1. Microscope
Microscope – an instrument that produces an enlarged image of an
object.
• Biologists use microscopes to study cells, cell parts, and organisms
that are too small to be seen with the naked eye.
• Microscopes magnify and show details of the image.
2. What Is Microscope?
Microscopes provide the observer with:
• Enhanced resolution (ability to observe two nearby objects as distinct objects)
• Contrast (ability to detect different regions of the specimen on the basis of
intensity or color)
• Magnification (ability to make small objects visible).
The human eye can resolve objects of the order of 0.1 mm, while the light
microscope can resolve objects on the order of 0.2 µm (200 nm) with a
magnification of 1000. The transmission electron microscope, can resolve objects
on the order of 0.1nm (100 A ˚ units). we require a microscope with appropriate
resolution, contrast and magnification to see them. For example, the typical linear
dimensions of few things are: Animal cell (20–30 µm), a red blood cell (7.8 µm ), a
mitochondrion (2–5µm), a nucleus (3-10 µm), microvilli (1 µm), a cell membrane
(10 nm), a microfilament (8– 10 nm), a bacterium )0.5-5 µm) and a virus (10–100
nm)
3. Three factors determine the quality of an
optical image
a. Magnification
b. Resolution
c. Contrast.
4. Magnification
Magnification is the apparent increase in size affected by a convex lens. A
compound microscope uses two sets of lenses, with differing focal lengths, to
facilitate magnification. The total magnification achieved by the lens array is the
product of each individual lens.
Magnification (total) = magnification (obj. lens) x magnification (ocular. lens)
Example: Mag (obj) = 40X and Mag (ocular) = 10X
Then Mag (total) = (40X) (10X) = 400X.
It is much easier to make two lenses with average magnifying powers and put them
together in a compound microscope than to make a single lens with a very high
magnifying power.
Compound microscopes are usually designed to give a highest possible
magnification of only 1,000-1,500X.
5. Resolution
The most important part of the microscope is the objective, which must
produce a clear image, not just a magnified one. Resolution is the ability to
separate points (in other words, to observe fine detail). or the ability of a
lens to distinguish between small objects that are close together. Resolution
is not the same thing as magnification. One of the ways to increase the
resolution of an image is to increase the amount of light that enters the
objective lens by using immersion oil. Refraction is the deflection of a light
ray that occurs when it passes between substances that have different
densities. A convex lens exploits refraction to focus light at a specific point.
However not all refraction results in the convergence of light, in many cases,
the rays of diverge, which results in an overall decrease in the intensity of
light. This typically occurs as light passes through the air, in the space
between the specimen and the objective lens.
6. Immersion oil (with a density closer to glass) can be used to reduce the
refraction of light rays (compared to air) and allows more light to enter
the objective. This improves resolution. Only the highest power (100X)
objective on the microscope is designed for use with immersion oil. DO
NOT USE OIL WITH THE LOWER POWER (10X AND 40X) OBJECTIVES.
Another way to increase resolution of an image is to decrease the
wavelength of light that is used to illuminate the specimen (use blue
light instead of white light).
7. Contrast
Microbes are composed of water, nucleic acids, proteins, and lipids.
Most appear colorless against a colorless background when observed
using bright field microscopy. Therefore in order to see them, we must
devise a way to increase the contrast. Direct staining of the
microorganisms and Indirect (negative) staining of the background In
order to stain a specimen, it must first be fixed to the slide and
chemically altered. This results in the death of a specimen.
Additional microscopic techniques have been developed to increase
contrast of living microorganisms:-
8. Brightfield microscopy
This is the commonly used type of microscope. In brightfield
microscopy the field of view is brightly lit so that organisms and other
structures are visible against it because of their different densities. It is
mainly used with stained preparations. Differential staining may be
used depending on the properties of different structures and
organisms.
9. Dark-field microscopy
• In darkfield microscopy the field of view is dark and the organisms are
illuminated. A special condenser is used which causes light to reflect
from the specimen at an angle.
• is one such technique that is often used to observe living, unstained
cells and organisms. Dark field microscopes illuminate the sample in
such a way that unreflected, and unrefracted light does not enter the
objective, only light that has been reflected/refracted by the
specimen passes through the objective and forms the image. This
results in a specimen that is brilliantly illuminated on a dark field, The
field surrounding the specimen appears black.
10. Phase-contrast microscopy
• Phase-contrast microscopy allows the examination of live unstained
organisms. For phase-contrast microscopy, special condensers and
objectives are used. These alter the phase relationships of the light
passing through the object and that passing around it.
• Phase-contrast microscopes: Convert slight differences in refractive
index and density into variations in light intensity
11. Fluorescence microscopy
• In fluorescence microscopy specimens are stained with
fluorochromes/ fluorochrome complexes. Light of high energy or
short wavelengths (from halogen lamps or mercury vapour lamps) is
then used to excite molecules within the specimen or dye molecules
attached to it. These excited molecules emit light of different
wavelengths, often of brilliant colours. Auramine differential staining
for acid-fast bacilli is one application of the technique; rapid
diagnostic kits have been developed using fluorescent antibodies for
identifying many pathogens.
• Specimens are treated with dye molecules called fluorochromes
which brightly fluoresce when exposed to light of a specific
wavelength.
12. Parts of a Compound Light Microscope:
1. Eyepiece Lens/ Ocular lens: The first lens in a compound
microscope, the lens at the top that you look through
(usually 10x).
2. Tube: Connects the eyepiece to the objective lenses
3. Arm:Supports the tube and connects it to the base. It is
used along with the base to carry the microscope
4. Base: The bottom of the microscope, used for support
5. Illuminator: A steady light source (110 volts) used in place
of a mirror
6. Stage: The flat platform where you place your slides.
Stage clips hold the slides in place.
13. 7. Revolving Nosepiece or Turret: This is the part that holds two or more objective
lenses and can be rotated to easily change power. Allows one to change between
the objective lenses.
8. Objective Lenses: The second set of lenses in a compound microscope (usually
4x, 10x, 40x). Usually you will find 3 or 4 objective lenses on a microscope. They
almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X
(most common) eyepiece lens, we get total magnifications of 40X (4X times 10X),
100X , 400X and 1000X. The shortest lens is the lowest power, the longest one is
the lens with the greatest power. The high power objective lenses are retractable
(i.e. 40XR). This means that if they hit a slide, the end of the lens will push in
(spring loaded) thereby protecting the lens and the slide.
9. Rack Stop: This is an adjustment that determines how close the objective lens
can get to the slide. It is set at the factory and keeps students from cranking the
high power objective lens down into the slide and breaking things.
14. 10. Diaphragm or Iris: Adjusts the amount of light that hits the slide from the light
source. Many microscopes have a rotating disk under the stage. This diaphragm
has different sized holes and is used to vary the intensity and size of the cone of
light that is projected upward into the slide. There is no set rule regarding which
setting to use for a particular power. Rather, the setting is a function of the
transparency of the specimen, the degree of contrast you desire and the particular
objective lens in use.
11. Coarse adjustment: This is used to focus the microscope. It moves the stage up
and down very quickly. It is used first, and it is used only with the low power
objective.
12. Fine adjustment: This is used to focus the microscope. It moves the stage up
and
13. down very slowly. It is used with the high power objective to bring the
specimen into better focus.
15. Electron Microscope
Resolution is the limiting factor to a light microscope since the greater the
magnification is, the less it is able to resolve the image. At magnifications
beyond 2,000x, the image becomes blurry, but electron microscopes can be
used at greater magnifications
Characteristics of the Electron Microscope
• A beam of electrons is used to produce an enlarged image of the specimen
(it does not use light).
• This electron beam and the specimen must be placed inside a vacuum
chamber so that the electrons in the beam do not bounce off gas
molecules in the air.
• Since living things cannot survive in a vacuum, the electron microscope
cannot be used to view living cells.
• Much more powerful than light microscopes.
16. Types of Electron Microscope
There are two types of electron microscopes:
1. Transmission Electron Microscope (TEM)
• Uses a beam of electrons transmitted through a very thinly sliced specimen. Magnets
guide the beam of electrons toward the specimen, and the image is produced to view.
• Magnification to 200,000 times
2. Scanning Electron Microscope (SEM)
Provides detailed 3-D images.
▪ The specimen is sprayed with a fine metal coating (it is not sliced to view as in the TEM).
▪ As the beam of electrons is passed over the specimen’s surface, the metal coating emits a
shower of electrons, and a 3-D image of the specimen’s surface is produced to view.
