GENERAL BIOLOGY I

BIOL 1101

M.N. MULUNGU

0882786742

[email protected]

M.N. Mulungu
MICROSCOPY
• Micro refers to tiny, Scope refers to view or look at.
• A microscope is defined as a tool used to enlarge an image of a
small object so as it can be studied.
• While on the other hand microscopy is the technology of making
small things visible to the human
• A microscope enlarges organisms so that they can be easily seen.
• Microscopes range from a simple magnifying glass to the expensive
Electron Microscope.
History of microscopes
 The first vision aid was invented (inventor unknown) called a
reading stone. It was a glass sphere that magnified when laid
on top of reading materials.
 In 1590 – Two Dutch eye glass makers, Zaccharias Janssen and
son Hans Janssen experimented with multiple lenses placed in a
tube.
 The Janssens observed that viewed objects in front of the tube
appeared greatly enlarged, creating both the forerunner of the
compound microscope and the telescope.
 In 1609 – Galileo Galilei developed a compound microscope
with a convex and concave lens.
• In the 1660s – the extensive use of microscopes in research
began in Italy, Holland and England.
• Anton Van Leeuwenhoek (1632-1723) was the first to invent a
microscope powerful enough to explore the world of
microorganisms (microbes).
• Cells were first seen in 1665 by Robert Hooke using a primitive
microscope.
• The first electron microscope was invented in 1931 by Max Knoll
and Ernest Ruska. While commercial scanning electron
microscopes came ashore in 1965.
Magnification and Resolving power
Magnification
• By using more lenses microscopes can magnify by a larger amount,
but this does not mean that more detail can be seen.
• Total visual magnification of the microscope is derived by
multiplying the magnification values of the objective and the
eyepiece.
• For instance, using a 5X objective with a 10X eyepiece yields a total
visual magnification of 50X
Resolving power
• The amount of detail depends on the resolving power of a
microscope.
• resolving power of a microscope is the smallest separation at which
two separate objects can be distinguished (or resolved).
• It is calculated by the Ernst Abbe formula:
• from the equation, (nsinθ) is the numerical aperture which ranges
between (1.4 to 1.6) for high quality lens, Where λ (lambda) is the
wavelength of light
• So resolving power of a microscope is ultimately limited by the
wavelength of light (400 – 600 nm for visible light).
• To improve the resolving power a shorter wavelength of light is
needed
• The numerical aperture of a microscope objective is the measure
of its ability to gather light and to resolve fine specimen details
while working at a fixed object distance
• Numerical aperture is calculated using this formula:
• NA = η(sinθ) where θ equals one half of objective’s opening
angle and η is the refractive index of the immersion medium
used between the objective and the coverslip protecting the
specimen (η = 1 for air; η = 1.51 for oil or glass
Sample problem
• The Numerical Aperture for a high-dry (400x) lens is 0.65. What
is the resolving power (d) of this objective when viewing a
wavelength of 550nm?
d = 0.61 λ / NA
d = (0.61)(550nm) / 0.65
d = 516.15nm
d = 0.52µm
Types of Microscopes
1.The Compound Light Microscope
• Also called light microscope or optical microscope
• Light passes through a specimen (the object) and then through two
sets of glass lenses, the objective lens and the ocular or eye piece lens.
• The lenses refract (bend) the light to give a magnified image
• The image may be projected directly into the viewer’s eye or onto
photographic film.
• A photograph taken through a light microscope is called a
photomicrograph or light micrograph
 Types of light microscopes
i. Bright Field Microscope:
In bright field microscope, illumination light passes through the
sample and the contrast is generated by the absorption of light in
dense area of the specimen
The advantages are
• convenience
• relatively inexpensive
• widely available.
Disadvantages include
• Low resolving power, 0.2 micrometers at best
• can recognize cells but not fine details
• needs contrast since cells are mainly water and do not contrast
with their medium.
ii. Dark field microscope
• this microscope is very rare.
• It has a condenser that prevents light from being transmitted
through the specimen but causes light to reflect off the
specimen at an angle
iii.Phase contrast microscope
• Cells are mostly water, very little contrast from surrounding
medium, so not very visible in light.
• Phase scope converts slight differences in refractive index and
cell density into variations.
• The microscope uses annular stop below condenser
• Advantage is that one can see live material without staining.
iv.Fluorescence microscope
• Fluors are chemicals that adsorb light to produce excited electrons,
later radiate light = fluorescence.
• To use this it needs special type of microscope.
• Needs filters to remove this light traveling to ocular lens; only
fluoresced light emitted from object will then appear to the eye.
• Need dark field condenser to create dark background.
• Requires correct microscope, fluors and technical skills
2.Scanning tunneling microscope (atomic force microscope)
• This uses a very fine needle to scan the surface of a specimen.
• It has a resolution of about 10 pm, and has been used to observe
individual atoms for the first time.
3. X-ray microscope
• It uses electromagnetic radiation
• This is an improvement to the light microscope, since x-
rays have wavelengths a thousand times shorter than
visible light and so could even be used to resolve atoms.
• Unfortunately, there are no good x-ray lenses to easily
deflect the radiation and radiation is invisible to human
eye, therefore x-ray microscope uses charge coupled-
device as a detector.
4.Electron Microscope
• Electron microscopes use a beam of electrons instead of a beam of
light.
• Electron beams have a much smaller wavelength than light rays,
so electron microscopes have greater resolving powers and can
produce much higher effective magnification than light
microscopes.
• Basic design of Electron Microscope (EM) is similar to Light
Microscope.
• To avoid electrons from being scattered by air molecules, air is
removed from microscope with vacuum pump.
• And water in specimen must be removed by dehydration after
fixation.
• As a result of this you cannot view living specimens under
electron microscope.
• There are two types of Ems.
I. Transmission Electron Microscope (TEM)
II. Scanning Electron Microscope (SEM).
i. The Transmission Electron Microscope (TEM)
• The TEM is used to study the details of the internal structure of cells (ultra-
structure).
• electron beam passes through the specimen and is scattered according to the
density of the different parts.
• Due to the limited penetrating power of the electrons, extremely thin
sections must be cut, using a diamond knife.
• To allow this, the specimen must be fixed and dehydrated, a process that
can introduce shrinkage and distortion to its structure if not correctly
performed.
• After being magnified by an objective ‘lens’, an image of the specimen is projected onto a

fluorescent screen or photographic plate.

• More dense areas, which scatter the beam, appear dark, and those where it has passed

through are light.

• A photograph taken through an electron microscope is called an Electron Micrograph.

• The most modern TEMs can distinguish objects as small as 0.2nm.

• This means that they can produce clear images magnified up to 250,000 times.

• The magnification is varied by changing the strength of electromagnets

Advantages of TEM

• High resolution (0.5 nm in practice)

Disadvantages of TEM:

• The specimen must be dead because it is viewed in a vacuum.

• It is difficult to be sure that the specimen resembles a living cell in all its details because
preservation and staining may change or damage the structure.

• Expensive to buy and run

• Preparation of materials is time consuming and requires training

• The specimen gradually deteriorates in the electron beam.

• Photographs must therefore be taken if further study is required.

ii. The Scanning Electron Microscope (SEM)
• The SEM is used to produce three-dimensional images of the surface of
the specimens.

• Electrons are reflected from the surface of a specimen stained with heavy
metal.

• This enables the SEM to produce images of whole specimens: cells,

tissues or even organisms.
Advantages of SEM
• Surfaces of structures are shown
• Great depth of field, meaning that a large part of the specimen is in focus at
the same time.
• This gives a very striking three-dimensional effect.
• Much larger samples can be examined than with TEM

Disadvantages of SEM
• Resolution (5 – 20 nm) is not as great as with a TEM (0.5 nm).
Table 1.4: Comparison of light and Electron Microscope

Features TEM Light microscope

Radiation source Electrons Light

Wavelength About 0.005nm 400 – 700 nm

Max. Resolution in practice 0.5 nm 200 nm

Max. useful Magnification X 250000 (on screen) X 1500

Lenses Electromagnets Glass

Techniques of light microscopy
• A microscope has to be used in order to study microorganisms.
• Microorganisms of interest are placed on the stage of the
microscope.
• This is called mounting the specimen.
• There are two ways in which that can be done.
• Wet mount
• Fixed mount
I) Wet mount
• This is the kind of mounting where a drop of medium containing
the microorganisms is placed onto microscope slides.
• Then a cover slip is used to prevent any contact between the
specimen and the objective lens of the microscope.
• This method can be used to observe living microorganisms.
II) Fixed mounts e.g. Smears
• This technique involves making a smear of microorganisms
• A sample of microorganisms is taken from a Petri dish using a loop.
• The loopful of microorganisms is spread onto the surface of a glass
slide.
• The smear is then fixed onto the slide with heat by moving the slide
swiftly across an open flame.
• This process called heat fixing ensures that microorganisms adhere
to the slide.
Disadvantages using this method.
• It cannot be used to study living microorganisms
• Sometimes when you leave the slide longer over the flame, the
shape of the microorganisms can be altered.
Staining
• Staining means any procedure that applies colored chemicals
called dyes to specimens.
• To study microorganisms, other techniques are employed to
enhance the visibility of microorganisms.
• Dyes or stains bind to cellular structures of microorganisms and
hence gives it color so it can easily be seen.
• This enables the microorganisms to stand out against the
microscope slide background.
Staining Techniques
• There are two basic staining techniques used depending on how the
dye reacts with the specimen and these are
• negative staining
• positive staining techniques.
• In positive staining the dye actually sticks to cells and give them
a colour.
• In Negative staining, is where the dye does not stick to the cells
or specimen, but settles around its outer boundary, forming a
silhouette.
• Negative staining “stains” the glass slide to produce a dark
background around the cells.
Types of positive staining techniques
1. Simple stain (basic stain)
• In this technique, a single dye is used to reveal the basic cell
shape and cell arrangements.
• Examples are Methylene blue, Safranin, Carbolfuchsin and
Crystal violet
2. Differential stain
• This technique uses two or more dyes.
• The dyes are used to distinguish between two kinds of organisms or
different parts of an organism.
• Examples are Gram stain, Zeihl- Neelsen acid-fast stain and Schaefffer-
Fulton spore stain.
3. Special stains
• "Special stains" are processes that generally employ a dye or
chemical that has an affinity for the particular tissue
component that is to be demonstrated.
• They allow the presence/or absence of certain cell types,
structure to be viewed microscopically.
• These types of stains are used to emphasize certain cell parts that
are not revealed by conventional staining methods e.g. capsule
staining and flagella staining.
 Reading Assignment

• Functions of the parts of light microscope

• Explain how a light microscope works

• Storing and caring a microscpe