Nano-instrumentation

Resolution
The resolution of an optical microscope is defined as the shortest distance between two points on a
specimen that can still be distinguished by the observer or camera system as separate entities

Unresolved

Resolved
 Numerical Aperture

The numerical aperture of a microscope objective is a measure of its ability to resolve fine
specimen detail.

The value for the numerical aperture is given by,

Numerical Aperture (NA) = n sin α

where n is the refractive index and equal to 1 for air and α is the half angle subtended by rays entering the
objective lens.

Numerical aperture determines the resolving power of an objective, the higher the numerical aperture of
the system, the better the resolution.
 Wavelength and resolution

The relationship between numerical aperture, wavelength, and resolution:

Resolution (r) = λ/(2NA)

NA is a general term for the microscope numerical aperture,

λ is the imaging wavelength
 Wavelength and Resolution

Louis de Broglie

where λ is the wavelength of a particle, h is Planck’s constant

(6.626 x 10-34 J seconds), and p is the momentum of a particle

The velocity of the electrons is determined by the accelerating voltage, or electron potential where

Mass of electron = 9.1×10−31 kg

Electric charge = 1.6×10−19 C
Unit of V (J/C)
 Resolution of electron microscope

100 kV λ = 1.23 x 10-9 / V1/2 m

λ = 1.23 x 10-2 nm

Resolution (r) = λ / (2NA)

Resolution (r) = λ / 2nsinα

angle α is usually very small, for example 10-2 radians

sinα = 0.0099 ≈ 9.9 x 10-3

Resolution (r) = 1.23 x 10-2 nm / 2 x 1 x 9.9 x 10-3 = 0.62 nm

 History

➢EM constructed in 1931

➢Von Ardenne first EM in 1938 by rastering the electron beam in a EM
➢Zworykin et al. 1942, first EM for bulk samples
➢1965 first commercial EM by Cambridge Scientific Instruments

Resolution at that time ~ 50 nm : Today < 1 nm

Morphology only at that time : Today analytical instrument

 Type of Electron Microscope

1. Scanning Electron Microscope (SEM)

2. Transmission Electron Microscope (TEM)

➢All the measurements need to be done in vacuum

 What is SEM?
➢ It is a microscope that produces an image by using an electron beam that scans the surface of a
specimen inside a vacuum chamber.

➢ Voltage use in SEM : 1 – 50 kV

What can we study in a SEM?

• Topography and morphology (How the surface look like)
• Topography : The surface features of an object and its texture
• Morphology : The shape and size of the particles making up the object
• IC and c
• Crystallography
• Orientation of grains
• In-situ experiments
• Reactions with atmosphere
• Effects of temperature
 What does it looks like….

AFM Cantilever Tip Ant Head Blood Cells

Diamond Thin Film Microstructure of a plain carbon Calcium Phosphate

(Numerous Multifaceted Micro- steel that contains 0.44 wt% of Crystal
crystals) carbon
The instrument in brief
 HOW THE SEM WORKS?
• The SEM uses electrons instead of light to
form an image.

• A beam of electrons is produced at the top

of the microscope by heating of a metallic
filament.

• The electron beam follows a vertical path

through the column of the microscope. It
makes its way through electromagnetic
lenses which focus and direct the beam
down towards the sample.

• Once it hits the sample, other electrons (

backscattered or secondary ) are ejected
from the sample. Detectors collect the
secondary or backscattered electrons, and
convert them to a signal that is sent to a
viewing screen similar to the one in an
ordinary television, producing an image.
 Electron beam Source
LaB6

W or LaB6 Filament
Thermionic or Field Emission Gun
 Thermionic electron gun

• A tungsten filament heated by

DC to approximately 2700K or
LaB6 rod heated to around
2000K
• A vacuum of 10-3 Pa (10-4 Pa
for LaB6) is needed to prevent
oxidation of the filament
• Electrons “boil off” from the tip
of the filament
• Electrons are accelerated by
an acceleration voltage of 1-
50kV
 The Condenser Lens

• For a thermionic gun, the diameter of the

first cross-over point ~20-50µm
• If we want to focus the beam to a size < 10
nm on the specimen surface, the
magnification should be ~1/5000, which is
not easily attained with one lens (say, the
objective lens) only.
• Therefore, condenser lenses are added to
demagnify the cross-over points.
 The Objective Lens - Focusing

• By changing the Objective

current in the lens
objective lens, the
magnetic field
strength changes
and therefore the
focal length of the
objective lens is
changed.

Out of focus in focus out of focus

lens current lens current lens current
too strong optimized too weak
 traditional detectors
Backscattered electron
detector:
(Solid-State Detector)

Secondary electron detector:

(Everhart-Thornley)

 Secondary electrons: Everhart-Thornley Detector

 Backscattered electrons: Solid State Detector
 X-rays: Energy dispersive spectrometer (EDS)
How do we get an image?
Electron gun
156
288 electrons!
electrons!

Detector

Image
 Where does the signals come from?

Incoming electrons
Secondary electrons
Auger electrons
Backscattered Cathodo-
electrons luminescence (light)

X-rays

Sample
 Secondary electrons (SE)
 Generated from the collision between the
incoming electrons and the loosely bonded outer
electrons
 Only electron generated close to surface escape
(topographic information is obtained)
 Charging in SEM

if a specimen is nonconductive, the

electrons stop in the specimen. That is
the charging

If the charging occurs, the electron probe that scans over the specimen
is deflected by the repulsive force from a charged potential, resulting in
a positional shift of the electron probe.
Distortion of a SEM image caused by the charging Anomalous contrast due to the charging
 To Prevent Charging

❖ Coating This method coats a nonconductive specimen with a highly

conductive thin metal film.
A thin film with a thickness ranging from a few to 10 nm of a noble
metal (for example, Au, Pt, Au-Pd, Pt-Pd) is coated on the specimen

❖ Low accelerating-voltage observation

As the accelerating voltage of the incident

electron beam is decreased, the
secondary-electron yield is increased
Example of use of SEM
What is a Transmission Electron Microscope?

electron source

condenser
system

specimen (thin)

objective lens

projector lens

Voltage in TEM: 80 – 300 kV

Beam and Specimen Interaction
3mm
 Comparing SEM and TEM

TEM SEM
Beam focused to fine point;
Electron Beam Broad, static beams
sample is scanned line by line
Accelerating voltage much lower (5
TEM voltage ranges from
Voltages Needed to 10 kV); not necessary to penetrate
60-300,000 volts
the specimen
Wide range of specimens allowed;
Interaction of the
Specimen must be very thin simplifies
beam electrons
sample preparation
Information needed is
Electrons must pass through and
Imaging collected near the surface
be transmitted by the specimen
of the specimen

Transmitted electrons are

Image Rendering Beam is scanned along the surface of
collectively focused by the
the sample to
objective lens and magnified to
build up the image
create a real image

Resolution
Usually in angstrom scale Subnanometer scale
Bacteriophage morphology Hepatocytes (HC) in the mouse liver
 Specimen Preparation for TEM
Overview
➢ TEM specimens must be:

• Very thin
• Electron dense
• Stable in the vacuum
ie; everything that most biological samples are not!

➢ The degree of specimen preparation for biological TEM depends on the

specimen

• Particulate samples (eg: protein, DNA and viruses) can be stained and viewed quickly
• Cells and tissue samples require extensive preparation for TEM
 Particulate samples
Negative Staining:

• Apply the particulate specimen on the grid

• Stain with heavy metal solution, eg: uranyl acetate,
phosphotungstic acid, sodium silicatungstate
• Blot dry and view in the TEM
Cells and tissues sample preparation
Fixation
A process used to preserve the structure of freshly killed material in a state that most closely resembles the structure and/or
composition of the original living state.

Chemically fixing biological samples

•Pieces should be small to allow rapid and complete penetration of the fixative.
•The volume of fixative should exceed the volume of tissue by factor of 10
eg. - plants - small pieces may be excised and dropped directly in fixative. Very
small plants and algae may be fixed whole
• Insects, other invertebrates can be dropped into the fix
• Larger tissues should be perfused with the fixative

TEM images of kidney tubules

Typical fixatives - aldehydes

Formalin
▪ Low MW - makes it one of the best penetrating of all the fixatives, widely used in
fixation of resistant materials, such as seeds, spores, plant material
▪ Formalin contains many impurities, so formaldehyde for use in EM, prepared from
paraformaldehyde

Glutaraldehyde
▪ Glutaric acid dialdehyde, a 5 carbon dialdehyde, is the most widely applied
fixative in both scanning and transmission electron microscopy.
▪ Most highly cross-linking of all the aldehydes. Glutaraldehyde fixation is
irreversible.

Action of aldehydes on proteins

• causes the cross-linking of proteins. Amine and amide
groups react with the fixing agent
Other fixative (and membrane marker) - Osmium Tetroxide

• Non-polar tetrahedral molecule MW 254, solubility in water and a variety of organic

compounds
• Mode of action
▪ Preferentially stain unsaturated fatty acids by reacting with double bonds
▪ React with thiol groups of proteins, causing major conformational changes in the
structure of proteins
• Very poor rate of penetration
• Vapors rapidly fix exposed mucous membranes such as those in the eyes (eventually
causing blindness) and respiratory tract (lung edema).
 Dehydration & resin infiltration

Embedding in resin

Epoxy resin - araladite

Acrylic resin - methyl methycrylate
Ultramicrotomy
Immunocytochemistry

Immunocytochemistry is a means of localizing particular molecular complexes (antigens) antigens within tissue and cells using
antibodies
Schematic drawing of a single mitochondria
Atomic Force Microscopy (AFM)
Atomic Force Microscope
 Atomic Force Microscope

➢ In atomic force microscope, a sharp tip is

attached to the extremity of a flexible cantilever
➢ The tip interacts with the sample surface while the
sample is scanned under the tip
➢ The interaction forces between the tip and the
sample cause the bending of the cantilever
➢ When the sample is scanned under the tip, the
tiny movements of the cantilever are detected by
an optical lever system
➢ This produces a spot of light whose position is
measured, and they are used as the basis to
reconstruct a pseudo-3D image of the sample
surface
The Lennard-Jones potential Repulsion:
At very small tip-sample distances (a
few angstroms) a very strong repulsive
force appears between the tip and
sample atoms. Its origin is the so-called
exchange interactions due to the
overlap of the electronic orbitals at
atomic distances.

Attraction (Van der Waals):

A polarization interaction between
atoms: An instantaneous polarization of
an atom induces a polarization in
nearby atoms – and therefore an
attractive interaction.
 Imaging mode

❖ Contact mode
❖ Non contact mode
 Contact mode

Laser

Detector Z
– tip in continuous contact with sample
– preferably used for hard samples
– imaging in air and liquid
Tip Cantilever
– high resolution X,Y

Feedback: Deflection of cantilever

Biological samples are challenging to study in contact mode because they are generally soft, weakly bound to the surface, and
damaged easily.
 Tapping Mode AFM

Detector
Piezoelectric material
drives oscillations

10-100nm

Cantilever oscillates can be driven

at a resonant frequency ~10-500 KHz
The surface acts to damp the resonance

Tapping mode is the most used operating mode in biology

 Resolution in AFM

Tip
trace

Image quality depends on tip size and shape

contact point
Imaging of isolated molecules in air

Endothelial cells

Cardiac Cells
 Advantages and Disadvantages
Advantages

➢ Image resolution limited by probe-sample interaction volume - not by diffraction

➢ Unlike the electron microscope, which provides a two-dimensional projection or a
two-dimensional image of a sample, the AFM provides a three-dimensional surface
profile.
➢ Only technique to allow measurement of height in nanodimensions
➢ Samples viewed by AFM do not require any special treatments (such as
metal/carbon coatings) that would irreversibly change or damage the sample, and
does not typically suffer from charging artifacts in the final image.
➢ While an electron microscope needs an expensive vacuum environment for proper
operation, most AFM modes can work perfectly well in ambient air or even a liquid
environment
➢ Interaction can modify surface – nanolithography possible

Disadvantages
➢ Scanning technique quite slow

➢ Limited maximum image size.