanning, i.e. STEM
Contrast is created in a totally
different manner in EM
Bottom photo - http://www.mos.org/sln/sem/velcro.html
Top photo - http://emu.arsusda.gov/snowsite/default.html
SEM of Anthophyllite Asbestos: A Typical Image
Bar
represents
50 m
Image from wikimedia commons (www.wikimedia.org)
Electron Microscopy: Basic Design
 Basic layout of an electron microscope:
Vacuum chamber
Electron
gun
(1-30 keV)
From www.jeol.com
Computer
Magnetic
lenses and
scanning
coils
Detectors
electrons
photons
Sample
electrons
Detectors
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Jeol JSM-7401F highresolution SEM (high
vacuum)
Electron Microscopy: Overall Design

Overall layout of a scanning
electron microscope (SEM):

TEM design is similar –
however, nowdays, TEM
systems usually include a
“cryo-stage” for keeping
samples extremely cold
during analysis
See pg. 551 of Skoog et al. and the references at the end of this lecture for details about SEM design.
Electron Optics: Electron Source (Gun)
Thermionic electron gun – how it works:
 Positive electrical potential applied to the anode
 The filament (cathode) is heated until a stream of
electrons is produced
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
The electrons feel the positive potential at the
anode (can be up to 30 kV) and move to it
A negative electrical potential (~500 V) is applied
to the Wehnelt cap
Electrons are forced back towards 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
Figure from http://www.unl.edu/CMRAcfem/interact.htm
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
Electron Optics: Focusing and Scanning
 Electron optics produce a
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
focused, monochromatic
(.e. same energy and
wavelength) electron beam
Apertures: usually made of
platinum foil, with circular
holes of 2 to 100 um.
Magnetic lenses:
Image from http://www.twi.co.uk/j32k/twiimages/eb_physicsf5.gif
– 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.).
– Used to focus and steer electrons in an EM
Electron Microscopy: Electron Detectors
 Electron detectors – don’t 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). They are only for
detecting the presence of electrons to form images.
The actual detector is usually a scintillator (doped glass,
etc…) that generates a photon burst when hit by an ethat is then detected by a photomultiplier tube.
– Semiconductor transducers are now becoming more common, since
they can be placed closer to the sample.
 But…the Everhart-Thornley detector is used to alternately
detect secondary and backscattered electrons based on
their energy (will be shown in detail in a few slides)
– Used as a “screen”, or basically a simple energy analyzer…
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:
1
2
mv 2  eV
2eV
v
m


h
m
h

m 2eV
2meV
6.63  10 34 J s
Note: In practice
resolution is
limited by lens
aberrations and
electron
scattering in the
sample, not by
wavelength!
2(9.11 10-31 kg)(1.60 10-19 C)(10 4 V)
  1.23 10 11 m  0.0123 nm
EM can see >10000x more detail than visible light!
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?
Using the
relativistically
corrected form of the
previous equation:


h
m
h

m 2eV
2meV (1  2eV
)
mc 2
6.63 10 34 J s
2(9.1110 kg)(1.60 10 C)(10 V)(1 
-31
-19
4
(1.6010-19 C)(10 4 V )
2 ( 9.1110-31 kg)( 3108 m / s ) 2
  3.7 10 3 nm
At high potentials, EM can see atomic dimensions
)
Electron Microscopy: Sample-Beam Interactions
 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)
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

Caused by an incident electron colliding with an
atom in the specimen which is almost normal to
the incident electron’s 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)
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
don’t 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 Microscopy: Image Formation
Light spots are zirconium (Z=40) aggregates
in an aluminium (Z= 13) matrix
BE
SE
From JEOL Application Note, V. E. Robertson, ca. 1985.
From wikimedia commons (www.wikimedia.org)
 BE and SE can be distinguished (and switched between)

using the Everhart-Thornley detection
SE shows topology, BE shows elemental distribution
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 weaklybound 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.
SEM: Sample-Beam Interactions – X-rays
SEM: Sample-Beam Interactions
Cathodoluminescence (CL)
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

The electron beam can excite electrons to the
valence band, leaving a hole behind. When an
excited electron recombines with a hole,
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 detectors
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:
–
–
–
–
–
Defects in semiconductors (GaAs, GaN, Si)
Differentiation of anatase and rutile
Studying ferroelectric domains in sodium niobate
Location of subsurface crazing in ceramics
Forensic analysis of glasses
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.
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
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
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 Probe Microscopies in Chemistry,” J. Phys. Chem., 1996, 100, 13103-13120.
Scanning Tunneling Microscopy (STM)
Rastering
Piezo actuators Z
DC
bias
X
control
electronics
computer
Y
display
tunnel
current
amp
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
Besocke-beetle style STM head
Slide courtesy of B. Mantooth and the Weiss Group at Penn State
Scanning Tunnelling Microscopy
 Tunnelling current is caused by
quantum mechanical phenomena
(confinement of an electron to a
“box” with finite walls)
 The tunnelling current It is given by:
Where:
V is the bias voltage
I t  Ve
 Cd
C is a constant based on the
conducting materials
d is the spacing between the atom
at the tip and the sample atom

Tips are prepared by cutting and
electrochemical etching – atomic
scale can be achieved because the
tunnelling current falls off
exponentially with increasing gap.
R. J. Hamers, “Scanned Probe Microscopies in Chemistry,” 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.
SPM Applications
 Numerous chemical and biochemical
applications where atomic-scale
magnification is useful
 Example: an AFM image of DNA
replication
R. J. Hamers, “Scanned Probe Microscopies in Chemistry,” J. Phys. Chem., 1996, 100, 13103-13120.
Further Reading
Skoog, Holler and Nieman, Chapter 21, “Surface Characterization by Spectroscopy and Microscopy”
Electron Microscopy and Electron Microprobe/X-ray Emission Analysis
1. J. I. Goldstein et al., Scanning Electron Microscopy and X-ray Microanalysis, 3rd Ed., Kluwer Academic,
2003.
2. J. J. Bozzola et al., Electron Microscopy: Principles and Techniques for Biologists, 2nd Ed., Jones 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. Johnson, “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).