Fundamentals of
Nano-biotechnology
Course Number: UBE08E02
SEM
Tridib Kumar Bhowmick
Dept. of Bioengineering
tbhowmick.be@nita.ac.in
What Can You See….
http://nobelprize.org/educational_games/physics/microscopes/powerline/index.html
Introduction to Electron Microscopy
• Ultrastructural study = Electron microscopy
• Two types of EM: Transmission (TEM) and
Scanning (SEM)
• Resolution of TEM : best at about 0.2 nm
(nanometer = 10^-9 m), which is about 1000x
better than ordinary light microscope
• TEM is far more useful for medical investigations
than SEM
Merits of EM
• high magnification at high
resolution
• technique largely standardized
• some ultrastructural features are highly
specific for certain cell types or diseases
Drawbacks of EM
• equipment expensive
• procedures time consuming (staff costly)
• small samples lead to possible sampling error
and misinterpretation
• optimum tissue preservation requires special
fixative and processing
• much experience is needed for interpreting the
results
when electrons hit matter ..
when electrons hit matter ..
(1) they may collide with an inner shell electron, ejecting same
> the ejected electron is a low-energy, secondary electron
- detected & used to from SEM images
> the original high-energy electron is scattered
- known as a ‘back-scattered’ electron (SEM use)
> an outer-shell electron drops into the position formerly
occupied by the ejected electron
> this is a quantum process, so a X-ray photon of precise
wavelength is emitted - basis for X-ray microanalysis
when electrons hit matter ..
when electrons hit matter ..
(2) they may collide or nearly collide with an atomic nucleus
> undergo varying degree of deflection (inelastic scattering)
> undergo loss of energy - again varying
> lost energy appears as X-rays of varying wavelength
> this X-ray continuum is identical to that originating from
an X-ray source/generator (medical, XRC etc)
> original electrons scattered in a forward direction will
enter the imaging system, but with ‘wrong’ l
> causes a ‘haze’ and loss of resolution in image
when electrons hit matter ..
when electrons hit matter ..
(3) they may collide with outer shell electrons
> either removing or inserting an electron
> results in free radical formation
> this species is extremely chemically active
> reactions with neighbouring atoms induce massive change in the
specimen, especially in the light atoms
> this radiation damage severely limits possibilities of EM
> examination of cells in the live state NOT POSSIBLE
> all examinations need to be as brief (low dose) as possible
when electrons hit matter ..
when electrons hit matter ..
(4) they may pass through unchanged
> these transmitted electrons can be used to form an image
> this is called imaging by subtractive contrast
> can be recorded by either
(a) TV-type camera (CCD) - very expensive
(b) photographic film - direct impact of electrons
Photographic film
> silver halide grains detect virtually every electron
> at least 50x more efficient than photon capture !
when electrons hit matter ..
‘beam damage’ occurs:
• light elements (H, O) lost very rapidly
• change in valency shell means free radicals formed
• . . .& consequent chemical reactions causing further damage
• beam damage is minimised by use of
• low temperatures (-160°)
• high beam voltages
• minimal exposure times
Specimen-Beam Interaction
Auger
electron
Incident
electron beam
Backscattered
electrons
X-ray
Secondary
electrons
Light
Specimen
Absorbed electrons
Inelastically
scattered
electrons
Unscattered
electrons
Elastically
scattered
electrons
Types of Microscopy
Electromagnetic
lenses
Glass lenses
Direct observation
Video imaging (CRT)
Comparison of LM and TEM
Light Source
Electron Source
Glass Lenses
EM Lenses
– Light has different
speeds in different
mediums (refraction)
– Light bends due to
refraction
Image
– Formed by transmitted
light
– Charged electrons bend
due to magnetic field
Image
− Formed by transmitted
electrons impinging on
phosphor coated
screen
 Both glass and EM lenses subject to same distortions and aberrations
 Glass lenses have fixed focal length, change objective lens to chang mag., move objective
lens closer to or farther away from specimen to focus
 EM lenses to specimen distance fixed, focal length varied by varying current through lens
 Light wavefront moves in a straight line while electrons move in helical orbits, EM lenses
change trajectory but no huge change in electron velocity
THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE
FEATURE
Electromagnetic
spectrum used
Maximum
resolving power
Maximum
magnification
Radiation
source
Lenses
Interior
Focussing
screen
© 2007 Paul Billiet ODWS
LIGHT MICROSCOPE
ELECTRON MICROSCOPE
Visible light
Electrons
760nm (red) – 390nm
Colours visible
app. 4nm
Monochrome
app. 200nm
0.2nm
Fine detail
x1000 – x1500
x500 000
Tungsten or quartz
halogen lamp
High voltage (50kV)
tungsten lamp
Glass
Air-filled
Magnets
Vacuum
Human eye (retina),
photographic film
fluorescent (TV) screen,
photographic film
THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE
FEATURE
Preparation of
specimens
Fixation
Embedding
LIGHT MICROSCOPE
Temporary mounts
living or dead
ELECTRON
MICROSCOPE
Tissues must be
dehydrated
= dead
Alcohol
OsO4 or KMnO4
Resin
Sectioning
Wax
Hand or microtome
slices  20 000nm
Whole cells visible
Microtome only.
Slices  50nm
Parts of cells visible
Stains
Water soluble dyes
Heavy metals
Glass slide
Copper grid
Support
© 2007 Paul Billiet ODWS
Microscopy
Structure determines properties
We have discussed crystal structure (x-ray diffraction)
But consider now different level of structure
Microstructure - also can be 'art'
Scanning Electron
Microscope (SEM):
• Is capable of higher resolution than the
light microscope
• An electron beam is “bounced off” the
specimen to a detector, instead of being
passed through it
• It produces a detailed image of the surface
of the specimen, but not its internal
structure
Scanning Electron
Microscope (SEM):
http://www.engr.uky.edu/emc/facilities/sem.html
SEM Image:
(Emiliania huxleyi, a haptophyte alga)
http://starcentral.mbl.edu/
Electron microscopy
SEM - scanning electron microscopy
tiny electron beam scanned across surface of specimen
backscattered or
secondary electrons detected
signal output to synchronized display
Electron microscopy
SEM - scanning electron microscopy
Magnification range 15x to 200,000x
Resolution of 50 Å
Excellent depth of focus
Relatively easy sample prep
Louis de Broglie showed that every particle or matter propagates like a
wave. The wavelength of a particle or a matter can be calculated as follows.
where λ is the wavelength of a particle, h is Planck’s constant (6.626 x 1034 J seconds), and p is the momentum of a particle. Since the momentum is
the product of the mass and the velocity of a particle,
Because the velocity of the electrons is determined by the accelerating
voltage, or electron potential where
The velocity of electrons can be calculated by
Therefore, the wavelength of propagating electrons at a given accelerating
voltage can be determined by
Since the mass of an electron is 9.1 x 10-31 kg and e = 1.6 x 10-19,
Thus, the wavelength of electrons is calculated to be 3.88 pm when the
microscope is operated at 100 keV, 2.74 pm at 200 keV, and 2.24 pm at 300
keV.
However, because the velocities of electrons in an electron microscope reach
about 70% the speed of light with an accelerating voltage of 200 keV, there are
relativistic effects on these electrons. These effects include significant length
contraction, time dilation, and an increase in mass. By accounting for these
changes,
where c is the speed of light, which is ~3 x 108 m/s. Therefore, the wavelength
at 100 keV, 200 keV, and 300 keV in electron microscopes is 3.70 pm, 2.51 pm
and 1.96 pm, respectively.
SEM - scanning electron microscopy
Electron gun
Don't make x-rays - use
electrons directly
Wavelength:
NOT l = hc/E
(massless photons)
l = h/(2melectronqVo)
(non-relativistic)
l = h/(2melectronqVo + q2Vo2/c2)1/2
(relativistic )
SEM - scanning electron microscopy
l = h / (2melectronqVo + q2Vo2/c2)1/2
l = 1.22639 / (Vo + 0.97845 · 10-6Vo2)1/2
l(nm) & Vo(volts)
10 kV ——> 0.12 Å
100 kV ——> 0.037 Å
SEM - scanning electron microscopy
Electron gun
Electron emitter
SEM - scanning electron microscopy
l = h/(2melectronqVo + q2Vo2/c2))
Effects of increasing voltage in
electron gun:
Resolution increased (l decreased)
Penetration increases
Specimen charging increases
(insulators)
Specimen damage increases
Image contrast decreases
SEM - scanning electron microscopy
Lenses
electrons focused by Lorentz force from electromagnetic field
F = qv x B
effectively same as optical lenses
Lenses are ring-shaped
coils generate magnetic field
electrons pass thru hollow center
lens focal length is continuously variable
apertures control, limit beam
SEM - scanning electron microscopy
Specimen
Conducting little or no preparation
attach to mounting stub
for insertion into
instrument
may need to provide
conductive path with
Ag paint
Non-conducting usually coat with conductive very thin layer (Au, C, Cr)
SEM - scanning electron microscopy
Specimen
Can examine
fracture surfaces
electronic devices
fibers
coatings
particles
etc.
SEM - scanning electron microscopy
Specimen
What comes from specimen?
Backscattered electrons
Secondary electrons
Fluorescent X-rays
composition - EDS
high energy
compositional contrast
Brightness of regions in image
increases as atomic number increases
(less penetration gives more
backscattered electrons)
low energy
topographic contrast
SEM - scanning electron microscopy
Backscattered electron detector - solid state detector
electron energy up to 30-50 keV
annular around incident beam
repel secondary electrons with
— biased mesh
images are more sensitive to
chemical composition (electron
yield depends on atomic number)
line of sight necessary
SEM - scanning electron microscopy
Secondary electron detector - scintillation detector
+ bias mesh needed in front of
detector to attract low energy
electrons
line of sight unnecessary
SEM - scanning electron microscopy
Composition - what elements present at a particular spot in specimen?
Use solid state detector
Do energy scan for fluorescent X-rays
The instrument in brief
MENA3100
How do we get an image?
Electron gun
156
288 electrons!
electrons!
Detector
Image
MENA3100
Topography and morphology
• High depth of
focus
Image: Christian Kjølseth, UiO
MENA3100
Image: Camilla Kongshaug, UiO
depth of focus - depth of field
The depth of focus, Dim is the extent of the region around the image plane
in which the image will appear to be sharp. This depends on
magnification, MT.
depth of useful focus (in the specimen) is primarily limited by chromatic
aberration effects
• the absolute depth of focus is larger than this: for all practical purposes,
everything is in focus to same level
• . . . So one cannot rack through focus (as in a light or even scanning
electron) microscope.
• The depth of field, Dob is the range of distance along the optical axis in
which the specimen can move without the image apppearing to lose
sharpness. This obviously depends on the resolution of the microscope.
• depth of field (in the image plane) is infinite - for all practical purposes
Depth of Field
• In optics DOF is defined as
Which depends on the focal length of the lens, the f-number of the
lens opening (the aperture), and the camera-to-subject distance.
f is the lens focal length, N is the lens f-number, and c is the circle of
confusion.
• in SEM systems DOF is dependent to two
variables
1.the final aperture size (radius R)
2.the working distance (W)
Some effects
• DOF will be larger when the emission disc is smaller
• DOF will be larger when the aperture is smaller
• DOF will be larger higher when the working distance
is longer
• DOF will be larger when the SEM is at lower
magnifications
Example
• for a specimen At working distance of
10mm we Can find D in different
conditions
Decreasing the size of the aperture
• can produce an increased depth of field
• a decrease in probe current
• a possible improvement in the probe
resolution
• a change in astigmatism (needs to be
corrected again)
increasing the distance W
* Can potentially increased depth of field
* lower attainable limits for low magnification
* some loss of resolution
* a possible decrease of signal strength
* astigmatism will worsen at long W
*
Sample Preparation and Mapping
• Steps
– Cleaning and drying of bulk samples
– Initial Preparation. Cutting, impregnation,
cast-making, fusing
– Mounting. Stubs, embedded samples, thin
sections, grain mounts
– Polishing. For quantitative analysis
– Cleaning
– Mapping
– Coating
– Handling and storage
Preparing Bulk Samples
• Initial Prep:
– Remove organic material with oxidizing
agent such as K-permanganate or H2O2
– Rinse sediments or soils to remove soluble
salts and fines if desired
– Carbonate can be removed with HCl
– Remove hydrocarbons by soaking in
trichlorethane
• Drying:
– Wet samples must be oven- or freeze-dried
before introduction into the instrument. Be
aware that temperatures of over 50oC can
remove structural water from clay minerals.
– Rock samples that have been cut using a
saw should be thoroughly dried prior to
mounting.
Initial Preparation
• Cutting
– Large samples must be cut to thin-section size, or cut or
broken to a appropriate size for an SEM stub. Typically,
diamond-blade circular saw is used. Friable samples may
need to first be impregnated.
• Impregnating
– Friable or porous samples should be vacuum impregnated
prior to sample prep. Loose material on the sample surface
can cause contamination of the electron column, and
unimpregnated porous samples can cause poor vacuum by
prolonged outgassing.
• Cast-making
– SEM visualization of pore structure can be facilitated by
vacuum impregnating a sample, followed by dissolution of the
sample material using hydrofluoric acid. Topographic details
of small fossils can also be facilitated by producing latex cast
Mounting
• SEM Stubs:
– SEM samples can be mounted onto Al or C stubs. C can be used for
low X-ray background in the case of particulate analysis. Mounting can
be done using epoxy, quick-setting glue, double-stick tape (mostly for
particulates) or wax. Small grains can be mounted onto double-stick
tape or partially dried carbon or silver paint.
Polishing
•
Polishing a sample to a flat, unscratched surface is CRITICAL for good
quantitative analysis. An uneven, or scratched sample surface can
lead to uneven production of x-rays from the sample surface, errors in
absorbtion correction, and spurious results
.
Cleaning
•
Any contaminating material, particularly skin oil, on the sample surface
will end up contaminating the column. Also, contaminating material
under the conductive coating of a sample can cause the conductive
coating that will be placed on the sample to bubble and crack, making
the sample impossible to analyze
•
KEEP THE SAMPLE CLEAN AND OIL FREE!!!
•
1. After final polish, clean the sample ultrasonically with deionized water
•
2. Wipe the sample surface with petroleum ether.
•
3. Following the petroleum ether cleaning, handle the sample as carefully
as possible. Glove handling is ideal, but often inconvenient. An
alternative is to handle the sample with a kimwipe, or other lint-free papr
or cloth.
•
4. Blow the sample off with air prior to coating.
Mapping
Finding analysis areas on a sample using the optical microscope or electron imaging
can be difficult because of the small field of view. The maximum optical field of view
for our instrument is 1750 microns (1.75 mm), and the maximum electron image field
is around 2500 microns (2 mm). So, having a good sample map, particularly for
quantitative analysis, is very important. Sample mapping is much less important for
SEM work.
Two types of map may be useful, depending on you sample.
1. Macro-map. Map of entire section. Depending on sample type, this may be
hand-sketched, made with a slide copier, or with an enlarging xerox machine.
2. Micro-map. Map of areas of analytical interest. This needs to be produced with
a camera-equipped microscope, and can be used to locate and document exact
analytical spots. A micromap can also be produced using the microprobe.
You can also mark areas of interest on your sample, but this must be done on a
clean sample surface prior to applying the conductive coat.
Sample Coating
Many geological samples are nonconductors of electricity. Therefore, if an uncoated
sample is placed in the path of the electron beam, the sample will charge, causing
deviation of the electron beam, as well as catastrophic decharging. The sample
must be coated with conductive material, such as carbon, gold, or gold-palladium
alloy.
CARBON:
For quantitative analysis and X-ray mapping, carbon is the coat of choice. Because
of its low Z, it has a minimal effect on the X-ray spectrum, either in terms of
producing X-ray lines or absorbing X-rays. A carbon coat is applied using a vacuum
evaporator at pressures of less than 10-4 torr. The ideal coating thickness is 20 nm.
GOLD:
Gold can be a better choice for SEM coating because of its higher secondary
electron yield.