EXERCISE 1

Electron Microscopy and Cell Ultrastructure

by Reamillo, MCS, Mendoza, JC, Diaz, MGQ, and Manuel, MCC. 2009.
A. ELECTRON MICROSCOPY
Principle of Electron Microscopy
As the science of Cell Biology and related fields progressed, it became
evident that there are smaller and more intricate structural details of
the cell that need to be probed. Though light microscopy can produce
highly magnified images with the aid of modern computing, it has
practically reached the limit of its usefulness due to the inability to
produce a corresponding increase in its effective resolution.
To review, the light microscope uses sunlight or a light bulb as a source
of illumination. A condenser lens concentrates the light and directs the
concentrated light to the specimen to produce an image. The light
then passes through the specimen and is captured by the objectives,
which consists of a series of lenses that initially magnify the image.
The image is then further magnified by the ocular (i.e. eyepiece) before
its light rays enter the eye and is perceived by the brain. Depending on
the objective and ocular used, a final magnification of roughly 20-2000
times the original size can be achieved. With the aid of modern
technology, images from light microscopes can now be relayed to
computer systems allowing digital magnification without limits.
An image is formed when light is differentially absorbed by the
specimen. This image may be coloured if the specimen is stained.
Unfortunately, clarity and magnification are not synonymous. Not
only must the microscope be able to magnify the image but it must also
be able to resolve the image so as to show the different of the image as
distinct and separate from one another.
The resolution (or resolving power) of a microscope refers to its ability to
distinguish two adjacent point as separate and distinct. The distance
between two adjacent points is referred to as the limit of resolution (r).
The smaller the limit of resolution, the higher is the resolving power of
a microscope, and vice versa. However, the limit of resolution depends
on two factors; namely 1) the wavelength of radiation () used to
illuminate the specimen, and 2) the numerical aperture (NA) of the
objective used. This relationship is depicted in the following formula:

0.61
NA

From the given formula, there are two ways by which the limit of
resolution of a microscope can be reduced; either is decreased or NA
is increased. The shortest wavelength of visible light is approximately
0.4 micrometer (m) and the biggest numerical aperture is that of an oil
immersion objective measuring 1.40. Using these values on the above
equation would yield a limit of resolution of 0.17 m. Thus, two points
that are less than 0.17 m apart cannot be resolved anymore by any
light microscope.
Since the numerical aperture is limited by the refractive index of the
imaging medium between the specimen and front lens of the
objectives, this determined of the limit of resolution cannot be further
increased to achieve higher resolution. To circumvent this limitation,
efforts were focused on achieving higher microscopic resolution
through the use of shorter wavelengths of radiation. This is where
electrons entered the picture.
Elections have corpuscular and vibratory properties similar to light.
However, their wavelength is not only much shorter than visible but
light but are also variable relative to the voltage used to excite them.
For example, elections emitted at 300,000 volts (V) have a wavelength
of 0.000004 (0.004 nanometer, nm). The relationship between and the
wavelength of an electron beam is depicted in De Broglies equation
below:
=

12.2 x 0.1m
V

where V is the working voltage.

By providing a much smaller
wavelength, electron beams could then immensely increase the
resolving power of the microscope (i.e. by decreasing r) making further
magnification more meaningful.
In the 1920s, experimental work on electron microscopy was conducted
using electromagnet to focus an electron beam. By the early 1030s,
Knoll and Ruska developed the prototype election microscope, which,
thought difficult to operate, had greater magnification and better
resolution.
Their work further inspired the production of the first
commercial electron microscope by Simmens Co. (Germany) in 1939.
From then on, many improvements in the design of the election
microscope and in the technique of election microscopy have been seen
by the scientific community.

To further appreciate the principles of electron microscopy, it is

profitable to look at it side by side with light microscopy. Figure 1
shows a comparison of the principles of image formation in a typical
compound light microscope, a transmission election microscope (TEM)
and a scanning electron microscope (SEM).
In contrast to light microscopy, a cathode filament serves as a source of
radiation (i.e. electrons) in a typical electron microscope. Since glass
lenses are opaque to electron beams (i.e. electrons cannot pass
through glass), electromagnetic coils are used to direct the electron
beams through a vacuumeven gas particles can deflect electrons and
affect the quality of image produced.
Thus, the emitted electrons
from cathode filaments are concentrated into laminar electron beams
by a condenser coil, which directs them to the specimen. These electron
beams then produce an image of the specimen, which is magnified by
objective coils. In some electron microscope, a projector coil is present
providing further magnification of the image produced. These
electrons are then converted into electrical signals that are amplified
and are displayed as grayscale images on either a cathode-ray tube
(CRT) or a liquid crystal display (LCD) monitor.
Image formation, however, differs in different types of electron
microscope. This exercise will focus on the two most common types,
namely the transmission electron microscope (TEM) and the scanning
electron microscope (SEM).

Figure1. Image formation in (a) light microscope, (b) a transmission

electron microscope, and (c) a scanning electron microscope. Image
Lifted from: http://www.jeol.co.jp/en/science/product_file/file/en_sc7-2.jpg
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In transmission electron microscopy, an image is produced based on

differential electron absorbency of a specimen. Electrons cannot pass
through thick specimens and specimen region with dense atoms and
therefore scattered; this result in the formation of a uniformly dark
image. This, therefore, necessitates that specimens for TEM should be
extremely thin (50-100 nm in thickness). Electrons that manage to pass
through the specimen are then directed to a zinc sulphide (ZnS) screen
by the projector coil. This ZnS screen then emits white light as
electrons strike its surface. Hence a dark image is produced from dense
regions due to the absence of transmitted electrons from that region
onto the ZnS screen. The image becomes lighter as the density of the
specimen decreases due to a corresponding increase in the number of
electrons hitting the ZnS screen. The image produced on the ZnS
screen can then be observed through a built-in viewer or be
photograph thru a built-in camera.
In scanning electron microscopy, an image is produced based on
different electron scatting of a specimen. Primary electrons from a
cathode filament are directed to a specimen coated with heavy metals.
This causes the release of secondary electrons from the heavy metal
coating. The amount of secondary electrons released is dependent on
the angle at which the specimen is struck by primary electrons. As a
rule of thumb, the higher the altitude of an area of the specimen the
more secondary electrons are released. These secondary electrons
pass through a scintillator where they are converted into light
signals proportional to the number of detected electrons. The light
signal released is magnified or amplified by a photo-multiplier
before they are projected on a cathode-ray or TV tube for viewing for
image capture. The resulting image shows bright regions representing
elevated surface, and dark regions representing depressed surfaces
creating a three-dimensional image. There are built-in adjustments in
the SEM that the specimen allowing its observation from different
angles.
The TEM is used to study cross-sectioned specimens and produces
two-dimensional images. Three-dimensional effects, though possible,
require special sample preparation techniques. On the other hand, the
SEM has been designed to produce 3-D projections of surface areas of
cell and organism. In other words, the SEM is not for studying cross
sections but for studying topography. Compared to a mapmaker, the
TEM simply marks on the map where the mountains and valleys are,
while SEM shows what the mountains and valleys look like.
In terms of dimensions and capabilities, the SEM is usually smaller
than the TEM and has a relatively lower resolving power. With special
electron emitters such as lanthanum hexaborite, however, the
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resolution could be improved to as low as 3.0 nm. Magnification is in

the order of 20,000x. Newer models could magnify up to 100,000x but
this is not very helpful due to poor resolution. Hence, with this
limitation, the best application of the SEM is from single cell to small
organisms.
So how does the resolution of the electron microscope compare with
the light microscope? The great aberrations of the electromagnetic coils
render NA quite small. But the very small wavelength of electron
beams more than compensates for that, such that, at 100,000 V, a limit
of resolution as low as 0.1 nm is achieved. However, for biological
specimens, other problems lower the effective resolution to around
2nm. These include among other sample preparation and contrast.
Despite this, the resulting effective resolution of an electron is still
more than a hundred times better than of the best light microscope.
Image magnification in electron microscopy can reach up to about
20,000 times without any significant loss of resolving power, with
power models being capable of magnification of up a million times!
In summary, though the electron microscope has the same basic plan
as the light microscope, the use of electron beams necessitated several
changes that rendered it quite different. These differences are
summarized in Table 1.
Table1. A comparison of the light microscope (LM), the transmission electron
microscope (TEM), and the scanning electron microscope (SEM).
CRITERION

LM

TEM

SEM

Radiation

light

electrons

electrons

Lenses

glass
lenses

Electromagnetic
coils

electromagnetic
coils

Casing

open to air

vacuum

vacuum

Image
formation

Differential
light
absorption

Differential
electron
absorbency

Differential
electron
scattering

Viewing

naked eye ZnS

screen CRT monitor
monitor
monitor
0.17 m
0.10 nm (2 nm 3nm
for
Biological
samples)
20-2,000x
20,000-160,000x 20,000-100,000x

Resolution

Magnification

REMARKS
electrons
have shorter

glass is
opaque to
electrons
air deflects
to electrons
dense
regions
scattering
electrons

Specimen Preparation for Electron Microscopy

By virtue of the ultra-thin sections needed for transmission electron
microscopy and the vacuum requirement, the specimen must be thinly
sectioned and dried. To meet these criteria, several steps are routinely
employed in ordinary transmission electron microscopy.
These are
fixation, washing and dehydration, embedding, sectioning, and
staining.
1. FIXATION
The goal of fixation is to skill the cells instantly and with minimal
structural alterations. This is done by applying non-coagulant
fixatives, which form cross-links between neighbouring molecules.
Glutaraldehyde, for example, covalently groups proteins while
osmium tetroxide groups lipids and tissue proteins.
2. WASHING AND DEHYDRATION
This has to be done because the embedding medium for the next
step (the resin) is immiscible with aqueous fixatives. One has to
use an intermediate solvent miscible with both water and resin.
Here the tissue is first washed with deionized water to remove
excess fixative and then the water is removed either by passing it
through increasing concentrations of ethanol or acetone until pure
dehydrating agent replaces water completely.
3. EMBEDDING
The embedding medium is made up of acrylic monomers or epoxy
resins, which, in the liquid states, is allowed to replace the
dehydrating agent until it has seeped into the interstices of the
tissue. After such infiltration, the monomers or resins are
polymerised to harden into a solid block or plastic. Hardness of an
embedded specimen is crucial to sectioning.
4. SECTIONING
This is done to provide ultra-thin cuts that would be penetrable to
electrons. Sections around 50-100 nm thin or around 1/200 the
thickness of a cell should be made. A special cutting device called
an ultramicrotome cuts the specimen with its very sharp glass or
diamond blade. Two crucial factors should be noted namely proper
embedding and sharpness of the cutting edge. The procedure
showing the tip of the specimen block being sectioned by the
cutting edge is shown Figure 2.
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RIBBON ON
WATER

Figure
2.
Ultrathin
sectioning.
Image
lifted
from
http://www.biologydiscussion.com/wp-content/uploads/2014
/09/clip_image014_thumb4.jpg

The sections formed (usually in a ribbon-like strip) are allowed to

float on water in the specimen boat. The thickness of the section is
gradually decreased to the desired range. This thickness is
accurately indicated by the colour reflected by sections as they are
exposed to white light under a built-in binocular microscope.
Yellow or gold indicate the correct range. The correct section are
picked up using a small circular copper grid (about 2.5 mm in
diameter), which serves as the slide in electron microscopy. This
grid was previously coated with collodion film and overlaid with a
thin supporting carbon film using evacuated carbon evaporation.
Both films support sections when picked up.

5. STAINING
After sectioning, the specimen is made up of its original atoms (C,
H, O, H, P, etc.), which have low atomic weights. Since light atoms
could hardly deflect electrons, the specimen should be stained.
Staining is done by exposure of the specimen to salts of heavy
metals that have specific affinities to certain cellular components
and that increase the electron-scattering capacity of those
components. Uranyl salts. For example, combine with proteins and
nucleic acid while lead salts combine with lipid-containing cell
structure like membranes. Usually, a double staining technique is
used. The specimen is stained first by uranyl acetate, which is a
general stain, and then followed by lead citrate, which is specific for
membranes.
Specimen preparation in SEM is less elaborate than in TEM. After
fixation, the specimen is dried completely using a critical point
dryer. Here the fixative is first replaced with alcohol and then the
alcohol is sublimed thus drying the specimen. (Sometimes e.g. in
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insects which have a dry and hard chitinous exoskeleton, critical

point drying is not essential.) Once dried, the specimen is coated
with a thin layer of gold or palladium using an evacuated coating
unit. This coating prevents the build-up of negative charges on the
surface of the specimen, which can distort the image badly. The
coated specimen is finally place on specimen stand for observation.
Special Electron Microscopic Techniques
A side from routine produces performed in electron microscopy,
other
special techniques have been developed to expand its application and refine
its results. Some of these special techniques are discussed below.
1. NEGATIVE STAINING
In this special staining technique the empty spaces are stained and thus
appear dark. The sections are suspended in a droplet of dense material
like phosphotungstate, which penetrates these spaces thus staining
them. This procedure is done on the copper grid itself so that, after
drying, the specimen is ready for observation. It is very quick and
convenient for staining bacteria.
2. CRYOFIXATION
Also termed physical fixation, this technique was developed because
chemically fixed preparation tend to be disturbed by artifacts and also
undergo certain ultra structural changes.
Cryofixation aims to
preserve the cellular structure just as it existed in the living state-much
like stopping a moving film to produce a photograph. This is done
through rapid freezing of the specimen in dry ice, alcohol baths, or
liquefied nitrogen or helium gases. One method, called freeze-drying,
requires freezing of the specimen in liquid nitrogen (77 K or - 196C or
- 321F) or helium (near 0 K) and dehydration by distilling the water
out in vacuum. Another method, called freeze substitution, uses the
same freezing procedure but substitute water with anhydrous solvents
like methanol, ethanol, or acetone for dehydration. These produces
eliminate the problem of chemical fixation.
3. SHADOWING
Ordinarily stained electron micrographs are two-dimensional.
Shadowing or shadow casting is done to create a three-dimensional
effect on the specimen. Here the specimen is placed in an evacuated
chamber and a heavy metal is evaporated (from a tungsten filament) at
an angle above it to strike it only on one side. The material is thus
deposited like a shadow. The shadowed preparation is coated with a
8

thin supporting carbon film and if the specimen is too thick, it is

digested away by a strong solvent. This produce is illustrated in Figure
3.

Figure3. Steps in casting shadows in electron micrographs of a

stained specimen.

Figure 4. Fracturing a cell under the EM specimen block.

4. FREEZE-FRACTURING
This procedure is especially useful in viewing the interface between the
lipid bilayer of cell membranes. It consists of several steps namely:
a. Freezing in liquid nitrogen below 100C in the presence of a
cryoprotectant (20% glycerol in buffer) to prevent formation of ice
crystals that would disturb the shape and appearance of cells.
b. Cracking of the frozen block with a relatively blunt knife where
the fracture usually passes through the hydrophobic middle of the
lipid bilayers (Figure 4.)
c. Shadowing with platinum.
d. Viewing of the shadowed replica under the EM.

Figure5. Etching a cracked specimen block.

5. FREEZE-ETCHING
This technique is very much related to freeze fracturing because it also
involves freezing and cracking of the specimen. However, it is deal in
examining the true exterior of cells and to an extent, the exterior (not
middle) of the lipid bilayers of organelles. Here, fracturing has to be
done in an evacuated chamber. Then the ice level, and to an extent, the
cytoplasm of the cracked specimen is lowered by sublimation of water
in a vacuum to reveal the said surfaces of the specimen block (Figure
5). The it is also shadowed and the platinum replica studied.

Before, freeze etching has been limited by ice crystal formation while is being
substituted. This has been overcome by very rapid freezing which is done b
slamming the cells against a copper block cooled to 260C with helium. This
cools the specimen at a rate of around 20C per millisecond and prevents ice
crystal formation while etching. This allows very deep etching of the
specimen.
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Interpretation of Ultrastructures
The accurate interpretation of electron micrographs entails intensive analysis
and experience skill. To the uninitiated, an important detail may be
overlooked or dismissed as insignificant, while an artifact is interpreted as
ultrastructure. In electron micrographs (especially from TEM), the observed
image may not really be what it appears to be due to distortions incurred
during the elaborate preparation of the specimen. Thus, certain guidelines
must be followed to discriminate between artifacts and genuine
ultrastructure.
1. DIMENSION
The transformation of the appearance of objects from the original
specimen to the final image is illustrated in Figure 6. It can be readily
noted that the projected image is strictly two-dimensional.

Figure 6. A diagram of how internal object appear after ultrathin sectioning

and image projection. The native appearance of internal objects in the solid is
shown in (a): vesicles of variable sizes (1-4); a cistern (5) with perforations (610) and an attached polyribosome (11); microtubules (12, 13); free ribosomes
(14-16); and a bundles of rods or fibrils (17). Those portions that are included
within the thickness of a section (b) generate the projected image (c).

11

Spherical structures like the nucleus and the vesicles (4 and 2) will then
appear as circles while sheet of endoplasmic reticulum membrane (5) becomes
a straight line. It must be remembered, though, that there are several threedimensional figures from which a plane figure can be derived (e.g., a circle
can be the cross section of a sphere, a cylinder, or cone). Figure 7 clearly
illustrates this consideration.

Figure7. Possibilities of the same two-dimensional sections from different

three-dimensional structure.

2. ORIENTATION
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Shapes of projected images depend on the orientation of objects with

respect to the plane of the section. Tubules (13) perpendicular to the
plane of section will have a circular cross section but when they are
oblique they will appear oblong and dense (12). When a membrane is
cut transversely, it appears as a sharp black line (5). As the plane is
almost horizontal with the membrane, the latter becomes a smudge.
Oblique section of membranes will be like pale ribbons. Therefore,
they are indistinct if they are close to the plane of the section and
become more o less dense as they come closer to being perpendicular
to the latter.
3. SUPERIMPOSITION
Even ultrathin sections are of finite thickness, and not everything that
is within the section is projected. Objects that are in reality separate
may be superimposed so as to appear close together, e.g., three
ribosomes (14), (15), and (16), at the bottom, middle, and top players of
the section, respectively, appear to be side by side. Some objectives
disappear through superimposition, e.g., the image shows only 3 of the
4 rods (17) and only 3 ribosomes in the polyribosome (11) instead of
the four that are actually included in the section.
4. SECTION THICKNESS
The thicker the section, the more material are to contribute to the
image. As shown in Figure 6, the appearance of structures depends
upon whether they are entirely within the section (1) and (10) or if they
have been cut through (2), (4), (6), and (9).
In the case of membrane systems, there will be more membrane
segments within a thick section, and there will be a low chance of them
all lying at right angles to the plane of the section. Depending on the
angle at which these segments were sectioned, their appearance under
the microscope would differ. Image of pores through cisternae are
especially subject to these effects and more so if their diameter is nearly
the same as the section thickness will, if perfectly centered, appear to
have a narrow diaphragm traversing it. Looking once more at Figure
6, it becomes evident that if the pore diameter is larger than the
thickness (6), the cistern is interrupted, but the gap underestimates the
pore diameter. Two pores that are, in realty, of the same size may
appear to be of different sizes if they lie at different levels (9) and (6). A
pore that is entirely within the section has no clear cut edges and is
seen only as done within the cisterna (10).
OBJECTIVES
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At the end of the exercise, the students should be able to:

1. differentiate the important feature of light and election microscope;
2. discuss the importance of the steps involved in specimen preparation
for election microscopy;
3. describe special electron microscopic techniques;
4. compute actual and total magnification of specimens in electron
micrographs;
5. calculate the limit of resolution of given microscope; and
6. compare the resolving power of the a light microscope and a TEM
given micrographs of the same specimen at the same magnification.
ACTIVITY
Answer the following questions in your worksheet.
1. Bacterial cells, which in general have dimensions lying between 1-4
m, are resolved only with difficulty under the light microscope. At
magnifications of 400 times natural size, large bacteria stained in
methyl violet can just be seen as small, dark specks. Providing the
resolution of the light microscope is good, higher magnifications (e.g.,
1000x or 1500x) do reveal a little more of the internal structure of
bacteria cells.
The light micrograph (Figure 8) shows a single type of bacteria cell
treated with Giemsa stain. Measure the length of cell X, and assuming
the actual length to be 3 m, calculate the magnification.
2. Take into consideration the magnification computed in number 1.
How has magnification of this number of the times been achieved if the
light microscope itself (used in taking the photograph) only magnifies
1500 times?
3. With reference to Figure 8, what internal organization can be
distinguished in cell X?

Figure 8. photomicrograph of bacteria cells stained with Giemsa.

14

4. Can you see a limiting membrane? Can you deduce its presence?
From what feature?
5. In Figure 8 some cells appear longer or shorter than cell X. Account for
the differing lengths of cell A, B, C, and D by matching each cell with
the possible explanation from the list below:
a. natural variation in light
b. a cell in the process of division
c. two contiguous ( neighboring) cells
d. damage or distortion in preparation
6. Figure 9 is an electron micrograph of the same type of bacterium as
shown in Figure 8 (i.e Escherichia coli). The picture has been obtained
by cutting a very thin section of the cell, and assuming the actual
length to be 2.1 m, calculate the magnification.
7. Again with reference to Figure 9, what are the major differences
between the inclusions found in Figure 8 and way they appear in
Figure 9?
8. What other structural features can be resolved?
9. Viruses among the smallest entities known. Figure 10 is an electron
micrograph of polio virus, magnified 85,000 times. Measure the
diameter of virus particle X and calculate its actual diameter.

Figure 9. Electron micrograph of an Escherichia bacterium.

10. Could this virus be resolved under the light microscope? Support
your answer with values of resolution.

Figure 10. Electron micrograph of polio virus particles.

11. The superior resolving power of the electron microscope is well
illustrated by the two (Figure 11 and 12). They show two nearly
identical cells from an onion root tip, both magnified 1000 times.

15

However, one is taken using a light microscope and the other using an
electron microscope.

Figure 11. Electron micrograph of an onion root tip cell magnified 1000 times.
Key to labelling the electron micrograph:
N
Nucleus
NM Nuclear membrane
M Mitochondria (mesh-like appearance)
D Dictyosome or Golgi Apparatus
(small stack of sacs)
P Plastic

Ch Chromatin
Nu Nucleolus
R Ribosomes (minute particles)
CW Cell wall
ER Endoplasmic Reticulum
(like dark threads)

Using intersecting lines in conjunction with the numbers and letters

surrounding Figure 11. Identify the cell inclusion at the point of intersection
with reference to the key above.
a. 10H
f. 2C
b. 10D
g. 11A
c. 8Q
h. 3E
d. 16G
i. 11Q
e. 2G or 2H
j.
13P

16

12. Now look at Figure 12 in which certain features have been labelled
from a to g. Try to identify the labelled structures as far as you can.
What structures (give the letter) do you find difficult to identify to
identify and why?

Figure 12. Photomicrograph of an onion root tip cell magnified 1000 times.
B. CELL ULTRASTRUCTURE
There is an extremely wide variety of cells that comprise a complex
multicellular organism. Much more so are great variations among cells
from different organisms. Despite this, however, certain cell
components can be identified to invariably occur in most cells.
Cells are broadly classified into two groups based on the complexity of
their structural organization: the prokaryotic and the eukaryotic cells.
Prokaryotic cells are relatively small (1-10 m) and simple, possessing
only one membrane, the plasma membrane. Thus, there is no distinct
nucleus and no membrane-bound organelles. The genetic material lies
in a region in the cytoplasm- the nuclear zone. To this group belong
bacteria under the kingdom Monera.
Eukaryotic cells, on the other hand, are larger (10-100 m), more
complex, and exist singly in unicellular organisms or form tissues in
multicellular organisms. They are readily distinguished by the
17

localization and the compartmentalization of metabolic reactions in

membrane-bound organelles. The DNA, enclosed in a nuclear
membrane is associated with histones and nonhistone chromosomal
proteins.
Eukaryotes can further be divided into the other four
kingdoms which include the animals, plants, fungi, and protists.
Ultrastructure of Prokaryotic Cells
A diagrammatic sketch of a generalized prokaryotic cell indicating its
parts is shown in Figure 13.

Figure 13. the general internal structure of a prokaryotic cell.

The subcellular components of a prokaryotic cell include:
1. CELL WALL
The outermost structure of prokaryotics cells is the cell wall, which
appears as a fuzz under the EM. It is primarily composed of
peptidoglycan, a rigid framework of polysaccharide chains
crosslinked with short peptide chains. It is now known that
differences in chemical and structural characteristics of the cell wall
account for the specific reaction to Gram staining which divide
bacteria into Gram-positive and Gram-negative.
Several functions have been attributed to the cell wall. These are:
limits cell growth, protects the cell against injury and infection,
prevent swelling and bursting under hypotonic condition, and
imparts immunochemical compatibility or pathogenecity.

18

2. CAPSULE
This is an amorphous envelope of organic polymers found outside
the cell wall of many bacteria. The capsule or the slime layer is
composed of polysaccharides such as glucan. It provides some
protection against antibacterial agents and it is believed t play a role
in cell-cell interaction.
3. PLASMA MEMBRANE
This is evident under the EM as a thin black outline just beneath the
cell wall. It is a lipid bilayer embedded with proteins. It constitutes
a molecular barrier against the outside environment thereby
regulating what substances enter and leave the cell.
Prokaryotes have special infoldings of the plasma membrane such
as mesosomes and photosynthetic lamellae. The mesosomes, also
called chondrioids or plasmalemmasomes, are invaginations found
near the cell division zone. Several possible functions are attributed
to the mesosome and these include cell membrane synthesis,
endospore formation, transverse septum formation, replication cell
division and DNA segregation. The photosynthetic lamellae or
chromatophores on the other hand, are found only in
photosynthetic bacteria. They contain bacteriochlorophyll and
other light trapping pigments for photosynthesis.
Aside from the functions mentioned above, the bacterial plasma
membrane contains permeases necessary for the uptake of various
nutrients, election transport chains for respiration and/or
photosynthesis, ATP synthase, enzymes for the biosynthesis of cell
wall components and binding sites for the bacterial chromosomes.

4. FLAGELLA AND PILI

Flagella are thin, rigid, curved rods protruding from the cell that
are found in many kinds of bacteria to which they confer motility.
They are made up of single filaments of globular protein called
flagellin. A basal body anchors the filament to the cell membrane
and serves as motor for rotating the flagellum.
Pili are also rod-like protein structures protruding from the cell but
are shorter and thinner compared to flagella and are not responsible
for bacterial motility. They are composed of structure proteins
called pilins that are arranged to form a cylinder. One type of pili,
the common pili, function for bacterial attachment to the host cell
surface or surface or the substratum. Another type, the sex pili,
19

allow attachment to other bacteria for DNA exchange during

bacterial conjugation.
5. NUCLEAR ZONE
The isolated bacterial chromosome consists of a single large
molecule of double- stranded circular deoxyribonucleic acid
(DNA). This is folded and packed within the nuclear region, and as
stated earlier, is not bound by a nuclear membrane. The nuclear
zone appears to be a lighter region than the cytoplasm under the
TEM.
Aside from the bacterial genome, there are small, independently
replicating DNA molecules called plasmids that confer additional
traits to the bacteria such as resistance to certain antibiotics.
6. RIBOSOMES
Scattered in the intracellular fluid are dense particles or granules
rich in RNA and proteins-the ribosomes. These are sites of active
protein synthesis. Prokaryotic ribosomes have a small (30S) and a
large subunit (50S). These subunits assemble together to form a
70S complex. The granular appearance of the cytoplasm is largely
due to the presence of 70S ribosomes. The most popular model of
the prokaryotic ribosome is shown in Figure 14.

Figure 14.The structure of a prokaryotic ribosome. Image lifted

from http://classes.midlandstech.edu/carterp/Courses/bio
225/chap04/04-19_Ribosome_1.jpg
Several ribosomes may form a single function unit, transcribing a
messenger RNA. This assembly is called polysome or
polyribosome.

20

7. STORAGE GRANULES
Storage granules consist of nitrogen stores (cyanophycin), or carbon
reserves (glucans, poly--hydroxybutyrate). Sugar polymers can be
anzymatically degraded to yield free glucose or -hydroxybutyric
acid. These molecules can then be utilized as immediate fuel
substance to provide energy.
8. CYTOSOL
This is the soluble portion of the cytoplasm where enzymes are
dissolved and where metabolic intermediates and inorganic salts
are found. It is highly viscous and provides a medium where the
various metabolic processes can occur.
Ultrastructure of Eukaryotic Cells
Figure 15 presents diagrammatic sketches of generalized animal and plant
cells Notice that there are many organelles common to both animal and plant
cells. There are also organelles, which by virtue of their functions are
exclusive to one or the other. All these organelles organelles are briefly
described in the following discussion.

Figure15. A comparison of the general internal structures of animal and plant

cells.
The following are the various organelles/ultrastructures found in eukaryotic
cells Most of these are found in animals and plant cells (Figure 15).

21

1. CELL WALL
Plan cell walls consist of thick, rigid material deposited immediately
after the plasma membrane. In plans it is composed of cellulose
microfibrils cemented together by a embedding matrix of
hemicelluloses, pectin and extension. Accessory materials such as
lignin, suberin, cutin, etc. may reinforce the structure. On the other
hand, fungal cell walls are chitinous in nature. The cell wall provides
mechanical support and protects the cell from swelling and bursting
under hypotonic conditions.
2. PLASMODESMATA
These are opening in plant cell walls, which act as bridges of
cytoplasmic material, thus establishing continuity between adjacent
cells. Moreover, a plasmodesma (sing.) allows for the free circulation
of fluids essential to the maintenance of plant cell tonicity, and allows
passage of solutes and macromolecules.
3. CELL COAT
Also called the glycocalyx, this is found at the periphery of some
animal cells and is composed of mucopolysaccharides, glycolipids and
glycoproteins. It is important in cell adhesion and cell recognition,
since the highly specific structures of oligosaccharide chains allow one
cell to recognize another. It is therefore responsible for tissue
organization.
4. PLASMA MEMBRANE
This is the semipermeable bounding membrane of the protoplast. It is
a lipid bilayer, interspersed with proteins, partially or completely
embedded in the layer, in a mosaic pattern. The other component in
some membranes is carbohydrate, linked covalently either to lipid
(glycolipids) or to proteins (glycoproteins).
The membrane is
dynamically involved in the transport of small molecules either by
active or passive means and large molecules through membrane flow.
5. NUCLEUS
This is the organelle in eukaryotic cells that contain the DNA-the
genetic material. Two concentric membranes separated by a perinuclear
space, 10-15 nm in width, bound the DNA. Each membrane has basic
unit structure similar to the plasma membrane. The outer membrane is
occasionally continuous with the endoplasmic reticulum and has
ribosomes attached to it. At certain points, pores formed by the fusion
22

of the fusion of the outer and inner membranes interrupt the nuclear
membrane. A highly organized annular material (annulus) consisting
of eight protein granules surrounds a pore. The pore and the annuals
comprise the pore complex. The pore functions in the exchange of
molecules between the nucleus and the cytoplasm, while the annulus
regulates the entry and exit of molecules by occluding the passageway
(Figure 16).

Figure16. The nucleus.

The nucleolus, a suborganelle of the nucleus can be seen as a
prominently staining region of the nucleoplasm. It is composed of
groups of ribosomal genes (rDNA) surrounded by their rRNA
transcripts, together with many proteins. It is the site of ribosomal
RNA transcription and the assembly of ribosomal subunits. Electron
micrographs indicate four recognizable components in its structure
(Figure 17):
1. Granular zone - occupies the peripheral region and is surrounded by
chromatin. It is made up of granules, 15-20 nm in diameter and
contains RNA.
2. Fibrillar zone occupies the central regions. It is made up of fine
fibers 5-10 nm in diameter and contains RNA.
3. Matrix or Pars Amorpha the amorphous background of the
nucleolus, consisting of many proteins.
4. Nucleolar Associated Chromatin chromatin situated around and
even extending into the nucleolus. It is composed of both DNA and
RNA.

Figure 17.The nucleolus.

23

6. RIBOSOMES
Like the prokaryotic ribosome, the eukaryotic ribosomes are made up
of RNA and proteins and consist of a small a large subunit which also
associated to permit translation. In contrast, however they have 40s
and 60s subunits which associate to form 80s complex. Ribosomes
appear as dense granules that may be free in the cytoplasm or bound to
the endoplasmic reticulum or nuclear membranes via proteins on the
surfaces of the these organelles. Functionally related ribosomes, or
polyribosomes, are formed in large amounts during times of the
protein synthesis.
Ribosomes have also been located in the mitochondria and chloroplasts
which synthesize some of the proteins needed by these organelles.
Chloroplast ribosomes are 70S while mitochondrial ribosomes are a
combination of 70S and 80S complexes.

7. ENDOPLASMIC RETICULUM
This organelle forms the most extensive membrane system in most
cells. It is occasionally continuous with the outer nuclear membrane
and the plasma membrane. It may have ribosomes in which case it is
called rough ER (rER) but when it lacks ribosomes, it is called smooth
ER (sER). The rER functions in the synthesis, modification, and
transport of secretory proteins, while the sER is linked with the
synthesis, of lipids and hormones, as well as, the detoxification of
drugs. The rough ER consists of a series of interconnected flattened
sacs (cisternae) while the smooth ER is generally more tubular in
appearance; these two are interconnected to form a single membrane
system.
8. GOLGI APPARATUS
The Golgi apparatus or complex is a pile of platelike membrane
structure called cisternae. The major function of this organelle is the
modification and packaging of secretory proteins and the formation of
primary lysosomes. Moreover, it is involved in the synthesis and
secretion of glycoproteins.
9. LYSOSOMES AND MICROBODIES
Hydrolytic enzymes (e.g., proteases, nucleases, lipases, etc) are stored
in single-membrane lysosomes. There substance aid in the digestion of
biological molecules. Thus, the lysosome can be considered as part of
the cells digestive system. It also plays a part in protecting the cell
24

against foreign materials by engulfing and degrading them. Aging cell

structure are eaten up (autophagy) by this organelle and during
conditions of starvation, it digests subcellular parts to be used as
immediate fuel.
Microbodies are a heterogenous group of small, vesicle-like, singlemembaned organelles containing enzymes for oxidation. Two types
deserve mentioning. First, peroxisomes are those which contain flavin
oxidases and catalases and function for oxidation of substrates.
Second, glyxisomes (which are found only in plants) are those which
contain enzymes for the glyoxylate cycle aside from peroxisomal
enzmes. The glyoxylate cycle is a specialized metabolic pathway that
facilities conversion of fats into carbohydrates.
10. PLANT VACUOLES
Because plant vacuoles are not only larger but also more significant
than animal vacuoles, the following discussion focuses on them. Each
is enclosed by a single membrane, the tonoplast, and contains the cell
sap containing dissolved gases, sugars, organic acids, salt of organic
acids, mineral salts, and other water soluble materials. Aside from the
storage of metabolically useful products and wastes, they have an
essential function for space filling as the cell grows, and for
intracellular digestion much similar to lysosomes. As the cell matures,
the concentration and volume of the vacuoles contents increase and
this affects a great increase in the size of the vacuole. A mature vacuole
may occupy as much as 80% of the total cell volume.
11. MITOCHONDRIA
Though classically depicted as elongated cylinders or ovoid bodies,
mitochondria can actually move about the cytoplasm along the
microtubules, and in the process, dynamically change to diverse
shapes. Two unit membranes are present (Figure 18), a smooth outer
membrane and an inner one that form infolding called cristae are
present. The respiratory chain and phosphorylation systems are
located in the inner membrane. Inside the inner membrane is a matrix
containing DNA, and granules, which are binding sites for magnesium
and calcium cations. Expect for succinate dehyrogenase, enzymes of
the TCA cycle are located in the matrix.

25

Figure18. The internal architecture of a mitochondrion.

12. PLASTIDS
These are membrane-bound organelles unique to plant cells. The most
important plastid is the chloroplast (Figure 19) where photosynthesis
occurs. A chloroplast has two membranes, the outer and the inner
membranes. Within the inner membrane is a fluid or matrix called
stroma, which contains DNA and soluble enymes. Alsod found within
the stroma is a third membrane system of flattened discs called
thylakoid discs which are either piled together to form grana (granum,
sing.) also called grana lamella, or spanning two or more grana to form
stroma lamellae. The internal aqueous compartment enclosed by the
thylakoid membrane is called the lumen.
Plastids other than chloroplasts are also double-membraned and
possess an internal genome. These generally function for storage of
metabolically important substances like starch grains (amyloplasts),
oils (elaioplasts), proteins (proteinoplasts), and colored pigments
(chromoplasts).

Figure19. The internal architecture of a chloroplast.

26

13. CYTOSOL
The cytosol comprises about 55% of the cell volume.
It is
approximately 20% by weight protein and has a gel-like consistency.
Many of the cytosolic proteins are enzymes for cellular metabolism.
However, not all metabolic pathway occur in the cytosol (unlike the
majority in prokaryotic cells) since some of these are localized in the
membrane organelles. Another distinct feature of the cytosol in
eukaryotic cells is the presence of the cytoskeleton.
14. CYTOSKELETON
It was only in the 1970s, at the advent of high-voltage electron
microscopy that the accurate structure of the ground substance of the
eukaryotic cytoplasm was elucidated to be made up not of a
structureless fluid but of an orderly and extensive framework of
proteins: generally called the cytoskeleton. Three distinct structure
proteins make up the cytoskeleton: microfilaments, intermediate
filaments, and microtubules.
Microfilaments are 6-7 nm in diameter and are composed of the protein
actin. Intermediate filaments are 8-11 nm in diameter; they are
intermediate in diameter between microfilaments and microtubules.
Five proteins: vimentin, desmin, glieal fibrillary acidic protein (GFAP),
neurofilament (NF) and cytokeratin have been detected in different
intermediate filaments. Microtubules appear as hollow tubules, 25nm
in diameter and are made up of tubulin subunits.
The cytoskeleton provides mechanical support to the cell and has
important functions in cell motility, cell migration, change in cell
shape, the movement of organelles within the cell, and in the
separation of chromosomes during cell division.
15. CENTRIOLES
These can be found only in animals, protozoans, and some fungi.
Centrioles consist of two hollow cylinders lying at right angles to one
another (Figure 20). Each cylinder is about 0.4 m long by 0.5 m
diameter. The walls of the cylinder have nine sets of microtubules, each
set being a triplet of fibers. During the cell cycle, the centrioles divide
to form daughter centrioles that migrates to opposite poles during cell
division. The centrioles are involved in spindle fiber formation or
organization during cell division.

27

Figure20. Centrioles. Image lifted from https://upload.wikimedia.org

/wikipedia/commons/thumb/6/6f/Centriole-en.svg/2000pxCentriole-en.svg.png

16. CILIA AND FLAGELLA

Cilia and flagella are found only in certain eukaryotic cells. Both are
composed of microtubules with a diameter of 0.5 m, but while cilia
are 2-10 m long. Flagella are about 100-200 m in length. In contrast
to the 9+0 arrangement of microtubules in the centrioles, both cilia and
flagella are characterized by 9+2 motif (i.e with two additional tubules
at the center).
Some protozoans such as Paramecium use cilia for locomotion while
other eukaryotic cells such as spermatozoa use flagella.
OBJECTIVES
At the end of the exercise, the students should able to:
1. Differentiate prokaryotic and eukaryotic cells:
2. Identify the ultrastructures common to both prokaryotic and
eukaryotic cells;
3. enumerate the organelles common to both animal and plant cells and
those unique to each:
4. describe the structure and major functions of prokaryotic and
eukaryotic organelles; and
5. identify cellular organelles/ultrastructures from elcetromicgraphs.

28

ACTIVITY
Answer the questions (instructions) in your worksheet.
1. In terms of average size, included organisms, localization of the genetic
material, presence of membrane organelles, present of cytoskeleton,
tabulate the differences between prokaryotic and eukaryotic cells.
2. Enumerate four ultrastructures common to all cells whether
prokaryotic or eukaryotic. What particular significance can you
associate with the presence of these common ultrastructures in all
cells?
3. The electron micrograph in Figure 21 shows a section through a cell
from a fungus magnified 48,000 times. The section has been positively
stained with uranly acetate, but a different fixative from that used to fix
the onion root cell has been used, i.e.
KMnO4 (potassium
permanganate). When comparing Figure 21 here with Figure 11, what
will be the three important differences to bear in mind?

Figure 21. An electron micrograph of a section of a fungal cell

magnified 48,000 times.
4. Tabulate the major functions of the eukaryotic organelles. Use short
phrases only.

29

5. In terms of organelles, tabulate the similarities and differences of plant

and animal cells.
6. Identify the numbered structures in Figure 21.
This is Exercise was lifted from:
Reamillo, MCS, Mendoza, JC, Diaz, MGQ, and Manuel, MCC. 2009. Cell
Biology Laboratory Manual. 6th Ed. Genetics and Molecular Biology Division,
Institute of Biological Sciences, College of Arts and Sciences, University of the
Philippines Los Baos, College, Laguna
REFERENCES
ALBERTS, B., D. BRAY, J. LEWIS M. RAFF, K. ROBERTS and J.D. WATSON. 1994.
3rd ed. Molecular Biology of the Cell. New York: Garland Pub., Inc.
CHESCOE, D. and P.J. GOODHEW. 1984.The Operation of the Transmission
Electron Microscope. New You: Oxford University Press.
DARNELL, J., H. LODISH, and D. BALTIMORE. 1990. Molecular Cell Biology. 2nd
ed. New York: Sci. Am. Book, Inc.
DE ROBERTIS, E.D., F. A. SAEZ, and E.M.F DE ROBERTIS. 1975. Cell Biology. 6th ed.
California: Wadsworth Pub. Co., Inc.
GRAY,P. The Use of the Microscope. New York: McGraw-Hill.
HAYAT, M.A. 1972. Basic Electron Microscopy Techniques. New York: Van
Nostrand Reinhold Co.
JENSEN, W.A. and R.B. PARK. 1967. Cell Ultrastructure. California: Wadsworth Pub.
Co., Inc.
SHEELER, P. and D. BIANCHI. 1993. Cell Biology. New Your: John Wiley and Sons,
Inc
SMITH, C.A. and E.J. WOOD. 1992.Cell Biology: Molecular and Cell Biochem.
London: Chapman and Hall.
STEER,M.W. 1981. Understanding Cell Structure. Cambridge: Cambridge Univ. Pres.
TRIBE, M.A., M.R ERAUT, and R.K. SNOOK. 1975. Electron Microscopy and Cell
Structure. Cambridge: Cambridge Univ. Press.
WINTERS, C. 1988.(Personal Communication). Suggested procedures for the
preparation of sections for transmission electron microscopy and tissue
preparation for scanning
electron microscopy.

30

WORKSHEET
EXERCISE 1

Electron Microscopy and Cell Ultrastructure

Name: ______________________________ Date Submitted: ______________
Lab Section: _________________________ Lab Instructor: ______________
A. ELECTRON MICROSCOPY
1. Calculations:

2.
_____________________________________________________________________
3.
_____________________________________________________________________
4.
_____________________________________________________________________
_____________________________________________________________________
5.
a. ______________________
c. ______________________
b. ______________________
d. ______________________
6. Calculations:

31

Page 2 0f 5
Exercise 1 Electron Microscopy and Cell Ultrasturcture

7.
_____________________________________________________________________
_____________________________________________________________________
8.
_____________________________________________________________________
_____________________________________________________________________
9. Calculations:

10.
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
11.
a.
f.
b.
g.
c.
h.
d.
i.
e.
j.
12.
a.
f.
b.
g.
c.
h.
d.
i.
__________________________________________________________________
__________________________________________________________________

32

Page 3 of 5
Exercise 1 Electron Microscopy and Cell Ultrastructre

B. CELL ULTRASTRUCTURE
1. Table
1.1_____________________________________________________________
CRITERIA

PROKARYOTES

EUKARTOTES

Average size (in m)

Included organisms

Location of the
genetic
Material

Presence of
membranebound organelles

Presence of
cytoskeleton

2.

a. ________________________
b. ________________________

c. _________________________
d. _________________________

_____________________________________________________________________
_____________________________________________________________________
3. Differences to remember:
a.
_________________________________________________________________
b.
_________________________________________________________________
c.
_________________________________________________________________

33

Page 4 of 5
Exercise 1 Electron Microscopy and Cell Ultrastructure

4. Table 1.2
___________________________________________________________
ORGANELLE
1. Cell wall
2. Plasmodesmata
3. Cell coat
4. Plasma membrane
5. Nucleus
6. Ribosomes
7. a. Rough ER
b. Smooth ER
8. Golgi apparatus
9. a. Lysosomes
b. Microbodies
i. Peroxisomes
ii. Glyoxisomes
10. Vacuoles
11. Mitochondria
12. Plastids
13. Cytosol
14. Cytoskeleton
15. Centrioles
16. Cilia and Flagella

FUNCTION

34

Page 5 of 5
Exercise 1 Electron Microscopy and Cell Ultrasture

5. Table 1.3
__________________________________________________________
ORGANELLES
COMMON

6.

a.
b.
c.
d.

EXCLUSIVE ORGANELLES
In plants only

__________________________
__________________________
__________________________
__________________________

In animals only

e. _________________________
f. _________________________
d. _________________________
h. _______________________

35