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FOUNDATIONS OF ZOOLOGY
LECTURE
MICROSCOPES
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Standard microscopes = 200 nm resolving power.
LIGHT MICROSCOPE
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Simple microscope
Compound Microscope
o Monocular
o Binocular
COMPOUND MICROSCOPE
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Basic Parts:
o Stand (Base)
o Body (Arm/Limb)
o Optical System
o Light/Illumination System
Stand (base) + Body (arm/limb)
o Body Tubes
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External Tube
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Internal Tube (Draw Tube)
o Stage
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Substage
o Adjustment Knobs
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Coarse
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Fine
o Optical System
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Eyepiece
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Objectives
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Condenser
o Light/Illumination System
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Mirror (plane and concave)
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Built-in electrical illumination
PREPARING MICROSCOPIC SPECIMEN: WET MOUNT
USING AND MAINTAINING A MICROSCOPE
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MAGNIFICATION AND RESOLVING POWER OF LIGHT
MICROSOCOPE
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Magnification Power
o Degree of image enlargement
o Depends upon the following:
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Length of optical tube
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Magnifying Power of Eyepiece
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Magnifying Power of Objectives
o Formula: M.P. of Eyepiece x M.P. of Objectives
Resolving Power
o Capacity of optical system to produce separate images
of objects very close to each other.
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Keep the microscope on stable eve surface and in
comfortable position.
Keep dust free.
Obtain appropriate illumination by adjusting the
mirror or intensity of light.
When examining colorless specimens, condenser
should be at the lowest position and iris diaphragm
closed or partially closed.
When using OIO, 100x objective lens should dip in
oil.
After using OIO, the lens of the objective should be
cleaned with tissue paper or soft cloth.
Hold the microscope firmly with one hand on the
arm while supporting the base firmly with one hand
on the arm while supporting the base with the other
hand.
Carry the microscope in front of you with its side
close to your body.
Do not tamper any parts of the microscope.
Never touch the lenses with your fingers.
Never allow liquids, particularly acids to come in
contact with any part of the microscope.
Always use cover slips when examining fresh
mounts.
Never force the lever of the iris diaphragm to the
full limit in either direction when regulating its
opening.
Keep both eyes open when looking into the
microscope.
Return the microscope in upright position with
LPO in place; stage is lowered.
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OTHER TYPES OF MICROSCOPY
DARK GROUND ILLUMINATION (DGI)
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Used for the examination of unstained living
microorganisms.
Principle:
o Microorganisms are illuminated by an oblique ray of
light which does not pass through the microorganism.
o The condenser is blackened in the center and light
passes through its periphery illuminating the living
microorganisms on a glass slide against a dark
background.
TRANSMISSION ELECTRON MICROSCOPE (TEM)
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Helps visualize cell’s cytoplasm and organelles.
Interprets atomic rather than molecular properties of the
tissue and gives two-dimensional image of the tissue.
SCANNING ELECTRON MICROSCOPE (SEM)
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Helps in the study of cell surface.
Produces three-dimensional image on a cathode-ray
oscillograph which can be amplified.
Can also be used for fluorescent antibody techniques.
PHASE CONTRAST MICROSCOPY
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Used for the examination of unstained structures, most
often living cells for assessing their functions at the level of
organelles.
Principle:
o Illumination of varying phase and amplitude is passed
through unstained cells which assesses the difference
in refractive indices of various organelles.
o The organelles shine differently based on what they
are dense/dark (higher refractive index) or less dark
(lower refractive index).
POLARIZING MICROSCOPE
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Used for demonstration of birefringence (e.g. amyloid,
foreign body, hair)
Principle:
o The light is made plane polarized.
o Two discs made up of prisms are placed in path of light:
polarizer and analyzer.
o Polarizer sieves out ordinary light rays vibrating in all
directions allowing light waves of one orientation to
pass through.
o The lower disc (polarizer) is rotated to make the light
plane polarized.
o During rotation, when analyzer comes perpendicular to
polarizer, all light rays are cancelled or extinguished.
o Birefringent objects rotate the light rays and therefore
appear bright in dark background.
RECENT ADVANCES IN MICROSCOPY
TEACHING MICROSCOPES
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POLARIZING MICROSCOPE
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Used for demonstration of naturally occurring fluorescent
material and other non-fluorescent substances or
microorganisms after staining with some fluorescent dyes.
Light source of low wavelength (UV light) is used, most
often mercury vapor lamp or xenon gas lamp.
Principle:
o Illumination of a substance with a low wavelength (e.g.
invisible spectrum) emits light at a higher wavelength
(e.g. visible spectrum), localizing the substance with
fluorescent in the visible range.
o Fluorescent dyes are used depending upon the type of
material to be visualized.
Penta-Headed Microscopes
o Multi-viewing microscope which allows simultaneous
use by one primary user and four other users seated
nearby.
o With the features of infinite optical system, effective
illumination, LED pointer and images coherence.
ELECTRON MICROSCOPE
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Used for the study of ultrastructural details of tissues and
cells.
Tissue is fixed in 4% glutaraldehyde at 4°C for 4 hours;
ultrathin microsections with thickness of 100 nm are cut with
diamond knives.
Principle:
o By using an electron beam of light, the resolving power
of the microscope is increased to 50,000 to 100,000
times, hence very small structures can be visualized.
IMAGE ANALYZERS AND MORPHOMETRY
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Microscopes are attached to video monitors and computers
with dedicated software systems.
TELEPATHOLOGY
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Provision of diagnostic, support, or educational services in
anatomic or clinical pathology by viewing gross or
microscopic images electronically.
Provides access to a generalist or specialist pathologist or
technologist not available on site.
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ROBOTIC TELEPATHOLOGY
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Uses a remote-control device to move the stage of the
microscope or change the microscope field or
magnification.
Image selection at remote site (robotic microscope) is
controlled by the consultant at the hub site.
Advantages:
o Robotic control of remote microscope allows distant
pathologist to view slides completely.
o Real-time
interaction
with
referrer/physician
associate/clinician.
o Allows performance of frozen sections.
Disadvantages:
o Most complex
o Most expensive – requires broad bandwidth
telecommunications.
ROBOTIC TELEPATHOLOGY
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Scans the images and uses high-speed internet server to
transmit the images to another station.
Static (still) images selected by referring provider is sent to
consultant.
Requires skillful image collection by referrer.
Advantages:
o Simple – minimal hardware needs (computer, camera)
o Inexpensive – hardware and image transmission
(internet)
o Adaptable – many computer systems can be adapted.
Disadvantages:
o Image selection – referrer
o Real time interaction – difficult
o Not suitable for primary diagnosis by surgical
pathologist at a distance.
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