B 1501- INTRODUCTORY CELL BIOLOGY &

MICROBIOLOGY

Lecture 2: Introduction to Microscopes

Dr Liteboho D. Maduna

Department of Biology
Introduction to Microscopes
 Microscopy reveals two fundamental cell types
 Prokaryotic cells (Bacteria, Archaea)
– Smaller size gives high surface area to low volume
– Facilitates rapid uptake of nutrients, excretion of wastes
– Allows rapid growth
– Disadvantages include vulnerability to threats including predators, parasites, and
competitors
 Eukaryotic cells (Eukarya)
– Larger, more complex, many cellular processes take place in membrane-bound
compartments
– Defined by the presence of a nucleus
Introduction to Microscopes

 Before microscopes were first used in the 1600s, no one knew that living
organisms were composed of the tiny units we call cells.
 A variety of microscopes have been developed for a clearer view of cells and
cellular structure.
 Microscopes make small objects appear bigger to the human eye.
 The most frequently used microscope is the light microscope (Compound
microscope)—like the one used in our biology laboratories.
– Light passes through a specimen, then through glass lenses, and finally light is projected
into the viewer’s eye.
– Specimens can be magnified up to 1,000 times the actual size of the specimen.
Light Microscope

 The lenses refract (bend) the light in such a way that the image of the
specimen is magnified as it is projected into the eye,
 In Light Microscope (LM), visible light is passed through the specimen
such as a microorganism or thin slice of animal or plant tissue and then
through glass lenses.
 LM has a series of lenses and uses light as its source of illumination.
– Objective lens
– Condenser lens
– Eyepiece (Ocular lens)
Parts of the Microscope
1. Eyepiece: Also known as ocular, located at the
top of the microscope. The eyepiece is the lens
through which the viewer looks to see the
specimen.
– It usually contains a 10X or 15X power lens.
2. Body tube: The body tube connects the
eyepiece to the objective lenses.
– Contains mirrors and prisms that direct the
image to the ocular lens
3. Nosepiece: Found on lower part of the arm and
holds the objective lenses of differing magnifying
power
Parts of the Microscope
4. Objective lens: Light microscopes contain
different types of objective lens. These are located
below the nosepiece and are closest to the
specimen
– 4x objective lenses (low power- red )
– 10x objective lenses (medium power- yellow)
– 40 x objective lenses (high power- blue)
– 100x objective lenses (Oil immersion- White)
– The objective lenses are color coded; red band is low
power, yellow is medium power, blue band is high
power, and white band is oil immersion
Parts of the Microscope
5. Stage:The flat metal platform located above the
condenser and below the objective lens. The slide
of the test specimen is placed over it.
– the control knobs on the mechanical stage allow the
operator to move the slide easily during the viewing

6. Stage clips: Above the stage, holds the slide

7. Condenser: It is located below the stage. It
gathers and focuses light from the illuminator
(light source) onto the specimen being viewed.
8. Iris diaphragm: It adjusts the amount of light
and intensity that is projected upward into the
slide, reaching the specimen.
Parts of the Microscope

9. Focusing knobes: located on the side of the microscope; outer (small) is the
fine focus knob and the inner (large) is the course focus knob.
10.Light source: They have built-in light source (this could be electrical source
or a mirror). The brightness control knob for electrical source is located on
the base and has a brightness range of 0-9.
11.Par focal: Microscopes are par focal, meaning that the image remains in focus
when the objectives are changed, requiring only a slight change in fine
adjustment for sharp image.
12.Field of vision: The area you can see at a given level of magnification.
Working Principle of Light Microscope
 Light microscopes have a combination of lenses that enhances both the
magnifying powers as well as the resolving power
1. The specimen or object, to be examined is usually mounted on a transparent
glass slide and positioned on the specimen stage between the condenser lens
and objective lens.
2. A beam of visible light from the base is focused by a condenser lens onto the
specimen.
3. The objective lens picks up the light transmitted by the specimen and creates a
magnified image of the specimen called the primary image inside the body tube.
This image is again magnified by the ocular lens or eyepiece.
Working Principle of Light Microscope

4. Occasionally very high magnification it required (e.g. for observing bacterial

cell). In that case, an oil immersion objective lens (usually 100X) is
employed.
5. The common light microscope is also called a bright-field microscope
because the image is produced amidst a brightly illuminated field.
– The image appears darker because the specimen or object is denser and
somewhat opaque than the surroundings. Part of the light passing
through or object is absorbed.
Parameters of a Microscope

 There are three important parameters in microscopy namely:

1. Resolution: Resolving power

2. Magnification and

3. Contrast or visibility
1. Resolution of a Microscope
 Resolution is a measure of the clarity of an image; it is the minimum distance at which
two points can be separated and still be distinguished as two points
 For example, what you see as a single star in the sky may be resolved as twin stars
with a telescope. Each optical instrument—be it an eye, a telescope, or a
microscope—has a limit to its resolution.
 The human eye can distinguish points as close together as 0.1 mm, about the size of
a very fine grain of sand.
 A typical light microscope cannot resolve detail finer than about 0.2 micrometer
(μm), about the size of the smallest bacterium
 No matter how many times the image of such a small cell is magnified, the light
microscope cannot resolve the details of its structure. Indeed, light microscopes can
effectively magnify objects only about 1,000 times.
1. Resolution of a Microscope (continued)
 Oil immersion: One way of increasing the optical resolving power of the
microscope is to use immersion oil between the front lens of the objective and the
cover slip.
 Most objectives in the magnification of 100x (and higher) are designed for use with
immersion oil.
 The oil that has a refractive index (measure of speed of light passing through medium) of
n = 1.51, which has been precisely matched to the refractive index of glass.
 Prevents refraction of light, keeps rays from missing opening in objective lens
 One or two drops of oil is usually placed on the cover slip and the 100x objective
lens is brought into position so that it touches the oil and creates a “bridge”
between the slide and the objective lens
2. Magnification

 Magnification is the increase in an object’s image size compared with its actual
size.
 It is the ratio of an object’s image size to its real size. It is the ability of the lens
to enlarge the object being viewed.
 Objective lenses form an enlarged real image within the microscope.
 The eyepiece further magnifies the image. The total magnification is obtained
by multiplying the objective and eyepiece magnifications.
Total magnification = magnification of the objective lens ×
magnification of the ocular (eyepiece) lens.
2. Magnification
 To achieve high magnification with good resolution an immersion oil lens is
usually used by introducing a drop of oil between the slide and the objective
les.
– This reduces loss of light rays after it passes through the specimen.
– Immersion oil has same refractive index as glass with same
effect as increasing the diameter of objective lens.

Class work
 What is the magnification if the eye lens is 10x and the objective lens is 25x?
 Which magnification would give you the smallest field of view?
– 25X; 50X; 200X ; OR 400X.
3. Contrast or Visibility

 Contrast emphasizes the differences in parts of the sample.

 In fact, most improvements in light microscopy has enhanced contrast such as
staining or labeling cell components to stand out visually.
– Transparent bacteria lack contrast, difficult to see against colorless background

 Staining will make objects to appear colored. The disadvantage is that staining
cannot be used with living cells because it kills the cell and cannot allow
movement of cells to be seen.
Figure 4 Light micrograph of the unicellular organism Paramecium, shows a micrograph
of a single-celled organism called Paramecium. The notation “LM 230X” printed along the right edge
tells you that this photograph was taken through a light microscope and that the image is 230 times
the actual size of the organism. This Paramecium is about 0.33 millimeter (mm) in length
Electron Microscopes

 Electron microscope (EM) focuses a beam of electrons through a specimen

or onto its surface.
 Electron microscopes can distinguish biological structures as small as about 2
nanometers (nm), a 100-fold improvement over the light microscope.
 This high resolution has enabled biologists to explore cell ultrastructure, the
complex internal anatomy of a cell.
 Types of Electron Microscopes
1. Scanning Electron Microscope
2. Transmission Electron Microscope
Electron Microscopes
1. Scanning Electron Microscope (SEM): Used to study the detailed
architecture of cell surfaces.
– The SEM uses an electron beam to scan the surface of a cell or other sample, which is
usually coated with a thin film of gold.
– The beam excites electrons on the surface, and these electrons are then detected by a
device that translates their pattern into an image projected onto a video screen

2. Transmission Electron Microscope (TEM): TEM is used to study the

details of internal cell structure.
– The TEM aims an electron beam through a very thin section of a specimen, just as a light
microscope aims a beam of light through a specimen.
– The section is stained with atoms of heavy metals, which attach to certain cellular
structures more than others.
Question? Which type of
microscope would you use to
study
(a) the changes in shape of a
living human white blood
cell;
(b) the finest details of surface
texture of a human hair;
(c) the detailed structure of an
organelle in a liver cell?
Preparing Specimens for Light Microscopy
 Wet mount uses a drop of liquid specimen
 Smear involves drying and fixing specimen before staining to visualize
 Basic dyes carry positive charge
– Attracted to negatively charged cellular components

 Acidic dyes carry negative charge and can be used on wet mounts
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