CHARACTERISATIONS OF

MATERIALS

OPTICAL
MICROSCOPY

ELECTRON MICROSCOPY

CONTENTS
3.0 Instrumentation
3.1 Sample Prep
3.2 Measurement Systems and Types
4.0 Examples
5.0 Correct Presentation of Results
5.1 Publication
5.2 Presentation

INSTRUMENTATIONS

Several important features

are visible:
Lenses
Eyepieces (oculars)
Light source

Let us watch video on parts of compound

microscope

Let us watch video on light sources in

microscope

Let us watch video on lenses and eyepiece

systems

Anatomy of a Modern Light Microscopes

Illumination systems
Provides visible light by which the
specimen is observed.
Three main types:
1. Low voltage tungsten filament
bulbs
2. Tungsten halogen bulb
3. Gas discharge bulbs

Let us watch video on How does the

Microscope Work

Light Microscopy: Basics in optics

Compound Microscope

Sample is placed in front of

objective focal plane. Intermediate
imagei s formed by objective and is
observed through eyepiece.

Two Sets Of Conjugate Focal Planes In The Kohler System

Illustrated In A Transmitted Light Microscopes

Type of Optical Microscope

Reflected-light microscopy
is used for a range of materials, including metals, ceramics and composites.
Contrast between different regions when viewed in reflected light can arise

from variations in surface topography and differences in reflectivity (e.g. of

different phases, different grain orientations, or boundary regions).
These features are revealed by a series of specimen preparation techniques

which, when carried out with care, can produce useful, high quality images.

Type of Optical Microscope

Transmission mode
can be used when the specimen is transparent.
The specimen is usually in the form of a thin slice (e.g. tens of microns thick).

Contrast arises from differences in the absorption of light through different

regions.
This method is used for the examination of minerals and rocks, as well as
glasses, ceramics and polymers. In addition, the transmission mode can often
be further enhanced with use of polarised light.
Polarised light microscopy
is a specialised use of the transmission mode, and contrast is due to differences

in birefringence and thickness of the specimen.

This can allow the observation of grains, grain orientation and thickness.

Type of Light Microscope

Brightfield Microscope
Brightfield microscopes are the most common type. A brightfield microscope
focuses light through a condenser lens and passes it through the specimen. Above
the specimen is a tube containing an objective lens and an eyepiece lens that
together focus the light to form an enlarged image. Images seen through a
brightfield microscope generally have poor contrast unless the specimen has been
stained with dyes or chemicals that adhere to certain types of structures in the cell

Dark Field Microscopes

Dark field microscopes are similar to brightfield microscopes but are equipped with
a dark field patch stop and a direct illumination block. Together, these two
components ensure that only light scattered by the specimen passes through the
objective lens. When viewed through a dark field microscope, a specimen appears
as if it were on a black background. Dark field microscopy can provide enhanced
contrast, although some of the features that are visible with bright field microscopy
may not be visible with the dark field technique.

Type of Light Microscope

Fluorescence Microscopes
Fluorescent materials absorb light or electromagnetic radiation of one wavelength
and re-radiate electromagnetic radiation of another wavelength (typically a longer
wavelength). In a fluorescence microscope, the specimen is first stained with
fluorescent dyes or molecules that adhere to specific molecules or structures in the
cell. Light from the light source shines on a dichroic mirror that reflects light of some
wavelengths and transmits light of other wavelengths. The light from the source is
reflected onto the sample, where the fluorescent dyes absorb it and emit light that
passes through the dichroic mirror into the objective lens. A fluorescent microscope
produces an image in which only the cell components tagged with fluorescent dyes
are visible.
Confocal Microscope
A confocal microscope is similar to a fluorescence microscope but incorporates
some additional features, most notably a pinhole aperture that excludes out-of-focus
light. Consequently, the confocal microscope can form a very sharp image of a
single plane in the sample. Confocal microscopes are powerful tools but require long
exposure times to form a good image.

Reflected Microscope Anatomy

Principle Of Bright Field Microscopy

Light from an incandescent source is aimed toward a lens beneath the stage
called the condenser, through the specimen, through an objective lens, and to
the eye through a second magnifying lens, the ocular or eyepiece.

The condenser is used to focus light on the specimen through an opening in

the stage.

After passing through the specimen, the light is displayed to the eye with an
apparent field that is much larger than the area illuminated.

Typically used on thinly sectioned materials

Principle Of Dark Field Viewing

To view a specimen in dark field, an opaque disc is placed underneath

the condenser lens, so that only light that is scattered by objects on the
slide can reach the eye.

Instead of coming up through the specimen, the light is reflected by

particles on the slide.

Everything is visible regardless of color, usually bright white against a

dark background

Principle Of Dark Field Viewing

Mirror Blocks For Reflected Light

Let us watch video on Darkfield Setup

How to calculate microscope image size

Quality of Image

When you look at a specimen using a microscope, the quality of the image
you see is assessed by the following:

Brightness
Resolution
Contrast

Quality of Image with different brightness

Image of pollen grain under good brightness (left) and poor brightness (right)

Brightness - How light or dark is the image? Brightness is related to the

illumination system and can be changed by changing the voltage to the lamp
(rheostat) and adjusting the condenser and diaphragm/pinhole apertures.
Brightness is also related to the numerical aperture of the objective lens (the
larger the numerical aperture, the brighter the image).

Quality of Image with different resolution

Image of pollen grain with good resolution (left) and poor resolution (right)

Resolution - How close can two points in the image be before they are no longer
seen as two separate points? Resolution is related to the numerical aperture of
the objective lens (the higher the numerical aperture, the better the resolution)
and the wavelength of light passing through the lens (the shorter the wavelength,
the better the

Quality of Image with different contrast

Image of pollen grain with good contrast (left) and poor contrast (right)

Contrast - What is the difference in lighting between adjacent areas of

the specimen? Contrast is related to the illumination system and can
be adjusted by changing the intensity of the light and the
diaphragm/pinhole aperture. Also, chemical stains applied to the
specimen can enhance contrast.

Example image from dark field and bright field

microscope

Measuring Image with Light Microscope

Estimating Viewing Field with a Ruler
Step 1: Use a clear ruler with a cm/mm scale to measure the diameter of your viewing field
at scanning (40x). On our scopes, we estimated the viewing field to be about 4 mm across.

Measuring Image with Light Microscope

Step 2: Repeat the process on low power (100x). We estimate our low power viewing field to
be about 2 mm across. Convert mm to microns for both the scanning and low power. There
are 1000 microns in a millimeter. So Scanning = 4000 microns; Low Power = 2000
microns.
The measurement is an estimation though, and probably not very accurate. For instance, if
you have a slide with the word are across it, use your measurement of the viewing field to
estimate the distance of each of the letters. Based on the ruler marks, the letter r is about
1100 microns, and the entire word, which takes up most of the viewing field is about 4000
microns across. In reality, you cannot place your slide over the ruler, so you have to make a
guess based on how large your microscopes viewing field is.

Measuring Image with Light Microscope

Calculating High Power Field of View
Measurements on High Power can be a little more complicated. If you try to use the clear
ruler technique, youll find that you cannot see the individual ruler marks. This is where
math comes in, the values you estimated above can be used to solve a ratio problem and
determine the size of your viewing field on high power.
Solving for High Power Field of View .
X / 2000 = 100/400
X (high power field of view) = 500 microns

Now that you have 500 microns as an estimate of your viewing field, any object you are
viewing under high power can be estimated based on that. I tell my students to look at a
paramecium and guess how many paramecia can fit end to end on their slide. They guess
they can fit two, then the length of their paramecium is about 250 microns.

Measuring Image with Light Microscope

Using a Stage (Slide) Micrometer

These are slides that contain a tiny ruler on
them. The ruler is marked as .01 mm,
which means each little etch mark on the
ruler is .01. The total length of the scale
on most stage micrometres is 1 mm, which
then means that there are 1000 microns
measured by that scale.
If each each mark is .01 mm then that is
equal to 10 microns. The picture side,
gives an idea of how the micrometer looks
under low power (100x). The image is
enlarged a little more due to the camera
used to photograph it, so you dont see the
full viewing field. The paramecium was
superimposed on the shot to illustrate the
scale.

Measuring Image with Light Microscope

Measuring grain Size through ASTM grain size number

Thank You

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