Microscopy

Three branches of Microscopy

Optical
Electron
Scanning Probe
Optical and Electron microscopy measure refraction, diffraction, and
reflection of the source radiation
Optical uses white light, fluorescent light, or lasers
Electron uses electromagnetic radiation/electron beams
Scanning uses a physical probe to interact with the surface of the
specimen
Imaging Techniques

Technique Image Formed By Lowest Resolvable Unit Approx Lower Limit

1 m
Optical Microscopy Light Rays Microns (m)
(monochromatic light)

.1 m
Confocal Microscopy Coherent Light Source (Laser) Microns (m)
(X-Y Direction)

Transmission
Electron Microscopy (TEM) 2
Electrons Angstroms ()
(high resolution TEM)

Scanning Electron Nanometers (nm) to 10 nm

Electrons (100 )
Microscopy (SEM) Angstroms ()

Atomic Force & Scanning

Tunneling Microscopies
40
(AFM/STM) Molecular Mechanical Probes Angstroms ()
(theoretical)
Units of Measure
m - Micrometer
1,000,000 micrometers = 1 meter
Strand of hair has a diameter of ~ 20-180 m
106
nm - Nanometer
1,000,000,000 nanometers = 1 meter
109
Wavelength of visible light (400-700 nm)
- Angstrom
10,000,000,000 Angstroms = 1 meter
1010
Used to measure the size of atoms/bond lengths
Length of a C-H bond in methane is ~1 Angstrom
Optical Microscopy
Properties of light
Reflection
Refraction
Numerical Aperture
Refraction
Change in the direction of a
wave (light) due to a change in
speed
The straw in the picture looks
bent because the light is bending
as it moves from the water to the
air
Refractive Index (RI)

RI of a material a measure of the speed of

light in material
RI is the ratio of the velocity of light in a
vacuum to the speed of light in the
specified material
Incident angle (1) is related to the
refraction angle (2) by Snells Law
n1sin(1)=n2sin(2)
Used in calculating focusing power of
lenses and dispersion properties of prisms
Reflection
Reflection is defined as a change
in direction of a wave at an
interface between 2 different
media so that the waveform
returns to the media from which
it came
Used in focusing light waves to
increase transmitted light
 Numerical Aperture
NA of a microscope objective is a measure of its
ability to gather light
The more light (higher NA) the better the resolving
power of the lens
Better resolution
NA = (n)sin()
n = Refractive Index
= the maximum cone of light than can enter
the lens
Usually the NA of an objective increases with its
magnifying power.
The smallest detail that can be resolved is
proportional to:
/NA
The Lens
Incident Ray Parallel

Refracted Ray

Transmitted
Notice that if the slab of
ray
glass is this, the incident
and transmitted rays are
almost the continuation
of each other.
Some Rays

Kirkpatrick/Francis Physics; A World View

Simple Lens
SPHERICAL SURFACE

Kirkpatrick/Francis Physics; A World View

Optical Microscope
1. Ocular lens
2. Objective turret
3. Objective
4. Coarse Adjustment
5. Fine Adjustment
6. Stage
7. Light source
8. Condenser
9. X-Y Control
 OPTICAL
MICROSCOPES

SCANNING ELECTRON
MICROSCOPES
 The object (O) is placed just outside Fo, the
Optical microscope principal focus of the objective lens.

Fe is the principal focus of the eye lens.

A real, inverted magnified image I1 is

formed. The magnified image I1 acts as an
object for the eye lens.

The final image I2 is virtual and is

magnified still further.

It is inverted compared with the object.

I2 may appear 1000 times larger than the
object
 Microscopy
It is used to create an enlarged view of an object such that we can observe details not otherwise possible with the human
eye.

Because of the enlargement, resolution is often confused with magnification, which refers to the size of an image.

Main function of a microscope:

Resolution enhancement
 Parts of an optical microscope

1. Objective lens
The objective lens of a compound microscope is a convex lens of very short focal length (fo<1 cm). The object to be seen
is kept very close to the objective lens.

2. Eye piece
The eye piece of a compound microscope is also a convex lens of short focal length fe. ( fe > fo).

3. Microscope tube
The objective lens and the eyepiece are mounted coaxially (having a common axis) at the ends of two brass tubes which
can be made to slide into each other so that the distance between the two lenses can be adjusted.

.
Optical microscope
The object (O) is placed just outside Fo, the
principal focus of the objective lens.

Fe is the principal focus of the eye lens.

A real, inverted magnified image I1 is

formed. The magnified image I1 acts as an
object for the eye lens.

The final image I2 is virtual and is

magnified still further.

It is inverted compared with the object.

I2 may appear 1000 times larger than the
object
 Characteristics of image formed by the objective lens
The objective lens forms a
Real
Inverted
Magnified image (I1) of the object.

The image I1 acts as an object for the eye piece.

The position of the eyepiece is so adjusted that the image lies within the focus of the eyepiece (Fe).

The eyepiece acts like a magnifying glass and forms a virtual erect and magnified image of the object.

Limitation of an optical microscope

Resolution is limited by the formula
d= l/2 AN
AN = Numerical aperture of the lens

Numerical aperture of a microscope objective is a measure of the ability to gather light and resolve finer details

The size of the smallest feature which we can distinguish under the microscope is of the order of wavelength
Electron Microscopy-Scanning electron Microscope (SEM)

An electron microscope is a microscope that uses a beam of

accelerated electrons as a source of illumination.

Because the wavelength of an electron can be up to 100,000

times shorter than that of visible light photons, the electron
microscope has a higher resolving power than a light
microscope and can reveal the structure of smaller objects
Working Principle of SEM
Interaction of sample with electron beam
Different modes of image formation in SEM
 Secondary electron imaging:

If the beam travels into a depression or hole in the sample, the amount of secondary electrons that can escape the
sample surface is reduced and the image processing places a corresponding dark spot on the screen. Conversely, if
the incident beam scans across a projection or hill on the sample, more secondary electrons can escape the sample
surface, and the image processing places a bright spot on the screen.

Backscattered electron imaging:

In backscattered imaging mode, as the incident beam scans across the sample's surface topography, backscattered
electrons are emitted from the sample. A low atomic weight area of the sample will not emit as many backscattered
electrons as a high atomic weight area of the sample. In reality, the image is mapping out the density of the sample
surface.
 Characteristics that could be viewed by SEM

Topology: The surface features of the object or how it looks and its texture.

Morphology: The shape and size of the particle making up the object.

Composition: The elements and compounds that the object is composed of and the relative amount
of them.
SEM images
 Transmission Electron Microscope (TEM)
Steps of image formation in TEM

A beam of electrons hits a very thin sample (usually no more than 100 nm thick).
The electrons are transmitted through the sample .
After the sample, the electrons hit a fluorescence screen that forms an image with the electrons that were
transmitted.
Parts of a Transmission Electron Microscope
 Working of a Transmission electron microscope

TEM works by emitting electrons from a cathode, then accelerating them through an anode.
The electrons pass through an aperture into the vacuum tube.

As it passes down through the tube the electron beam is controlled by electromagnetic lenses formed by
coils around the tube (whose effect is moderated by adjusting the electricity flowing through the coils).

These electromagnetic lenses direct the electron beam through the centre of the tube to a very
thin specimen located part-way down the tube.

Formation of an image of the specimen is possible because the electrons in the beam are affected by
different regions of the specimen in different ways:

Some parts of the specimen might allow electrons to pass through unaffected.

Other regions within the specimen may absorb some or all of the electrons that reach them
 Some tiny regions of the specimen may deviate the path of the electrons forming the beam so that the
electrons "bounce" off those precise positions or areas within the specimen in a range of directions

They eventually form an image when they reach an image plane where they are detected by a suitable sensitive material
such as a fluorescent film. The image produced is a greyscale image.

The darker areas represent regions of greater absorption of electrons by the specimen while lighter areas correspond
to parts of the specimen that absorbed fewer, if any, electrons
 Scanning Tunneling Microscope (STM)

When a conducting tip is brought very near to the surface to be examined, a bias (voltage difference) applied between the
two can allow electrons to tunnel through the vacuum between them.
The resulting tunneling current is a function of tip position, applied voltage, and the local density of states (LDOS) of
the sample.
Information is acquired by monitoring the current as the tip's position scans across the surface, and is usually displayed in
image form.

As the tunneling probability is exponentially dependent on the

distance, the contours of the surface can be mapped out by
keeping the current constant and measuring the height of the tip.
Image from a scanning Tunneling microscope
 Atomic Force Microscope

The atomic force microscope (AFM) is one kind of scanning probe microscopes (SPM).
SPMs are designed to measure local properties, such as height, friction, magnetism, with a probe.

To acquire an image, the SPM raster-scans the probe over a small area of the sample, measuring the local property
simultaneously.

The basic principle of AFM is whereby a fine tip is scanned across the surface of the sample to measure surface
morphology and properties to construct a 3-D image of the surface.

Resolution for AFM is of the order of fractions of a nanometer, more than 1000 times better than the optical diffraction
limit.
 Atomic force Microscopy
The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface.
The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometer.

When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection
of the cantilever.