Topic 9 Microscopy and Surface Analysis

7/29/2019 Topic 9 Microscopy and Surface Analysis

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SKA6014

ADVANCED ANALYTICAL CHEMISTRY

TOPIC 11Microscopy and Surface Analysis 1

Azlan Kamari, PhD

Department of ChemistryFaculty of Science and Mathematics

Universiti Pendidikan Sultan Idris


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Microscopy and Surface Analysis

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


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Why Study Surfaces?

Surfacethe 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 ofsystem, and the techniques available are extremely

powerful

The properties of surfaces often control chemicalreactions


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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 powerfulanalytical tools available


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Imaging Resolution and Magnification

Some typical values for microscopic methods:

Method ResolutionMagnification

(x)

Human Eye 0.1-0.2 mm -

OpticalMicroscopy

0.1-0.2 um ~1200

Electron

Microscopy

30-50 10-75,000

ProbeMicroscopy

500,000


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Optical Microscopy - History

An ancient technique the lens has been around for

thousands of years. Chinese tapestries dating from1000 B.C. depict eyeglasses.

In 1000 A.D., an Arabian mathematician (Al Hasan) madethe first theoretical study of the lens.

Copernicus (1542 A.D.) made the first definitive use of atelescope.

As glass polishing skills developed, microscopes becamepossible. John and Zaccharias Jannsen (Holland) made

the first commercial and first compound microscopes. Then came lens grinding, Galileo, the biologists, and

many great discoveries.


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Modern Optical Microscopy in Chemistry

As optical microscopy

developed, the compoundmicroscope was applied tothe study of chemicalcrystals.

The polarizing microscope(1880): can seeboundaries between

materials with differentrefractive indices, whilealso detecting isotropicand anisotropic materials.

http://www.microscopyu.com/articles/polarized/polarizedintro.html


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Optical Microscope Design

Objective lenses are

characterized NA(numerical aperture)

The numerical aperture of amicroscope objective is ameasure of its ability to

gather light and resolve finespecimen detail at a fixedobject distance

Large NA = finer detail =better light gathering

http://www.microscopyu.com/articles/polarized/polarizedintro.html

Diagram from Wikipedia (public domain)

Microscope design has notchanged much in 300 years

But the lenses are moreperfect free ofabberations


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The Diffraction Limit

The image of an infinitelysmall point of light is not apointit is an Airy disk withconcentric bright/dark rings

http://www.cambridgeincolour.com/tutorials/diffraction-photography.htm, http://www.olympusmicro.com/primer/java/mtf/airydisksize/

See Y Garini, Current Opinion in Biotechnology 2005, 16:312

minairy dNA

r 61.0

sinnNA The minimum distance between resolved point objects of equal intensity

is the Airy disk radius (rairy), since resolution of a conventional opticalmicroscope is limited by Fraunhofer diffraction at the entrance aperture ofthe objective lens

Airy disk

Resolved Not resolved
http://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.olympusmicro.com/primer/java/mtf/airydisksize/http://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.olympusmicro.com/primer/java/mtf/airydisksize/http://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.cambridgeincolour.com/tutorials/diffraction-photography.htmhttp://www.cambridgeincolour.com/tutorials/diffraction-photography.htm


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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 diffractionlimit

Newer techniques make use of near-field methods toovercome the diffraction limit. A fiber tip with an aperture


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Confocal Scanning Microscopy

Confocal imaging (or confocal scanning microscopy, CSM) was firstproposed by Marvin Minsky in 1957.

Confocal imaging: A technique in which a single axial point isilluminated and focused at a time. The light reflected (or producede.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)


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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


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Laser Confocal Scanning Microscopy

Laser confocal scanning

(LSCM) is the mostcommon type of CSM

Applications:

Biochemistry

(includingfluorescence probes)

Materials science

Can be used with afluorescent dye to stainbiological samples

Diagram from http://www.cs.ubc.ca/spider/ladic/images/system.gif


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A complete LCSM system:



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LCSM is often combined with fluorometry or with Raman

For fluorometry, there are numerous LCSM fluorophores:



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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 isof course limited by the diffration limit, which is even worse for IRwavelengths.

Figure from J. L. Koenig, S. Q. Wang, and R. Bhargava,Anal. Chem.,73, 361A-369A (2001).


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IR Microscopy: Image Analysis

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) ofevery point in the image vs. two

(or more) frequencies, followedby location of the center-of-gravityand possible statistical analysis

False colour images can then beconstructed (b)



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IR Microscopy: Polymer Chemistry Applications

FTIR microscopy can analyze compositional differences inmaterial science, chemical and biochemical applications

Example the study of time-dependent processes likedissolution of a polymer by a solvent



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IR Microscopy: Polymer Chemistry Applications

A complex, solvent-dependent dissolution, diffusion andmolecular motion process is observed for polymers (e.g.polymethylstyrene) above their entanglement mwt:



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Raman Microscopy

Raman microscopybetter inherent resolutionthan IR (uses lasers atshorter opticalwavelengths)

Not capable of imaging(must still scan the sample) this does have itsadvantages though

Often integrated withLCSM systems forcombined 3D visualizationand spectroscopy


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Raman Microscopy: Forensic Applications

Raman microscopy has many obvious applicationsone that is not so obvious is for forensic analysis ofcolored fibers.

The Raman spectra obtained from fibers acts as afingerprint, and the complex spectra obtained from

dye mixtures can be used to determine if two fibersare from the same origin

The individual dyes used in fabics are varied, andtheir 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).


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Electron Microscopy (EM)

Scanning electronmicroscopy (SEM) anelectron beam is scannedin a raster pattern andreflected effects are

monitored.

Transmission electronmicroscopy (TEM)transmitted electrons are

monitored. Most TEM areactually scanning STEM!

Contrast is created in a totallydifferent manner in EM

Bottom photo - http://www.mos.org/sln/sem/velcro.html

Top photo - http://emu.arsusda.gov/snowsite/default.html

Velcro (x35)Ice crystalsoptical SEM
http://www.mos.org/sln/sem/velcro.htmlhttp://emu.arsusda.gov/snowsite/default.htmlhttp://emu.arsusda.gov/snowsite/default.htmlhttp://www.mos.org/sln/sem/velcro.html


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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 thatare impossible to discern with optical microscopy?

Example calculate the wavelength of electronsaccelerated by a 10 kV potential:

nm0.0123m1023.1

)VC)(101060.1)(kg102(9.11

sJ1063.6

22

2

11

419-31-

34

2

21

meV

h

eV

m

m

h

meVv

eVmv

EM can see >10000x more detail than visible light!

Note:

Resolution islimited by lensaberations!


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Electron Microscopy: Resolution

What about relativistic corrections? The electrons inan EM can in some cases be moving pretty close to the

speed of light. Example what is the wavelength for a 100 kV potential?

nm107.3

)1)(VC)(101060.1)(kg102(9.11

sJ1063.6

)1(22

3

)/103(kg)109.11(2

)VC)(101060.1(419-31-

34

2

28-31

419-

2

sm

mc

eVmeV

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 Interactions

Sample-beam interactionscontrol how both SEM and TEM

(i.e. STEM) operate: Formation of images

Spectroscopic/diffractometricanalysis

There are lots (actually eight)types of sample-beaminteractions (which can beconfusing and hard to

remember!) It helps to classify these 8 types into two classes of sample-

beam interactions: bulk specimen interactions (bounce off samplereflected)

thin specimen interactions (travel through sample- transmitted)


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SEM: Sample-Beam Interactions

Backscattered Electrons (~30 keV) Caused by an incident electron colliding

with an atom in the specimen which isalmost normal to the incident electronspath. The electron is then scattered"backward" 180 degrees.

Backscattered electron intensity varies

directly with the specimen's atomicnumber. This differing production ratescauses higher atomic number elements toappear brighter than lower atomic

number elements. This creates contrast inthe image of the specimen based on

different average atomic numbers.

Backscattered electrons can come from awide area around the beam impact point(see pg. 552 of Skoog) this also limits theresolution of a SEM (along with

abberations in the EM lenses)

S S


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Secondary Electrons (~5 eV)

Caused by an incident electron passing "near"

an atom in the specimen, close enough toimpart some of its energy to a lower energyelectron (usually in the K-shell). This causes aslight energy loss, a change in the path of theincident electron and ionization of the electronin the specimen atom. The ionized electronthen leaves the atom with a very small kineticenergy (~5 eV). One incident electron canproduce several secondary electrons.

Production of secondary electrons is closelylinked to sample topography. Their low energy(~5 eV) means that only electrons very near to

the surface (


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Electron Microscope: Image Formation


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Auger Electrons (10 eV 2 keV)

Caused by relaxation of an ionized atomafter a secondary electron is produced.The lower (usually K-shell) electron thatwas emitted from the atom during thesecondary electron process has left avacancy. A higher energy electron from the

same atom can drop to a lower energy,filling the vacancy. This leaves extra energyin the atom which can be corrected byemitting a weakly-bound outer electron; anAuger electron.

Auger electrons have a characteristicenergy, which is unique and depends on theemitting element. Auger electrons haverelatively low energy and are only emittedfrom the bulk specimen from a depth ofseveral angstroms.


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X-ray Emission Caused by relaxation of an ionized atom

after a secondary electron is produced.Since a lower (usually K-shell) electronwas emitted from the atom during thesecondary electron process an inner(lower energy) shell now has a vacancy. Ahigher energy electron can "fall" into the

lower energy shell, filling the vacancy. Asthe electron "falls" it emits energy in theform of X-rays to balance the total energyof the atom.

X-rays emitted from the atom will have a

characteristic energy which is unique tothe element from which it originated.

X-ray (elemental) mapping of samplesurfaces is a common applications and avery powerful analytical approach.


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SEM: Sample-Beam Interactions X-rays

SEM S l B I t ti


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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 usuallyvery weak and covers a wide range ofwavelengths, and requires high beam currents,lowering resolution and challenging detector

systems!

CL signals typically result from small impurities inan otherwise homogeneous material, or latticedefects in a crystal.

CL can be used effectively for some analyticalproblems. Some random examples:

Differentiation of anatase and rutile

Studying ferroelectric domains in sodiumniobate

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 aretransmitted through the thinspecimen without any interactionoccurring inside the specimen.

Used to image - the transmission ofunscattered electrons is inverselyproportional to the specimenthickness. Areas of the specimenthat are thicker will have fewer

transmitted unscattered electronsand so will appear darker,conversely the thinner areas willhave more transmitted and thus willappear lighter.


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Elastically-Scattered Electrons Incident electrons that are scattered

(deflected from their original path) by atoms inthe specimen in an elastic fashion (withoutloss of energy). These scattered electrons arethen transmitted through the remainingportions of the specimen.

Electrons follow Bragg's Law and arediffracted. All incident electrons have thesame energy (and wavelength) and enter thespecimen normal to its surface. So allincident electrons that are scattered by thesame atomic spacing will be scattered by thesame angle. These "similar angle" scattered

electrons can be collated using magneticlenses to form a pattern of spots; each spotcorresponding to a specific atomic spacing,This pattern can then yield information aboutthe orientation, atomic arrangements andphases present in the area being examined.


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Inelastically-Scattered Electrons Incident electrons that interact with sample atoms

inelastically (losing energy during the interaction).These scattered electrons are then transmittedthrough the rest of the sample.

Inelastically scattered electrons have two uses:

1. Electron Energy Loss Spectroscopy (EELS): Theamount of inelastic loss of energy by the incident

electrons can be used to study the sample.These energy losses are unique to the bondingstate of each element and can be used to extractboth compositional and bonding (i.e. oxidationstate) information on the sample region beingexamined.

2. Kakuchi bands: Bands of alternating light and darklines caused by inelastic scattering, which arerelated to interatomic spacing in the sample.These bands can be either measured (their widthis inversely proportional to atomic spacing) orused to help study the elasticity-scatteredelectron pattern

O S (G )


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Electron Optics: Electron Source (Gun)

Positive electrical potential applied to the anode

The filament (cathode) is heated until a stream ofelectrons is produced

The electrons are then accelerated by the positivepotential down the column (can be up to 30 kV)

A negative electrical potential (~500 V) is appliedto the Wehnelt cap

Electrons are forced toward the column axis bythe Wehnelt cap

Electrons collect in the space between thefilament tip and Wehnelt cap (a space charge orpool)

Those electrons at the bottom of the space

charge (nearest to the anode) can exit the gunarea through the small (


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Electron Optics: Focusing and Scanning

Electron optics consist of several components:

Apertures usually made of platinum foil, with circularholes of 2 to 100 um.

Magnetic lenses: Circular electro-magnets capable of

projecting a precise circular magnetic field in a specifiedregion. The field acts like an optical lens, having thesame attributes (focal length, angle of divergence...etc.)and errors (spherical aberration, chromaticaberration....etc.). They are used to focus and steer

electrons in an EM (SEM and STEM).

Goal a focused, monochromatic (I.e. sameenergy/wavelength) electron beam!


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Electron Microscopy: Electron Detectors

Electron detectorsdont get them confused with other topics wewill discuss these are not for energy analysis! (We will discuss

energy analyzers/detectors with Auger and photoelectronspectroscopy.)

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 detectsecondary and backscattered electrons based on their energy (seeprevious slide)

Used as a screen, or basically a poor mans energy analyzer

El t Mi O ll D i


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Electron Microscopy: Overall Design

Overall layout of a scanningelectron microscope (SEM):

TEM design is similarhowever, nowdays, TEM

systems usually include acryo-stage for keepingsamples extremely coldduring analysis


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Transmission Electron Microscopy: Applications

Morphology

The size, shape and arrangement of the particles which make upthe specimen as well as their relationship to each other on thescale of atomic diameters.

Crystallographic Information

The arrangement of atoms in the specimen and their degree oforder, detection of atomic-scale defects in areas a fewnanometers 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


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Scanning Probe Microscopy

SPM, also known as profilimetry

The first form, scanning tunneling microscopy (STM), wasinvented by G. Binning and H. Roher (IBM) in 1982

Probe microscopies can achieve surface resolutions inthe 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 asample, recording an image based on the movement of

the tip and its associated cantilever

Scanning Tunneling Microscopy (STM)


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Scanning Tunneling Microscopy (STM)

Besocke-beetle style STM head

Rasteringcontrol

electronicscomputer

DC

bias

Piezo actuators

tunnel

current

amp

displayX Y

Z

Constant current imaging:A feedback loop adjusts the separationbetween tip and sample to maintain a

constant current. The voltages applied tothe piezo are translated into an image.

Image represents a convolution of

topography and electronic structure1/8 in


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Scanning Tunnelling Microscopy

Cdt VeI

Tunnelling current is caused byquantum mechanical phenomena

(confinement of an electron to abox with finite walls)

The tunnelling current Itis given by:

Tips are prepared by cutting andelectrochemical etching atomicscale can be achieved because thetunnelling current falls offexponentially with increasing gap.

Where:

Vis the bias voltage

Cis a constant based on the

conducting materials

dis the spacing between the atom

at the tip and the sample atom


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Atomic Force Microscopy

STM requires conducting samples. AFM scans a similar

cantilever across the surface, but instead of holding thetunnelling current constant (and watching the piezovoltages), the deflection of the tip is observed by asensitive apparatus.

In AFM the piezos just move the tip in x and y thedeflection in z is detected by a laser focused on thecantilever and a photodiode array.

Individual atoms can be moved (pushed) by the AFM tip.

For sensitive samples, tapping-mode AFM (with atapping frequency of ~100 kHz) can be used to take lessintrusive images.


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SPM Applications

Numerous chemical and biochemicalapplications where atomic-scalemagnification is useful

Example: an AFM image of DNAreplication

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Topic 9 Microscopy and Surface Analysis