Lab: 5
Microscopy
The type of light source used classifies microscopes:
1. Bright Field Microscopy:
The light source lies in the visible region.
Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece
usually contains a 10X or 15X power lens.
Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to
correct for any difference in vision between your two eyes.
Body tube (Head): The body tube connects the eyepiece to the objective lenses.
Arm: The arm connects the body tube to the base of the microscope.
Coarse adjustment: Brings the specimen into general focus.
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Fine adjustment: Fine tunes the focus and increases the detail of the specimen.
Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the
nosepiece to select different objective lenses.
Objective lenses: One of the most important parts of a compound microscope, as
they are the lenses closest to the specimen.
A standard microscope has three, four, or five objective lenses that range in power
from 4X to 100X. When focusing the microscope, be careful that the objective lens
doesn’t touch the slide, as it could break the slide and destroy the specimen.
Specimen or slide: The specimen is the object being examined. Most specimens are
mounted on slides, flat rectangles of thin glass.
The specimen is placed on the glass and a cover slip is placed over the specimen.
This allows the slide to be easily inserted or removed from the microscope. It also
allows the specimen to be labeled, transported, and stored without damage.
Stage: The flat platform where the slide is placed.
Stage clips: Metal clips that hold the slide in place.
Stage height adjustment (Stage Control): These knobs move the stage left and right
or up and down.
Aperture: The hole in the middle of the stage that allows light from the illuminator
to reach the specimen.
On/off switch: This switch on the base of the microscope turns the illuminator off
and on.
Illumination: The light source for a microscope. Older microscopes used mirrors to
reflect light from an external source up through the bottom of the stage; however,
most microscopes now use a low-voltage bulb.
Iris diaphragm: Adjusts the amount of light that reaches the specimen.
Condenser: Gathers and focuses light from the illuminator onto the specimen being
viewed.
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Base: The base supports the microscope and it’s where illuminator is located.
Important points around the light microscope
 Total magnification = Eyepiece magnification X Objectives magnification.
The image seen by the eye through a compound microscope is termed the virtual
image and is upside and reversed.
 The numerical aperture (NA):
Is a designation of the amount of light entering the objective from the microscopic
field.
Lens
NA = R sin 
c
B
Where R is the refractive index of glass
 is the angle made by one ray passing through
the edge of the lens with the other ray
passing the center of the lens.
Then NA depends on the radius of the lens.

Object
 Resolving power:
Is the useful limit of magnification, it is the ability of microscope, at specific
magnification to distinguish two separate objects situated close to one another and
the ability of the lens to reveal fine details.
The smaller the distance between the two specific objects that can be distinguished
apart, the greater the resolution power of the microscope.
Minimal distance between two objects = (0.612 X ) / NA
The larger NA, the smaller the resolvable distance and hence, the more efficient
the resolution power.
 Depth of field:
Is the capacity of the objective lens to focus in different planes at the same time.
This is largely dependent on the NA. Where the greater the NA, the smaller the
depth of the field, it is possible to increase the depth of the field slightly by closing
the iris diaphragm. Thus decrease the NA.
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 Chromatic Aberration:
Since light is formed of several wavelength then light component are not bent in the
same way as they pass through the lens and therefore are not brought to the same
focus.
 Spherical aberrations:
The light wave, as they travel through the lens, are bent differently, depending in
which part of the lens they pass through, rays passing through the peripheral
portions of the lens are brought to a shorter focal point than those rays passing
through the thicker part of the lens.
To correct chromatic and spherical aberration achromatic (brings 2 colors) and
apochromatic (brings 3 colors blue, yellow and red) lenses may be used which are
fine lenses produced to bring rays of several colors to a common focus.
The medium between the objective and the object is a factor that must be taken into
consideration for the most effective use of the microscope lens. The low power
objective 4x, 10x, and high dry objective 40x use air.
When oil immersion lenses are employed, a drop of oil should be used, otherwise
bending of the light waves occurs since oil has the same RI of glass, while air
increase diffraction.
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2. Ultraviolet Microscopy:
The shorter wavelength of UV can extend the limit of microscope resolution to
about 0.1 m. However, UV light is invisible to the human eye, so the image must
be recorded on a photographic plate or fluorescent screen. Because this light is
absorbed by glass, all lenses must be made of quartz, such microscopes are two
expensive for routine use.
3. Fluorescence microscopy:
A sample labeled with a fluorescent dye is illuminated with UV light, the location of
the dye in the specimen is revealed by its fluorescence or emission of visible light
4. Dark field Microscopy:
One sees a black background; against which suspended bacteria or element appear
bright. The dark field microscope uses a special condenser that illuminate the
sample with a hallow cone of light in such a manner the light is not directed into the
objective lens, revealing the shape of that object.
5. Phase contrast Microscopy:
Bacterial or animal cells are difficult to be seen using the light microscope unless
the sample is dried and stained. This microscope enhances the slight difference in
refractive index between the cells and the medium and thus can be used to visualize
the living bacteria and platelets, in which the slight differences in RI are converted
to differences in light intensity.
6. Electron Microscopy:
Since magnification greater than 1500X to 2000X are not practical with the light
microscope due to decreased efficiency in resolving power. The electron microscope
has come into use, where magnification of 50,000X may be obtained, with a high
degree of resolving power. There are two types of electron microscope:
1. Transmission Electron Microscope (TEM) {2 dimensional}
2. Scanning Electron Microscope
(SEM) {3 dimensional}
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