INTRODUCTION TO

MICROSCOPY

Manoj Chopkar
Assistant Professor
Department of Metallurgical Engineering, NIT Raipur
 Components of Microscope

Light source: light or electron

Condenser lens

Objective lens

Projector lens

Suitably prepared specimen

Image formation in reflected illumination
Image formation in transmission illumination
 Condenser Lens

 Collects light to direct it at the small area of the

object which is to be examined. It makes the object
brighter (better contrast) and enables to control
the angle at which the illumination reaches the
object.

 The condenser lens can converge the light beam on

object or can illuminate it with parallel rays.

 Condenser aperture: controls the area of specimen

to be illuminated
 Magnification
Objective lens provides an inverted image of object at B
with magnification,

M1 = (v1–f1)/f1

Projector lens: the lens gives a final upright image at a

further magnification,

M2 = (v2–f2)/f2

image distance (v) and focal length (f)

The total magnification: M = M1x M2

Comparison of OM, TEM and SEM
Transmission Electron Microscope

Similar to an optical instrument in that lenses are

used to form images

Scanning Electron Microscope

Not similar to an optical instrument (no image

forming lens) but uses electron optics to form a
fine probe on the specimen and the signals emitted
are detected and characterised.
 Resolution Limit
Abbe (Diffraction) limit:
The minimum resolvable distance between the image
of two point objects using a perfect lens; in any
magnifying system, a point object (i.e. zero dia) can
not be imaged a point but as a distribution of intensity
having a finite width:


 sin( )

 wavelength of the imaging radiation
 refractive index of the lens
 illumination semi angle
 Resolution
The ability to discern fine details. It is represented by
the minimum distance between two points such that
the two points are perceived as separated image

ρ = 0.61/N.A.

N.A. =  sin , Numerical

aperture
λWavelength of light
 - the half acceptance
angle of the lens.
Minimum distance between two diffraction
maxima still projected separately
 Resolution limit of a light microscope
 can decrease to 400 nm (green light)
µ sin  is limited to ~ 1.6
Thus R. P. =  0.61/N.A. = 0.61x 400/1.6 = 152 nm

The resolution is about 150 nm (0.15 µm)

Resolution limit of an electron microscope
can be as small as 0.001 nm; µ sin  is very small,
because µ is unity and  is about a degree.
0.61
R. P . 

For wavelength of 0.0037 nm and  = 0.1 radians,
the resolution is about 0.02 nm
 Important Features for Imaging
Wave length  of the electrons accelerated by a
potential of V volts is given by the expression,

h

2meV
h is Planck’s constant (6.626x10-34 Js), m is electronic
mass (9.11x10-31kg) and e is electronic charge
(1.602x10-19C).
 Important Features for Imaging (contd.)

For large values of V, electrons can attain velocities

comparable with the speed of light and relativistic
increase in mass should be taken into account.

This is done by replacing V by Vc (relativistic

accelerating voltage)

Vc = V[1 + eV/2m0c2]

where c is the velocity of light (2.998x108 m/s). For 100

kV electrons, wavelength is 0.04Å (without relativistic
correction) and 0.037Å with correction.
Variation of wavelength with applied voltage
Applied voltage, keV Wave length, nm

20 0.0086

50 0.0054

80 0.0042

100 0.0037

200 0.0025

500 0.0014

1000 0.00087
 Lens Aberrations

 Spherical aberration

 Chromatic aberration

 Astigmatism
 Spherical aberration
 Focussing action of outer regions of lens is more
than that of the regions close to the optic axis.
 Point in the optic axis is imaged as a disc in the
image plane.
 Radius of the disc of least confusion, Δrs, is given
by
Δrs = Csβ3
Cs is the spherical aberration coefficient (typically
1 – 5 mm) and β is the semi-aperture angle.
Note that even for a small value of β (~10-2),
Δrs ~ 1nm.
 Chromatic Aberration
 Arises from variations in accelerating voltage or
lens current and the consequent energy spread of
the electrons.
 Δrc, radius of the disc of least confusion, is given by
Δrc = Ccβ(ΔE/E).
 Typical values of Cc is 1-4 mm.
 Electrons from W filament have a spread of energy
of 0 – 3 eV and HT and lens current stability are ~
10-6/min.
 Electron energy loss from inelastic scattering (20-50
eV) would be the resolution limiting factor.
 Astigmatism
 A point in the object being imaged as a disc due
to differing focal lengths depending on the
plane of the ray paths.
 Radius of the disc of least confusion, ΔrA, is
given by
ΔrA = ΔfAβ,
where ΔfA is the maximum difference arising
from astigmatism.
 Aberration corrected by the use of
electromagnetic stigmators.
Scanning electron micrograph of magnetic tape (a) no
astigmatism, (b) & (c ) images with astigmatism,
(b) is overfocused & (c ) is underfocused
 Resolution of the Microscope

Resolution is given by the expression,

Δrdiff = (0.61λ/μsinβ) = (0.61λ/β),
since μ is 1 and β is small.

Compromise angular aperture size with diffraction

and spherical aberration into account is
β ~ 0.88 (λ/Cs)1/4.
Corresponding value for resolution Δr, is given by
Δr = 0.69λ3/4Cs1/4.
 Light VS Electrons

Light Microscope Electron Microscope

 = 0.5µm ~
150
 0.055 A (@ 50kV )
V
0

 = 1.5 (glass) = 1.0 (Vacuum)

 = 70°  = 1°
ρ = 0.2µm=2000  ρ = 0.00016µm
= 0.16 nm
 Depth of Field
The distance parallel to the optical axis of the
microscope that a feature on the specimen can be
displaced without loss of resolution
Optical Microscope Electron Microscope

 wavelength
 refractive index
 semi angle
M total magnification
NA numerical aperture of the lens
 Depth of
Field
Magnification Depth of field
Optical SEM
10 60µm 1000µm
100 8µm 100µm
1000 0.2µm 10µm
10000 -- 1µm
Micrographs of blood
corpuscles
(a) optical, (b) scanning
Dependence of
depth of focus
on probe diameter
and magnification
for SEM and OM
 Comparison of Optical and Scanning
Electron Microscopes

Characteristic SEM Optical

Illumination Electron beam Light beam
Wavelength ~ 0.05A 2000-7000A
Lens Electrostatic Optical
lens for demag lenses for
crossover and magnifi-
electromag lens cation
for
magnification
 Comparison of Optical and Scanning
Electron Microscopes (contd.)

Characteris- SEM Optical

tic
Resolution ~ 50 Å Visible region
2000Å UV
region 1000 Å
Depth of 30 m at ~ 0.1m
field 1000 X
Magnifi- Continuously 10X to 2000X
cation variable from
10X to
200,000X
 Comparison of Optical and Scanning
Electron Microscopes (contd.)

Characteris SEM Optical

-tic
Focusing Electrical Mechanical

Obtainable Several Transmitted and

images reflected
Contrast Shape and Colour and
chemical property brightness
controlled controlled
 IMPORTANT INSTRUMENTAL
FEATURES

Manoj Chopkar
Assistant professor
Department of Metallurgical Engineering, NIT Raipur
 Basic Electron Optics

 Electrons and ions are charged particles; they can be

accelerated in an electric field

 The trajectory of an accelerated charged particle can be

changed (deflected) by E and/ or B field.

 The accelerated particles also behave like waves

(de Broglie)
Schematic of TEM

Condenser lens: control beam

intensity, density, convergence

Objective lens: magnification,

introducing contrast

Intermediate lens: further

magnification, imaging or
diffraction

Projector lens: final

magnification
Schematic
of SEM
 Scanning electron microscope

 A scanning electron microscope is similar to a light

microscope being used in reflection.

 The major difference is that instead of imaging the

entire specimen at once, the electron beam is
scanned back and forth over the specimen, imaging
only one point at a time (much like how a television
works--there is only one electron beam, but it scans
every spot on the screen).

 The interactions of the electrons with the surface

are registered, and from this data an image can be
constructed.
Rastering
Magnification in the SEM
Scanning electron microscope
1. Electron gun
2. Electro-magnetic lenses to direct and focus the
electron beam inside the column
3. Vacuum pumps system
4. Opening to insert the object into the high-
vacuum observation chamber in conventional
SEM mode.
5. Operation panel with focus, alignment and
magnification tools and a joystick for positioning
of the sample.
6. Screen for menu and image display.
7. Cryo-unit to prepare (break, coat and sublimate)
frozen material before insertion in the
observation chamber in Cryo-SEM mode.
8. Electronics stored in cupboards under the desk
Schematic of SEM
Close-up of region near specimen chamber
Electron optical elements &attachments

 Electron source

 Lenses

 Deflection coils

 Stigmators

 Electron detectors

 Photon/X-ray detectors
 Electron
Source
Generation of electrons that can be accelerated by
high tension to obtain the illuminating electron beam.

Thermionic gun:

 triode or self-biasing gun,

 W, LaB6, CeB6

Field Emission Gun:

 Single crystal W
 Electron Sources

Thermionic Emitters

Field Emitters
 Thermionic Electron Gun

 Electrons are emitted from a heated tungsten

filament and then accelerated towards an
anode; a divergent beam of electrons emerges
from the anode hole.

 Commonly used electron source

 Robust, cheap and does not require relatively

high vacuum
Schematic of thermionic electron gun
 W

LaB6
Relationship between beam brightness and filament
heating current in a W-cathode self-biasing gun
 (a) (b)
Scanning electron micrograph of W filament: (a) unused,
(b) failed
Potential variation at the gun and beam configuration
 Utilizes a tungsten coil wrapped around the pointed end
of a long (around 2 cm) LaB6 rod
 Heat radiation and electron bombardment from the
tungsten coil heat up the tip end
 Conduction of heat through the structural mount helps to
minimise reactivity of LaB6.
 A short LaB6 rod heated directly by passing
current, perpendicular to the length of the rod
using rigid electrical connectors that also provide
the support for the rod.
 Pyrolytic graphite is used in between the
conductors and the rod to avoid the problems of
chemical reactivity of LaB6 and the conductors.
 A short LaB6 rod is supported by a ribbon or strip
through which an electrical current is passed for heating.
The rod is heated by conduction from the ribbon.
 The ribbon material is chosen to be chemically inactive
with the LaB6, such as graphite or tantalum
Electron Sources

Similar in design to a
tungsten filament
LaB6 Emitters
 Thermionic Gun Electron Source

Energy Spread:

filament imperfections
High tension instability
Surface temperature
Boersch effect (mutual interaction)

Source spot size

30 μm for W
5 μm for LaB6
 Field Emission Gun

 Very strong electrical fields (109V/m) used to

extract electrons from a metal filament.

 Temperatures are lower than those required

for thermionic emission

 Requires very good vacuum

Electron Sources

Field Emitter

Single oriented
crystal of tungsten
etched to a fine tip
 Electron Sources

Field Emitter

Single oriented
crystal of tungsten
etched to a fine tip

The emission of electrons that are stripped

from parent atoms by a high electric field
Electron Sources

A Field Emission
tip can be “cold” or
thermally assisted to
help overcome the
work function but
a high voltage
field of 3 KeV
is needed
 The FE tip is generally made of a single crystal
tungsten wire sharpened by electrolytic etching;
tip diameter of 100 to 1000 Å is used, with the
apparent source size much less than that.

 A simple tungsten tip can be very sensitive to

surface contamination. More than any other
cathode design, the field emission tip is extremely
sensitive to the size, shape and surface condition.

 The electrostatic anodes are also very susceptible

to contamination.
 The emission process itself depends on the
work function of the metal, which can be
affected by adsorbed gases. This is the reason
a very high vacuum is required.

 Sustaining high electrical field gradients is also

essential to emission, so a tip that is well worn
might not emit electrons at all.
 As with the tungsten filament gun, the
voltage difference or bias between the first
anode and the accelerating voltage on the
cathode determines the emission current.

 The second anode is at ground potential and

the voltage difference from here to the
cathode determines the acceleration given
the electrons.

 The shape of the anodes is carefully selected

to minimize aberrations.
 Electron Sources

Work Function

Energy (or work) required to

withdraw an electron
completely from a metal
surface. This energy is a
measure of how tightly a
particular metal holds its electrons
Electron Sources
Filament Current
(Heating Current)
Current running
through the emitter

Beam Current
Current generated
by the emitter
 Comparison of Electron
Sources
W LaB6 FEG

Max. current, nA 1000 500 300

Normalised 1 10-30 2500
brightness
Energy spread, eV 3-4 1.5-3.0 0.6-1.2

Source spot size 30-100 5-50 μm 15-30 nm

μm
 Comparison of Electron Sources (contd.)

W LaB6 FEG

Vacuum required, 10-3 10-5 10-7

Pa
Temperature, K 2700 2000 1800

Life time, h ~100 1000 >2000

Normalised price 1 10 100

Probe Current vs probe diameter for electron sources
Electromagnetic lenses
 Common Modes of Operation of TEM

 Bright Field (BF) Microscopy

 Selected Area Diffraction

 Dark Field (DF)

 Weak Beam (Special case of DF)

Formation of Diffraction
Formation Spots
of Diffraction from
Pattern
Different Planes
 (a) (b)

Formation of (a) Bright Field (BF) and

(b) Dark Field (DF) Images
Imaging in (a) BF mode, (b) DF mode with shift of
objective aperture and (c) DF mode with beam tiltin
Ray Diagram and Ewald Sphere Construction for (a) 2 Beam
BF, (b) High Resolution 2 Beam DF and (c) Weak Beam DF.
 Image disturbance and their causes

Lack of sharpness
 Choice of improper voltage
 Instability of gun emission
 Improper electron probe diameter
 Improper objective aperture setting/centering
 Insufficient astigmatism correction
 Improper focal depth
 Too high magnification
 Specimen charge-up and magnetization
 Defocus of camera system
 Image disturbance and their causes (contd.)

Poor Image Quality

 Improper choice of accelerating voltage
 Improper probe current setting
 Incorrect astigmatism correction
 Noise due to excessive PMT gain
 Improper contrast and brightness
 Improper specimen preparation
 Improper photographic material
Improper positioning between specimen and detector
No specimen tilt
Image disturbance and their causes (contd.)

Noise

 Instability of accelerating voltage and gun

emission
 Discharge of detector
 Charge-up of specimen surface
 Burnt CRT or dust on CRT screen
 Stray external magnetic field
 Mechanical vibrations
Image disturbance and their causes (contd.)

Image distortion and deformation

Specimen charge-up
Stray external magnetic field
Electron beam damage
Deformation of specimen durign rpeparation
Image drift caused by column interior charge-up
Specimen drift on heating and cooling stages
Effect of accelerating voltage
 Apertures

 Apertures in each lens limit the number of

electrons striking the specimen (protecting it from
excessive radiation) and limit the X-rays generated
from the specimen hitting parts of the microscope
column

 A thin (< 100 um thick) disc or strip of metal

(normally Pt) with a small (2 – 100 um) circular
through-hole.
 Apertures (contd.)

 Probe size and semi-angular aperture of

illumination determined by C1 and C2 apertures.

 Objective aperture is used to form bright field, dark

field or high resolution images

 Selector area aperture is used to define the area from

which the diffraction pattern is obtained.
 Electron vs optical lenses

Electrons do not touch the lens

Electrons rotate in the magnetic field
Electrons repel one another
Focus and magnification electronically controlled;
no physical movements
Electron lenses are only converging
Electron lens aberrations can only be minimised
Electrons operate at small apertures
Ray diagram showing the imaging process at the objective
Lens in a TEM
a) BF, (b) DF and (c) high resolution mode
(axial illumination) of imaging
 TEM sample preparation

Bulk ceramics
Mechanical grinding, polishing, dimpling
Ion erosion, focused ion thinning

Metals
Mechanical grinding, polishing
Electrolytic thinning

Organic Materials
Freeze drying, embedding,
ultramocrotomy
 Preparation of powder samples
Small particles:
Direct transfer to carbon film mounted on Cu grid
Ultrasonic dispersion of suspension on C/Cu
Membrane filtration and subsequent dissolution
of the filter
Large particles:
Embedding in resin (phenyl formaldehyde)

La2RuO5 particles
In resin
Powder Samples