LAB 1

Practical Histology
microscope
Definition: microscope means the little seer which discovered by Leeuwenhoek
in 1600. It allows researchers to see cellular and macromolecular details that are
not visible to the naked eye.
 Properties:
1. Resolution power is the smallest degree of separation at which tow objects can
still be distinguished as separate objects and based on wavelength of the
illumination.
2. Magnification is the enlargement of the image.
 Classification:
Microscopes classify according to the light source into tow types:
1. Light microscopes
2. Electron microscopes
 Light microscopes: employ transillumination and are used to examine living
and prepared specimens and specimens with inherent or applied fluorescent
properties.
A. Light microscope components: (see figure 1)
1. A light microscope has a built-in light source and an adjustable sub-stage
condenser, which project light into the objectives.
2. A mechanically operated stage carries the specimen.
3. Objectives magnify the specimen image, and oculars complete image
formation process.
B. Image formation:
1. Light microscopes have a resolution limit of 0.2-0.4 µm, or approximately
one-twentieth the diameter of a human erythrocyte.
2. The resolution power of light microscope depends on three variables:
objective magnification, objective numerical aperture (N.A), and the
wavelength of light used to illuminate the specimen.
Figure (1): Components of light
microscope.
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LAB 1
Practical Histology
microscope
C. Types of light microscopy:
1. Bright field microscopy:
a. It uses standard lenses and condensers and has a limit of resolution of
approximately 0.3 µm
b. It is called a compound microscope because it uses toe lenses,
objective and ocular, to form and magnify the image.
c. An image is formed by passing a beam of light through the specimen
and then focusing the beam using glass lenses.
2. Phase-contrast microscopy:
a. It use modified objective lenses and condensers to permit direct
examination of living cells without fixation or staining.
b. It has sub-stage condensers and objectives that convert slight
differences in the refractive indices of specimen structures and
domains into distinct differences in light intensity.
c. This type pf microscopy doesn't harm living cells and allows scientists
to examine cell behavior under many artificial circumstances.
3. Differential interference contrast microscopy:
a. This type is also called (Nomarski light microscopy).
b. It is used special condensers and objective lenses to transform
differences in refractive index into an image that appears to have a
three-dimensional character.
c. In this type of microscopy, the nucleus and various particulate
cytoplasmic inclusions appear in low relief.
4. Fluorescence light microscopy:

a. It is used to localize inherently fluorescent substance or substance
labeled with fluorescent tags.
b. This microscopy has a high intensity light source and tow special
filters.
1) The excitor filter: located between the light source and the
specimen, blocks all light wavelengths except those that excite the
fluorochrome.
2) The barrier filter: located between the specimen and the ocular,
blocks all light wavelengths except those emitted by the
fluorochrome.
Electron microscopes: illuminate specimens with an electron beam. They
have 1000 times the resolution power of light microscopes and provide resolution
to the threshold of atomic detail. This microscopy is used to view fixed and
sectioned or metal-coated specimens under magnification high enough to resolve
fine details of the specimen.
A. Electron microscope components and image formation:
1. In contrast to light microscopes, electron microscopes illuminate
specimens with a short wavelength stream of electrons rather than photons,
and they form images with magnetic lenses rather than glass lenses.
2. Specimens are illuminated in an evacuated column within the
microscope, because the electron beam would be scattered by air.
3. The magnetic lenses form an image that is displayed on a video screen.
Often, the image is photographed to generate a permanent record. These
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LAB 1
Practical Histology
microscope
electron micrographs show the fine details of a specimen, often to the
molecular level of resolution.
B. Type of electron microscopy:
1. Transmission electron microscope (TEM):
a. TEM uses thinly sliced, plastic-embedded sections that are stained with
heavy metal salts.
b. TEM is used to study the fine details of cell structure, such as the
morphology of cell surface and internal elements of cells; it can
resolve features as small as 0.5 nm.
c. An image is formed by passing a beam of electron through the
specimen and focusing the beam using electromagnetic lenses.
d. Similar arrangement of lenses is used as with optical microscopy;
magnification is up to 400,000 times, which is sufficient to visualize
macromolecules (e.g., antibodies and DNA).
2. Scanning electron microscope (SEM):
a. SEM uses whole specimens that are subjected to critical point drying
and then coated with a thin layer of gold and palladium.
b. SEM is used to study three-dimensional features of cell surface; it can
resolve features as small as 5 nm.
c. The image is formed by electrons that are reflected off the surface of a
specimen, magnification ranges from 1-1000 times.
 How used light microscope:
1. Put the microscope on flat table where the handle toward you.
2. Clean the microscope lenses by special papers and don't touch lenses by
hands.
3. Put the slide on the stage carefully and make sure the cover glass on the slide.
4. Examine the sample by low objective lens (10×) then by high objective lens
(40×) with fine adjustment but without using coarse adjustment to avoid the
breaking of slide.
5. Avoid the use of immersion oil lens without oil and directly clean the lens and
slide from oil after the use.
6. Don't use one eye during the examination of sample.
7. After the finish of examining rise the slide from stage carefully then clean it
and clean the microscope lenses then cover the microscope to keep out it from
dust.
8. During the drawing from microscope mention the magnification power which
is calculated by the formula:
Magnification = Ocular lens × objective lens
 Units of measure:
1.
2.
3.
4.
3
Millimeter (mm) = 1/ 1000 meter, 10-3 M
Micron, micrometer (µm) = 1/ 1000 mm, 10-3 mm, 10-6 M
Nanometer (nm) = 1/ 1000 µm, 10-3 µm, 10-9 M
Angstrom unit (A) = 1/ 10 nm, 10-10 M