MICROSCOPY AND OVERVIEW OF HISTOLOGY AND ITS METHODS

OVERVIEW OF HISTOLOGY - NA is engraved on the side of the

objective lens
AND ITS METHODS 4. Parfocal
I. MICROSCOPY - means that the microscope remains
Microscope focused when one switches from one
- an instrument that magnifies an image and objective to the next objective
allows visualization of greater detail than is 5. Total Magnification
possible with the unaided eye - The magnification obtained by the objective
multiplied to the magnification of the ocular
It as two major types:
Ex: low-power obj. (10X) x ocular (10X) = 100X
● Simple microscope (i.e., magnifying lens)
● Compound microscope
B. Parts of a Microscope

A. Concepts:
1. Resolution
- The ability to see clearly two items as
separate objects
- Refers to how clear the produced Optical parts
images of two distinct - used to view, magnify, and produce an image
objects/specimen are from a specimen placed on a slide
2. Resolving Power
- the ability of a microscope lens or an 1. Ocular - the lens closest to the eyes
optical system to produce separate - it magnifies up to 10X
images of two closely positioned
objects 2. Nosepiece
- refers to the size of the smallest object - a movable circular structure that houses all
that can be seen with the lens the objective lenses
- based on the wavelength of the light - connected to the body tube and lies just
used and the numerical aperture of the above the stage
lens - can be rotated clockwise or
3. Numerical Aperture counterclockwise to increase or decrease
- ability to gather light and to resolve the magnification
fine specimen detail while working at a - change in magnification results due to a
fixed object distance change in the objective lens
- Refers to the widest cone of light that
can enter the lens
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 MICROSCOPY AND OVERVIEW OF HISTOLOGY AND ITS METHODS
3. Objective lenses
- the lenses closest to the object and are
fitted on the nosepiece
- are color-coded and are of different sizes:
a. Scanning lens (4X)
b. Low-power lens (10X)
c. High-power lens (40X) -
d. Oil immersion lens (100X)
● Smallest lens is of the lowest power, and
gradually, the longest will be of the highest
power
● The high power lenses are retractable, i.e.,
their end can be pushed inward
4. Fine Adjustment Knob
- a smaller knob and is used to move the
stage up or down very slowly
- used to sharpen the image
- mostly used while viewing under high
power.
5. Coarse Adjustment Knob
- used for focusing the image under low
power magnification
- a larger knob and is used to move the
stage up or down very rapidly.
6. Stage - the section in which the specimen is
placed for viewing.
● Mechanical stage - the most
common type of stage
- allows the control of the slides by
moving the slides using the
mechanical knobs instead of
moving them manually
● Stage clips - hold the specimen
slides in place
7. Stage Control Knobs – used to move the
stage mechanically, in the field of vision.
There are two knobs:
● for moving left and right
● for moving forward and backward
8. Aperture - a hole in the microscope stage
through which the transmitted light from the
source reaches the stage.
9. Microscopic illuminator - a light source.
● Mirror - reflects the light from an
external source to the sample
● Electric light bulbs with low
voltages - used as a constant light
source
10. Condenser
- lenses that are used to collect and focus
light from the illuminator into the specimen
- condenses light rays to a strong beam
- play a major role in ensuring clear, sharp
images are produced with a high
magnification of 400X and above
- the higher the magnification of the
condenser, the clearer the image
- found under the stage next to the
diaphragm
11. Condenser focus knob – moves the
condenser up or down, thus controlling the focus
of light on the specimen.
12. Diaphragm
- located under the condenser; controls the
amount of light coming through it
- controls the the size of the beam and the
amount of light (intensity) that reaches the
specimen
13. Rack stop – controls how far the stages
should go, preventing the objective lens from
getting too close to the specimen slide
Structural Parts
1. Head
- a cylindrical metallic tube that holds the
eyepiece lens at one end and connects to
the nose piece (holds the objectives)
- also called a body tube or eyepiece tube
- light coming from objectives will bend
inside this tube
2. Arm
- connects the base to the head and the
eyepiece tube to the base of the
microscope
- supports the head of the microscope and is
also used when carrying the microscope.
3. Base
- lowermost part of the microscope that
supports the entire microscope structure
- provides stability for the microscope
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 MICROSCOPY AND OVERVIEW OF HISTOLOGY AND ITS METHODS

- useful in demonstrating specific bacteria,

C. Types of Microscope
(Light Microscopy)
1. Light Microscope - widely used by students
and researchers. Parts include: light source,
condenser lens, stage, objective lens, ocular
lens.
2. Phase Contrast Microscope
- enables examination of unstained cells and
tissue
- especially useful for living cells
- shows a halo around the specimen
- can highlight the special parts/morphology of
the subject
Two modifications of PCM:
a. Interference Microscope
- allows quantification of tissue mass
b. Differential Interference Microscope
- useful for assessing surface properties of
cells and other biologic objects
- uses Nomarski optics
particularly, Treponema pallidum (spirochetes that
cause syphilis)
4. Fluorescence Microscope
- uses UV light as a light source (this increases
the resolution of the object)
- used to display autofluorescent molecules:
● Vitamin A
● some neurotransmitters
- widespread application if on the detection of
antigens or antibodies in immunocytochemical
procedures
- Help identify unknown bacteria
- A higher level of microscopy
Example: The direct fluorescent antibody test of
brain tissues from animals suspected to be
infected with rabies. Apple green fluorescence
indicates positive dFA, brain tissues contain rabies
antigen. No fluorescence or no staining indicates
rabies virus is absent.

● Fluorescent antibody technique

- technique that employs fluorescent dyes
and antibodies to help identify unknown
bacteria

3. Dark-field Microscope Examples of Fluorochromes:

- useful in examining
autoradiographs, in
which the developed
silver grains appear
white in a dark
background
- contains a special condenser 5. Confocal Scanning Microscope
that scatters light and causes it
to reflect off the specimen at an angle
- useful in examining urine crystals
a. FITC (Fluorescein Isothiocyanate) -
Apple Green
b. Acridine - Orange
c. Rhodamine B - Red
d. Auramine O - Yellow
- Allows visualization of biological specimen in
three dimensions (3D)
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 MICROSCOPY AND OVERVIEW OF HISTOLOGY AND ITS METHODS

- Combines components of a light optical

microscope with a scanning system to dissect 2. Scanning Electron Microscope (SEM)
a specimen optically. - electron beam does not pass through the
specimen but is scanned across its surface
6. Ultraviolet microscope - whole objects are used, and gold or
- uses quartz lenses with a ultraviolet light palladium staining is employed
source - has lower magnifications compared to
- Image depends on the absorption of UV lights TEM, but permits three‐dimensional views
by the molecules in the specimen of microorganisms and other objects
- May achieve a 0.1m and resemble the
workings of a spectrophotometer (an
instrument that measures the amount of
photons or the intensity of light absorbed after
it passes through sample solution)
- useful in detecting nucleic acids, esp. Purine
and pyrimidine

7. Polarizing MIcroscope
- a simple modification of the light microscope
- polarizing filter (the polarizer) is located
between the light source and the specimen
- a second polarizer (the analyzer), which is an
additional source of light, is located between the
objective lens and the viewer

D. Types of Microscope
(Electron Microscope)
Two types of Electron Microscope:
1. Transmission Electron Microscope (TEM)
- uses the interaction of a beam of electrons
with a specimen to produce an image
- ultra thin slices of microorganisms or
viruses are placed on a wire grid and then
stains them with gold or palladium before
viewing
- densely coated parts of the specimen
deflect the electron beam, and both dark
and light areas show up on the image.

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 MICROSCOPY AND OVERVIEW OF HISTOLOGY AND ITS METHODS
II. TISSUE PREPARATION
The ideal microscopic preparation is preserved in Histopathologic techniques:
1. Fixation
- Small pieces of tissues are immersed in a
solution called fixative after removal from the
body
○ Fixative: 10% Neutral buffered
formalin
○ Ratio: 20:1
- To avoid tissue digestion by enzymes present
within the cells or bacteria
- To preserve cell and tissue structure
Most important step
2. Dehydration
- Tissue is transferred through a series of
increasingly concentrated alcohol solutions
called as dehydrating agents, ending in 100%,
which removes all water.
Regular interval: 75% → 95% → 100%
- sample is introduced first to a low-
concentrated alcohol solution, then to a higher
one.
- sample is immersed for 3-5 hours or even up
to 24 hours
○ Ratio: 10:1 (10 is the volume of the
solution)
○ Solution: Ethanol
3. Clearing (Dealcoholization)
- Alcohol is removed in the tissue by immersing
in a clearing agent called xylene.
○ Fluid to tissue ratio: 10:1
4. Infiltration (Impregnation)
Tissue is then placed in melted paraffin until it
becomes completely infiltrated with the substance.
❖ Paraffin wax - put in the spaces if the
sample for easy cutting in the next
procedures
5. Embedding
- The paraffin- infiltrated tissue is placed in a
mold with melted paraffin and allowed to
harden
- Note: The medium used in infiltration of tissue
is the same medium used for embedding.
6. Trimming
- The resulting paraffin block is trimmed to
expose the tissue for sectioning (slicing) on a
microtome.
- The 4th and 6h steps are preparatory steps for
the 6th (Trimming)
7. Section-Cutting
- Tissue block is sliced into thin films using a
microtome which are placed on glass slides
and allowed to adhere, deparaffinized, and
stained.
8. Staining
- Methods of staining have been devised that
not only make the various components
conspicuous but also permit distinctions to be
made between them.
- Cell components with a net negative charge
(anionic) stain more readily with basic dyes
and are termed basophilic
- Cationic components (positively charged) have
affinity for acidic dyes and are termed as
acidophilic
❖ H&E
H - Hematoxylin: dye the nuclei
Color: blue black or dark blue
E - Eosin: dye the cytoplasm
Color: Pale pink
9. Mounting
- Stained tissue slides are mounted with a
cover slip using a mounting media.
❖ Canada Balsam - preserves the sample;
looks like a clear nail polish
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 MICROSCOPY AND OVERVIEW OF HISTOLOGY AND ITS METHODS

10. Labeling
- Tissue slides are labeled on the frosted
areas with assigned tissue numbers or
codes
- Tissue slides should be properly labeled.

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