Prof. Dr.

Abdel-Nasser Ahmed Hussein

Professor of Zoology (Parasitology)
Zoology Department, Faculty of Science
South Valley University

Lecture No. 3 MICROSCOPY

3rd Lecture 1
Microscopy is any technique for
producing visible images of
structures or details too small to
be seen by the human eye, using a
microscope or other magnification
tool.
It is often used more
specifically as a technique of
using a microscope.
Microscopy has evolved
with the development of
microscopes.
3rd Lecture 2 2
3rd Lecture 3 3
A simple microscope by Leeuwenhoek The first compound microscope by
Zacharias Jansen

This microscope was made for the famous

British scientist Robert Hooke in the late
1600s, and was one of the most elegant
microscopes built during the period.

3rd Lecture 4
Categories of microscopes
Microscopes developed in the 17th century, allowed us to re-
evaluate our definition of life. There are two well-known
branches of microscopy:-
• Light (optical) microscopes (LM)
• Electron microscopes (EM).

The stereo microscope is used for low magnification (5X to 40X). It is

used to view specimens those are visible to the naked eye. The
compound optical microscopes have high magnification up to 2000X,
and used for microspecimens, it has:-
 Bright light source.
 Set of lenses arranged to focus the image.
 A thin enough specimen for the passage of light.
3rd Lecture 5 5
A- Microscopes use visible light as a source of illumination
(1)- Bright field light microscope
(2)- Dark field microscope
(3)- Phase- Contrast microscope
B- Microscopes use ultraviolet light as a source of illumination
(4)- Fluorescence microscope
C- Microscopes use ultraviolet laser as a source of illumination
(5)- Confocal microscope
D- Other types of light microscopes
(6)- Ultraviolet microscope
(7)- Differential interference contrast microscope

3rd Lecture 6 6
A- Microscopes use visible light as source of illumination

In this type of light

microscope (Bright field
microscope), bright light
source and set of lenses,
arranged to focus the
image. The specimen must
be thin enough for passing
the light.
A compound
microscope with a single
eyepiece is known as
monocular, while the one
with two eyepieces is It is best suited to viewing stained or
known as binocular. naturally pigmented specimens.
3rd Lecture 7
 Stereo microscope Compound microscopes

3rd Lecture 8
Sample illumination is via transmitted
white light entering the microscope from a
source in the base, and passes through a
condenser. A condenser converges the
light beam so that they pass through the
specimen
Best compound microscope have a
resolving power of about 0.2 micrometer
and a magnification power of about 2000
times the sizes of the objects. It the most
elementary microscopy technique.

3rd Lecture 9 9
 Paramecium image taken under
light microscope

3rd Lecture 10 10
Advantages of bright field Microscopes:
1- Simplicity of setup with only basic equipment required.
2- No sample preparation required, allowing viewing of
living cells.

Disadvantages (limitations) of bright field Microscopes:

1- Very low contrast of most biological samples.
2- Low apparent resolution due the blur of “out of focus”
material.

3rd Lecture 11 11
Dark field microscopy uses
a certain light source to
minimize the quantity of
directly transmitted light
(i.e. unscattered light)
entering the image, and
only collected light
scattered by the sample.
This is done by
confining the illumination
to a ring of light.
• Special condenser lens
• Opaque disc

3rd Lecture 12 12
In dark field microscope,
a special condenser lens is
used above an opaque
disc at the center, so that
direct rays don’t enter the
objective lens. Only light
scattered by the specimen
enter the objective lens to
form a bright image
against dark background.
Everything is visible
regardless of color. It
provides outline of
specimen with reduced
internal cellular detail
It is used in microbiology and in autoradiography.
3rd Lecture 13
 Paramecium image taken
under dark field microscope

3rd Lecture 14 14
Advantages of dark field microscope:
1- Clearly shows even transparent objects in the sample.
2- Simplicity of setup with only basic equipment required.
3- No sample preparation required, allowing viewing of
living cells.

Disadvantages of dark field microscope:

1- Low light intensity in final image of many biological
samples.
2- Low apparent resolution due the blur of “out of focus”
objects.

3rd Lecture 15
An electron microscope is a type of
microscope that uses electrons to
illuminate a specimen and create an
enlarged image.

The wavelength of the light limits the

resolution to around 0.2 micrometers
in light microscopy. In order to gain
higher resolution (0.005 um), an
electron beam with a shorter
wavelength is used in electron
microscopes.

3rd Lecture 16
 History of EM

3rd Lecture 17
 A sketch from Ernst Ruska's
laboratory book, depicting the
design of an early TEM prototype.
With this group developing the first
TEM with resolving power greater
than that of light in 1933 and the
first commercial TEM in 1939.

3rd Lecture 18
The General Principle of EM
 A stream of electrons is shot towards the specimen.
 The stream is focused into a thin beam using the
apertures and electromagnetic lenses of the microscope.
 The electron beam hits the specimen, and the
microscope records how the electrons react.
 The electrons may pass through the specimen, or bounce
off and scatter. Every interaction gives information
about the structure and shape of the specimen. The
microscope detects and measures this electron activity
and a picture is created from these informations.

3rd Lecture 19
Type of electron microscopes
There are two main types of EMs:-
Transmission electron microscopy (TEM)
It produces a higher magnification of structures by
sending an electron beam through a very thin slice
of the specimen.
Scanning electron microscopy (SEM)
It visualizes details on the surfaces of specimens and
gives a very good picture.

3rd Lecture 20
 Transmission electron microscopy (TEM)
Transmission electron
microscopy (TEM) is
principally quite similar to the
compound light microscope,
by sending an electron beam
through a very thin slice of the
specimen. A transmission
electron microscope can
achieve better than 50 pm
resolution and magnifications
of up to about 10,000,000X.

-‫ يستخدم لقياس أقطار الذرات واألبعاد الذرية‬:)Picometre( ‫بيكو متر‬

.‫ تعادل واحد من ترليون من المتر‬-‫ متر‬12−10 = ‫ بيكومتر‬1
3rd Lecture 21
 Transmission electron microscopy (TEM)
Structural parts of TEM

(a) Electron gun

It consists of a tungsten filament (or
cathode) that emits electrons on
receiving high voltage electric current
(50,000-100,000 volts). Near the top of
the tube there is an anode, which attracts
electrons.
(b) Ray tube (Microscope Column)
It is a high vacuum metal tube (2m. high)
through, which electrons travel.
(c) Condenser lenses
It is the electromagnetic coil, which
focuses the electron beam in the plane of
the specimen.
3rd Lecture 22
(d) Objective lens
It is the electromagnetic coil, which produces the first magnified
image.
(e) Projector lens
It is also an electromagnetic coil, which further magnifies the first
image formed by the objective lens and produces the final image.
(f) Fluorescent Screen or Photographic Film
Since unaided human eye cannot observe electrons, therefore, a
fluorescent screen is used for viewing the final image of the
specimen. The final image obtained is called an electron
micrograph.

Each electromagnetic coil has a coil of wire encased by a soft iron casing
3rd Lecture 23
 Microtubule-triplets in centriole- TEM An erythrocyte- TEM

Transmission electron microscopes produce two-dimensional, black and white images

3rd Lecture 24
Advantages of TEM
1. TEM has a high magnification and resolving power.
2. Without the aid of TEM, biologists would have never
known submicroscopic cell organelles (e.g., ribosomes,
micro-bodies, centrioles, microtubules, endoplasmic
reticulum) and internal structure of microscopic organelles
(e.g., chloroplasts, mitochondria).
3. Study of microorganisms and viruses have been possible
only with the aid of TEM.
4. It can discern even macromolecules. This has helped
scientists to know the arrangement of molecular
aggregates and even their components, e.g. nucleosomes.

3rd Lecture 25 25
Disadvantages of TEM
1- It is complicated and costly.
2- There is risk of radiation leak.
3- Require very high voltage electric current.
4- A cooling system is required.
5- The specimen or object has to be given special treatment
including complete dehydration. Sections of biological
specimens may require special treatment with heavy atom labels
in order to achieve the required image contrast.
6- The major disadvantage of the transmission electron microscope
is the need for extremely thin sections of the specimens,
typically about 100 nanometers. Electrons are unable to pass
through thick specimens. Biological specimens are typically
required to be chemically fixed, dehydrated and embedded in a
polymer resin to stabilize them sufficiently to allow ultrathin
sectioning.
3 Lecture
rd
26 26
7- Resolution of the TEM is limited primarily by spherical
aberration, but a new generation of aberration correctors has
been able to partially overcome spherical aberration to increase
resolution.
Hardware correction of spherical aberration for the high-
resolution transmission electron microscopy (HRTEM) has
allowed the production of images with resolution below 0.5
angstrom (50 pm) and magnifications above 50 million times.
The ability to determine the positions of atoms within
materials has made the HRTEM an important tool for
Nanotechnologies research and development.

Spherical aberration?

3rd Lecture 27 27
Spherical aberration is an optical effect observed in an optical
device (lens, mirror, etc.) that occurs due to the increased
refraction of light rays when they strike a lens.
Or a reflection of light rays when they strike a mirror near its
edge, in comparison with those that strike nearer the center.
On top is a depiction of a perfect lens without spherical aberration: all incoming rays are focused in
the focal point.
The bottom example depicts a real lens with spherical surfaces, which produces spherical
aberration: The different rays do not meet after the lens in one focal point. The further the rays are
from the optical axis, the closer to the lens they intersect the optical axis (positive spherical
aberration).

The blurring of an image that occurs when light from the margin of a lens or mirror with a
spherical surface comes to a shorter focus than light from the central portion.

spatial variation
Spatial variability occurs when a quantity that is measured at different spatial locations exhibits values
that differ across the locations. Spatial variability can be assessed using spatial descriptive statistics such
as the range.

3rd Lecture 28
Scanning electron microscopy (SEM)
Scanning electron
microscopy (SEM) uses
electrons that are scattered
or emitted from the
surface of the object. The
SEM allows viewing the
surfaces of specimens
without sectioning.
It is used to study
the three dimensional (3D)
images of the surfaces of
cells, tissues or particles.

3rd Lecture 29 29
Structural parts of SEM
Essential components of all SEM
include the following:
 Electron source ("Gun")
 Electron lenses
 Sample stage
 Detectors for all signals of
interest
 Display / Data output devices
 Infrastructure requirements
 Power supply
 Vacuum system
 Cooling system
 Vibration-free floor
 Room free of ambient
magnetic and electric fields.
3rd Lecture 30 30
 Blood corpuscles- SEM Schistosoma- SEM

3rd Lecture 31 31
Advantages of SEM
1- Most SEM's are comparatively easy to operate.
2- Many applications require minimal sample preparation.
3- For many applications, data acquisition is rapid.
4- Modern SEMs generate data in digital formats are highly portable.
5- Because the SEM image relies on surface processes, it is able to
image bulk samples up to many centimeters in size and has a
great depth of field, and so can produce images that are good
representations of the three-dimensional shape of the sample.
6- Another advantage of SEM is its variety called environmental
scanning electron microscope (ESEM) can produce images of
sufficient quality and resolution with the samples being wet or
contained in low vacuum or gas. This greatly facilitates imaging
biological samples that are unstable in the high vacuum of
conventional electron microscopes.

3rd Lecture 32 32
Disadvantages of SEM
1- Generally, the image resolution of an SEM is poorer than that of a
TEM.
2- Samples must be solid and they must fit into the microscope
chamber. Maximum size in horizontal dimensions is usually on
the order of 10 mm; vertical dimensions are generally much more
limited and rarely exceed 40 mm. For most instruments samples
must be stable in a vacuum.
3- Samples likely to outgas at low pressures (rocks saturated with
hydrocarbons, "wet" samples such as coal, organic materials or
swelling clays, and samples likely to decrepitate at low pressure)
are unsuitable for examination in conventional SEM's. However,
"low vacuum" and "environmental" SEMs also exist, and many of
these types of samples can be successfully examined in these
specialized instruments.
3rd Lecture 33
 Differences between light (optical) microscope and electron microscope

Electron microscope Light microscope

High level of technical skill needed Techniques are simple 1
Only dead and dried specimens can be seen Live or dead specimens may be seen 2
Specimen preparation normally often takes a several Specimen preparation normally takes a few 3
days minutes to few hours
Specimen requires special stains or treatment Not always specimen requires special stains or 4
treatment
Specimen place on holders or copper mesh Specimen place on glass slide 5
Operates at high vacuum No vacuum is needed 6
Electron beam produced by the cathode is used to Visible light rays are used to illuminate the 7
produce image specimen
All lenses are electromagnets Condenser, objective and eye piece lenses are made 8
of glass
It has high resolving power i.e., about 250 times more It has low resolving power i.e., below 0.25 µm to 9
than light microscope. Thus it can resolve down to 0.30 µm.
0.0001 µm.
Useful magnification 1,000,0000 or more Useful magnification 2,000X 10
The image formation is due to electron scattering The image formation depends upon of light 11
absorption in different zones of the objects
Good surface details (SEM) or internal details (TEM) Surface view of the specimen is poor 12
can be seen
The image is viewed through zinc sulphate fluorescent Image viewed by eyes through glass ocular lens. No 13
screen on photographic plate screen is needed
Image is black and white Image is coloured (natural colour of object is seen) 14
Very costly and heavy running cost Cheap and negligible running cost 15
Affected by magnetic field Not affected by magnetic field 16

3rd Lecture 34 34
 Outlines of lecture 3
1- Microscopy.
2- Light microscope and its types.
3- Electron microscope and its types.
4- Study of different types of light microscope
5- Student research projects (dark-field, phase-contrast microscopes)

Dark field microscopes

&
Phase contrast microscopes

3rd Lecture 35
 ‫المصطلح‬ ‫‪Expression‬‬
‫االش‪X‬وريين (آش‪X‬ور‪ ،‬أول دول‪X‬ة ق‪X‬امت في مدين‪X‬ة آش‪X‬ور في ش‪X‬مال‬ ‫)‪Assyrians (Assyria‬‬
‫بالد ما بين النهرين)‬

‫‪Glossary and Terminology‬‬ ‫يخترع‬ ‫‪Invent‬‬

‫براء اختراع‪/‬االختراع المسجل‬ ‫‪Patent‬‬
‫يوجه نحو نقطة‬ ‫‪Converges‬‬
‫قدرة تحليلية ‪ -‬قوة التبيين ‪-‬قوة التمييز‬ ‫‪Resolving power‬‬
‫غشاوة‪ -‬رؤية غير واضحة‬ ‫‪Blur‬‬
‫الضوء الصادر\ المقذوف‬ ‫‪Emitting light‬‬
‫غامض او يحجب‬ ‫‪Obscure‬‬
‫يشوه الصورة او يشوش الصوت‬ ‫‪Distort‬‬
‫تأثير الهاله‪ -‬هلة القداسة‬ ‫‪Halo effect‬‬
‫تاثتير الظل‬ ‫‪Shade-off‬‬
‫حصر‬ ‫‪Confining‬‬
‫يحدد قوة االنكسار‬ ‫‪Refractive index‬‬
‫تباين‬ ‫‪Contrast‬‬
‫المجهر الضوئي ذو األطوار المتباينة‬ ‫‪Phase contrast microscopy‬‬
‫ضوء فوق بنفسجى‬ ‫‪Ultraviolet light‬‬
‫فى الجسم الحى‬ ‫‪In vivo.‬‬
‫فى المختبر‬ ‫‪In vitro‬‬

‫‪3rd Lecture‬‬ ‫‪36‬‬

 Questions about lecture 3
Choose the best answer (MCQ)
1- Optical microscope consists of
A- Bright light source B- Set of lenses C- A thin enough specimen D- All of mentioned
2- To produce an image source of illumination in light microscopes is
A- Visible light B- Ultraviolet light C- Ultraviolet laser D- All of mentioned
3- To produce an image source of illumination in fluorescence microscopes is
A- Visible light B- Ultraviolet light C- Ultraviolet laser D- All of mentioned
4- To produce an image source of illumination in ultraviolet laser microscopes is
A- Visible light B- Ultraviolet light C- Ultraviolet laser D- All of mentioned
5- Consists of a tungsten filament that emits electrons on receiving high voltage electric
current, it is:
A- Electron gun B- Ray tube C- electromagnetic lens D- Fluorescent Screen
6- A high vacuum metal tube through which electrons travel, it is:
A- Electron gun B- Ray tube C- electromagnetic lens D- Fluorescent Screen
7- In TEM, to view the final image of the specimen uses:
A- Electron gun B- Ray tube C- electromagnetic lens D- Fluorescent Screen

3rd Lecture 37
Which of the following is true (√) or false (×) (Read and answer true or false )
1- Microscopy is any technique for producing visible images of structures or
details too small to be seen by the human eye.
2- Light microscopy employs visible light to detect small objects.
3- Electron microscopy uses of an electron beam with a far shorter wavelength to
gain higher resolution of small objects.
4- SEM get a higher magnification of fine structures
5- TEM visualizes details on the surfaces of specimens
6- One of limitation of compound light microscope is very low contrast of most
biological samples
7- One of limitation of compound light microscope is simplicity of setup
8- By dark field microscopy everything is visible usually bright white against a
dark background.
9- One of limitation of dark field microscope is low apparent resolution
10- TEM visualizes details on the surfaces of specimens
11- SEM produces a higher magnification of structures by sending an electron
beam through a very thin slice of the specimen
12- An electron microscope is a type of microscope that uses electrons to
illuminate a specimen.
338
rd
Lecture 38
3rd Lecture 39 39