Microscopy
MLS 201 Laboratory Instrumentation
Mrs Koto
Learning Outcomes
• To understand the optical principles and
construction of microscopes
• To understand the components, use, and care
of the compound brightfield microscope
• Practical use of the compound microscope for
visualization of cellular morphology from
stained slide preparations
• To carry out maintenance of a microscope
Definition
• A microscope (Greek: micron = small and scopos
= aim)
• MICROSCOPE - An instrument for viewing objects
that are too small to be seen by the naked or
unaided eye
• MICROSCOPY - The science of investigating small
objects using such an instrument
3
Historical background cont..
• Antony van Leeuwenhoek. In 1673, with the aid
of a crude microscope consisting of a biconcave
lens enclosed in two metal plates, Leeuwenhoek
introduced the world to the existence of
microbial forms of life.
• Over the years, microscopes have evolved from
the simple, single-lens instrument of
Leeuwenhoek, with a magnification of 300, to the
present-day electron microscopes capable of
magnifications greater than 250,000.
Historical background
• 1590 - Hans Janssen and his son Zacharias Janssen,
developed first microscope.
• 1609 - Galileo Galilei - occhiolino or compound
microscope.
• 1620 - Christian Huygens, another Dutchman,
developed a simple 2-lens ocular system.
5
Microscope made by Antony van
Leeuwenhoek.
• http://www.britannica.com/technology/microscope
What is a microscope?
Role of a microscopy
• The most used piece of equipment in the lab
• Microscopy is a basic part of work in many
areas of the lab i.e
– Hematology
– Urinalysis
– Microbiology
– Histology/ cytology
– Others
microscopy
macroscopy
Types of microscopes
Light microscopes
• use visible light or ultraviolet rays to illuminate
specimens
a)brightfield,
b)darkfield
c)phase-contrast
d)fluorescence
Electron microscopes
• use electron beams instead of light rays, and magnets
instead of lenses to observe submicroscopic particles.
Magnification
• Degree of enlargement
Magnification is defined by the
magnification by the objective
x
the magnification by eyepiece
BUT maximum magnification does not mean maximum
resolution!
Resolution
• It is the ability to distinguish closely
spaced points as separate.
www.miniaturehorizons.com
Relative resolving power
The closest distance between two objects that
when magnified still allows the two objects be
viewed as separate
• Human eye 0.25mm 0.00025m
• Light microscope 0.25µm 0.00000025m
• Electron microscope 0.5nm 0.0000000005m
Numerical aperture (NA)
• An index or measurement of the resolving
power of a lens
• The greater the NA the greater the resolving
power of a lens
Illumination
• The action of supplying or brightening with
light or the resulting state . www.merriam-webster.com/dictionary/illumination
-Bright light, dark field or florescence
• Effective illumination is required for efficient
magnification and resolving power
• Excessive illumination may obscure the
specimen because of lack of contrast
Contrast
• Contrast is commonly achieved by staining
which highlight the microorganisms or cells to
be differentiated from one another and from
the background or debris
Compound Light Microscope
Compound Light Microscope
• Modern compound light
microscopes, under
optimal conditions, can
magnify an object from
1000X to 2000X (times)
the specimens original
diameter.
Brightfield Microscopy
Brightfield Microscope
• Contains two lens systems for magnifying specimens: the
ocular lens in the eyepiece and the objective lens located in
the nose-piece.
• The specimen is illuminated by a beam of tungsten
• Light focused on it by a sub-stage lens called a condenser
• The specimen appears dark against a bright background.
• Limitation of this system is the absence of contrast
between the specimen and the surrounding medium,
which makes it difficult to observe living cells
• Most brightfield observations are performed on nonviable,
stained preparations
Brightfield Microscope
Components of the Microscope
Components of the Microscope
• Condenser: This is a system of different lens elements which is mounted
beneath the stage of the microscope. It contains an iris diaphragm which
controls the diameter of the light beam. The light beam should be
adjusted to be larger or equal to the numerical aperture of the objective in
use. Condensers can be moved up and down. The normal operating
position is up.
• Base: This is the bottom part of the microscope, it contains the lamp.
• Coarse Focus: Also referred to as rough focus, this knob raises and lowers
the microscope stage quickly. It should only be used in connection with
the low magnification lenses.
• Eyepiece Lens: Also known as ocular lenses, they magnify the image of the
objective. The eyepiece is the lens into which a person looks into when
observing. The total magnification of a microscope is calculated by
multiplying the magnification of the objective by the magnification of the
eyepiece. Many eyepiece lenses have a magnification of 10x ot 15x.
• Fine Focus: This focus knob moves the stage up and down in small steps. It
is used to focus at different layers of the specimens.
Components of the Microscope cont..
• Head: This is the top part of the microscope. It carries the eyepiece(s) and other
optical elements. There are several different types of heads: a monocular head is
designed to carry only one eyepiece, a binocular head carries two (but does not
give stereoscopic vision in compound microscopes) and a trinocular head is
designed to carry a camera as well.
• Mechanical Stage: This type of stage is equipped with a slide holder and two
knobs to turn. One knob moves the stage backwards and forwards, the other one
moves the slide sideways.
• Nosepiece (or revolving nosepiece, turret): This part carries the objectives. It can
be rotated.
• Objective Lens: This is a highly magnifying lens system; it is located close to the
specimen to be observed. The image of the objective is then magnified again by
the ocular lens which is close to the eye.
• Stage: This is the flat surface on which the slides are placed on. It can be moved up
and down for focusing.
• Stage Clips: These are clips that hold the slide.
• Trinocular Head: This microscope head has three exits, two for viewing (for
binocular vision) and a third exit to connect a camera. Some microscopes also
allow for taking photographs through a special adapter at the eyepiece, but a
trinocular head offers more stability and is to be preferred for photographic work.
OBJECTIVE LENS
Mounted on Nose piece
• It forms magnified real image.
Scan - 4X
Low Power - 10X
TYPES
High Power - 40X
Oil immersion - 100X
26
OIL IMMERSION OBJECTIVE
• Highest magnification
• Oil prevents refraction of light outwards and allows
it to pass straight into objective
G
FBEG - OIL D
ABCD - AIR
E
OIL
C GLASS
B
27
A F
28
Koehler illumination
• Köhler illumination was first described in 1893 by
August Köhler, a young zoologist in Giessen,
Germany, who later joined Carl Zeiss
• Koehler illumination is a microscope technique
that provides superior control over the light rays
during brightfield microscopy by aligning and
focusing the microscope,
• ensures the best resolution and contrast,
bright, evenly illuminated background for your
images.
Koehler illumination
• https://www.youtube.com/watch?v=dROpYb
Uj4xE
How to Set Koehler Illumination
1. Adjust the diopters on the
microscope eyepieces to
zero.
Adjust the interpupillary
distance of both eyepieces to fit
your eyes.
http://www.spotimaging.com/resources/whi
te-papers/koehler-illumination.php
Step 2
• Determine which objective
will be used most for your
detailed work. It is
recommended that you
start with a 10X. This will
be the target objective for
aligning the Koehler
illumination.
Step 3
• Place a sample slide on the
stage and bring it into focus with
the selected target objective.
You may have to adjust the
opening of the field diaphragm
to get an edge into the field of
view (this condenser focus point
tends to be near the specimen
end of the condenser focus
travel).
Step 4
• Adjust the condenser
focus knob until the
edge of the field
diaphragm comes into
sharp focus.
http://www.spotimaging.com/resources/white-
papers/koehler-illumination.php#sthash.OGl6CHXU.dpuf
Step 5
• Adjust the condenser
centering knobs until the
field iris is in the center of
the field of view
• Gradually open the field
iris and make final tweaks ttp://www.spotimaging.com/resources/white-papers/koehler-
illumination.php#sthash.OGl6CHXU.dpuf
of the centration with the
field iris at the very edge
of the field of view. Then
open the field iris just
outside the field of view.
Step 6
• The last step is to adjust the
condenser aperture iris to match the
NA (numerical aperture) indicated
on the objective. The aperture iris
can also be stopped down to 60% of
the NA setting (trading image
resolution for contrast.) An
alternative way to set the condenser
aperture is to remove the eyepiece
and look down the eye tube at the
back of the objective and adjust the
aperture diaphragm until
approximately 90% of the area is
illuminated (a setting that visually
looks very near the edge of the
objective but is not quite fully to the
edge
Using a microscope
1. Grip the microscope arm firmly with the right
hand and the base with the left hand, and lift
the instrument from the cabinet shelf. Carry
it close to the body and gently place it on the
laboratory bench
2. Uncoil the microscope's electric wire and
plug it into an electrical outlet.
3. Clean all lens systems lowest to oil
Use of a microscope
4. Place the microscope slide with the specimen
within the stage clips on the fixed stage. Move the
slide to center the specimen over the opening in
the stage directly over the light source.
5. Rotate the scanning lens or the low power lens
into position.
6. While looking through the ocular lens, turn the
coarse focus knob carefully, and slowly move the
stage away from the lens until the specimen comes
into vague focus. Then, use the fine focus knob to
bring the specimen into sharp focus.
Use of a microscope
7. Switch to the high power objective lens only after
adjusting condenser and iris diaphragm.
8. Place a drop of oil over specimen before using oil
immersion objective.
9. Lower the objective until oil makes contact with
objective.
10. Looking through the eyepiece, very slowly focus
the objective away from the slide i.e. by raising
the objective lens.
Shape
• Bacilli • Spiral
Spirillum volutans
• Cocci
• Vibrio
• Filamentous Vibrio cholerae
Shape
Arrangements
• Bunch • Chain
• Pair
• Rosette
Roseobacter spp.
• Sarcinae
Causes of error in focusing
• Revolving Nose Piece is off centre
• Preparation is upside down
• Thick cover slip
• Dirt or Dried oil over Lens
• Air bubble in immersion oil
• Poor illumination – Condenser not fully racked
up
44
Care of the microscope
1. Clean all lenses with dry, clean lens paper. If you
need to, you can use a drop or two of methanol
to help clean the lens.
2. Place the low-power objective in position and
bring the stage and objectives close together.
3. Center the mechanical stage.
4. Coil the electric wire around the body tube and
the stage.
5. Cover the microscope and carry the microscope
to its position in its cabinet in the manner
previously described
Some things not do in
microscopy
• Never clean the lenses of the objectives and
eyepieces with ethanol
• Never dip the objectives in xylene or ethanol
• Never use ordinary paper or cotton wool to clean
the lenses
• Never touch the objectives with your fingers
• Never clean the supports or the stage with xylene
• Never keep the microscope away with immersion
oil on the objective
Factors Affecting the Result of Microscopic
Exams
• Specimen quality
• Clinical presentation of the patients (Rx)
• Presence of the various cellular components
in the background smear
• Smear preparation and staining technique
Darkfield Microscope
Darkfield Microscope
• Similar to the ordinary light microscope; however, the
condenser system is modified so that the specimen is
not illuminated directly.
• The condenser directs the light obliquely so that the
light is deflected or scattered from the specimen
• Specimen appears bright against a dark background.
• Living specimens may be observed more readily with
darkfield than with brightfield microscopy
• Applications include detection of micro-organisms in
unstained smear preparations, blood analysis
Comparison of Light Pathways of
bright field and dark field Microscopy.
Dark field versus bright field
illumination of lens tissue paper
Darkfield Microscope applications
dark-field microscope is used for
• Examining live microorganisms that are either
invisible in the normal light microscope
• Can’t be stained by standard techniques or
• Are so distorted by staining that their
characteristics can’t be recognized
i.e. Treponema pallidum-syphilis
Phase-Contrast Microscopy
Phase-Contrast Microscope
• A unique part of the phase-contrast microscope, called the
phase-plate
• Its optics include special objectives and a condenser that
make visible cellular components that differ only slightly in
their refractive indexes.
• As light is transmitted through a specimen with a refractive
index different from that of the surrounding medium, a
portion of the light is refracted (bent) due to slight variations
in density and thickness of the cellular components.
• The special optics convert the difference between transmitted
light and refracted rays, resulting in a significant variation in
the intensity of light and thereby producing a discernible
image of the structure under study.
• The image appears dark against a light background.
• Observation of microorganisms in an unstained state is
possible with this microscope.
Phase-contrast microscope
Phase-Contrast Microscope
brightfield (left) and with phase
contrast illumination (right)
Phase-contrast microscope
applications
• To study the structure of yeasts , molds and
protozoa
Fluorescence microscopy
Fluorescence microscope
• A fluorescence
microscope is an optical
microscope used to study
properties of organic or
inorganic substances
using the phenomena of
fluorescence instead of,
or in addition to,
reflection and absorption
Fluorescence microscope
Principle
• The fluorescence microscope is based on the
phenomenon that certain material emits
energy detectable as visible light when
irradiated with the light of a specific
wavelength
• In fluorescence microscopy, the sample you
want to study is itself the light source.
• The technique is used to study specimens,
which can be made to fluoresce.
Fluorescence microscope
• Specimens are chemically tagged with a fluorescent dye.
• Light source: ultraviolet (UV) light obtained from a high-
pressure mercury lamp or hydrogen quartz lamp.
• The ocular lens is fitted with a filter that permits the longer
ultraviolet wavelengths to pass, while the shorter wavelengths
are blocked or eliminated.
• Ultraviolet radiations are absorbed by the fluorescent label
and the energy is re-emitted in the form of a different
wavelength in the visible light range.
• The fluorescent dyes absorb at wavelengths between 230 and
350 nanometers (nm) and emit orange, yellow, or greenish
light.
Fluorescence microscope
www.olympusmicro.com
Fluorescence microscope
www.olympusmicro.com
Fluorescence microscope applications
Fluorescent Staining in Tuberculosis
• The Auramine-rhodamine process uses a yellow
fluorescent dye to visualize Mycobacterium
tuberculosis under a fluorescence microscope.
Potassium permanganate or acridine orange can
be used as a counter stain.
• Under the lens, the bacterial cells will appear
green. The Auramine-rhodamine stain is more
sensitive than the Ziel-Neelson and more cost
effective
Fluorescent Staining in Tuberculosis
• Tb fluorescence
Fluorescence microscope
applications cont
Quantitative Buffy Coat Malaria Test
• Uses the Principles of fluorescence
• The QBC Malaria Test is a fluorescence
microscopy-based malaria diagnostic test that
speeds and simplifies malaria detection
Electron microscopy
Electron microscope
• Provides a revolutionary method of
microscopy, with magnifications up to one
million.
• This permits visualization of submicroscopic
cellular particles(i.e.bacteriolophages) as well
as viral agents.
• Transmission & scanning electron microscope
Electron microscope cont
• Specimen is illuminated by a beam of electrons rather than
light, and the focusing is carried out by electromagnets
instead of a set of optics.
• These components are sealed in a tube in which a complete
vacuum is established.
• Transmission electron microscopes require specimens that are
thinly prepared, fixed, and dehydrated for the electron beam
to pass freely through them.
• As the electrons pass through the specimen, images are
formed by directing the electrons onto photographic film
• Internal cellular structures visible.
• Used for visualizing surface characteristics rather than
intracellular structures
• A narrow beam of electrons scans back and forth, producing a
three-dimensional image as the electrons are reflected off the
specimen's surface.
Electron microscope cont
Scanning electron
microscope
• The specimen is put in the
vacuum chamber and
covered with a thin coat of
gold.
• The electron beam scans
across the specimens and
knocks the loose showers
of electrons that are
captured by the detector
and the image is build line
by line
http://www.lab.anhb.uwa.edu.au/hb313/ma
in_pages/timetable/lectures/Image6.gif
SUMMARY
Type Resolving Useful Advantages Disadvantages
power magnification
Bright field 0.2 µm 1000× cells / microorganisms can Cells cells /
be observed in a stained or microorganisms less than
unstained state 0.2 µm in diameter cant
be resolved
Dark field 0.2 µm 1000× Unstained organisms such May be difficult to
as spirochetes whose operate than a bright
diameter is at or just below field due to manipulation
resolving power of bright of condensers
field microscope can be
observed
Phase 0.2 µm 1000× Intensifies contrast between Operation more difficult
contrast dense structures and than bright field
transparent cytoplasm of
live eukaryotic cells and
prokaryotic microbes
Fluorescent 0.2 µm 1000× Important diagnostic tool in Requires training in its
clinical lab operation and evaluation
(immunoflorescence); of results
microorganisms can be
detected in various types of
specimens
Transmission 0.0005 µm 200,000× High resolving power Specimen in living state
electron plus molecular architecture of cannot be examined, 2
cells, organelles and dimensional image
microorganisms can be
identified
Scanning Up to 15,000 – 3-dimensional image, useful Low resolving power,
electron 0.007 µm 50,000× for evaluating surface of specimen must be
cells nonliving state
