MODULE 1: CELL AND MOLECULAR BIOLOGY

2.1. Differences between electron and light microscope and between resolution and
magnification.

MICROSCOPY

Hi guys! My name is Robert Hooke. I

am a Scientist. I lived in the 17th
century.

I made observations of cork tissue

from a tree under a microscope.

I found that they were made up of tiny

structures which I called CELLS.

A MICROSCOPE USED BY
CORK TISSUE
ROBERT HOOKE

Since the time that Robert Hooke observed cork tissue underneath a microscope, this
instrument has become an indispensable tool in observing cells, tissues and tiny living
organisms which are invisible to the naked eye (e.g. bacteria, microscopic fungi,
microscopic algae, protoctists) and even gametes.
Modern day microscopes are much more powerful than the one that Robert Hooke used,
thus they have helped us to gain in-depth knowledge about the structure and functions of
cells. This has had numerous applications in the field of biological, medical and even forensic
research.

THE LIGHT MICROSCOPE

The most common microscope used in the laboratory is the light microscope.
They use glass lenses to bend light rays to produce a magnified image of an object.
The diagram below shows the parts of a typical light microscope and their functions.
 A LIGHT MICROSCOPE SHOWING THE DIFFERENT PARTS AND THEIR FUNCTIONS

Lens that magnifies and focusses

the image from the objective
lens to the eye.

In between coverslip above it

Lens which collects light passing through and glass slide below it.
the specimen and produces a magnified
image.
Focusses the light onto the
specimen which is between
the coverslip and the slide.
Iris diaphragm- can be used to
change the diameter of the beam of
light.

HOW TO USE A MICROSCOPE TO OBSERVE A SPECIMEN

DIAGRAM SHOWING HOW A LIGHT MICROSCOPE WORKS

A) PREPARE THE SPECIMEN (MOUNTING THE SPECIMEN ON A SLIDE)

MOUNTING A SPECIMEN FOR VIEWING UNDER A LIGHT MICROSCOPE

 A specimen must be THIN and TRANSPARENT.

 It must be placed on a GLASS SLIDE.
 A drop of water can be placed over the specimen. It the specimen is
colourless, a drop of a coloured chemical substance called a STAIN can be
placed over it. Some stains can colourise different parts of the cell or
different cells more than others (e.g. a stain called methylene blue is taken
up more readily by nuclei than the cytoplasm. Thus nuclei will appear dark
blue and cytoplasm will appear light blue).

 Cover the specimen with a thin COVERSLIP made of very thin glass or
transparent colourless plastic.
 The method outlined above can be used to produce a TEMPORARY SLIDE.

A PERMANENT SLIDE can be made by staining the specimen, then placing a

special type of liquid such as Canada balsam over it. The coverslip is then
placed over the specimen, and the special liquid is allowed to solidify.

NB: The coverslip must be carefully placed over the specimen to avoid
trapping air bubbles.

VIEWING THE PREPARED SPECIMEN

After the diameter of the light from the source has been adjusted by the iris diaphragm and
focussed onto the specimen by the condenser lens, light passes through the specimen and
goes through the objective lens above it.

OBJECTIVE LENS
Objective lenses are mounted on a REVOLVING NOSEPIECE or TURRET.
They collect light from the specimen and produce a magnified image.
Each one has a different MAGNIFICATION.

You can choose the magnification of the objective lens that you want to view the object, by
rotating the revolving nosepiece until the objective lens you have chosen is directly over the
specimen (when that objective lens has been moved into its proper position by you, a click
will be heard.
The magnification of objective lenses found on most light microscopes are:

X 4 (LOW POWER); X 10, X 40, X 100 (HIGH POWER)

OBJECTIVE LENSES WITH

DIFFERENT MAGNIFICATIONS

OBJECTIVE LENSES MOUNTED ON A REVOLVING

NOSEPIECE OR TURRET
EYEPIECE LENS
This lens magnifies and focusses the image from the objective lens to the eye.

MAGNIFICATION
This is defined by the following formula:

SIZE OF IMAGE
MAGNIFICATION=
SIZE OF SPECIMEN
The physical quantity associated with size is usually LENGTH.
The image can be a photograph of the specimen (a photograph of a specimen as observed
under a light microscope is called a PHOTOMICROGRAPH), or a drawing.

Magnification has no units. It is written as follows: e.g. X 0.5; X 40.

7.0 cm 12.0 cm

QUESTION:
The actual length of the Hibiscus leaf is 7.0 cm. The length of the drawing of the Hibiscus
leaf is 12.0 cm. Calculate its magnification.
 UNITS OF MEASUREMENT OF LENGTH USED IN
MICROSCOPY

The following units of length are commonly used in microscopy:

o micrometre (µm)- sometimes called the micron.

o nanometre (nm)

With reference to the millimetre (mm):

MICROMETRE

1 micrometre (1 µm) = 1/1 000 mm = 1 x 10-3 mm

1 000 µm = 1 mm
To convert from mm to µm: multiply by 1 000.
e.g. Convert 50 mm to µm.
50 x 1 000= 50 000 µm
To convert from µm to mm: multiply by 1/1 000 OR divide by 1 000.
e.g. Convert 16 µm to mm.

16 x 1/1 000= 0.016 mm (1.6 x 10-2 mm) OR 16/1 000=

0.016 mm (1.6 x 10-2 mm) .
NANOMETRE

 1 nanometre (1 nm) = 1/1 000 000 mm = 1 x 10-6 mm

1 000 000 nm = 1 mm
 1 nanometre (1 nm) = 1/1 000 mm = 1 x 10-3 µm

1 000 nm = 1 µm
To convert from mm to nm: multiply by 1 000 000.

e.g. Convert 125 mm to nm.

125 x 1 000 000 = 125 000 000 µm
To convert from µm to mm: multiply by 1/1 000 000 OR divide by
1 000 000.
e.g. Convert 65 µm to mm.

65 x 1/1 000 000 = 6.5 x 10-5 mm OR

65/1 000 000 = 6.5 x 10-5 mm.

In some scientific literarture, the ångström unit is also used (Å).

1 angstrom unit (1 Å) = 0.1 nanometre (0.1 nm)

QUESTIONS:
Convert the following units:
i) 13.5 mm to nm.
ii) 70.2 nm to mm.
iii) 220 nm to µm.
iv) 133 µm to nm.
CALCULATION OF MAGNIFICATION OF SPECIMENS AS SEEN UNDER
A LIGHT MICROSCOPE
METHOD 1

A red blood cell was observed under a light microscope and it was measured to be 7 µm in
diameter.
A drawing of this red blood cell was made as shown below.

DRAWING OF A RED BLOOD CELL AS SEEN UNDER A LIGHT MICROSCOPE

STEP 1: MEASURE the diameter of the drawing of the red blood cell in millimetres.

Let’s say it is 35 mm in diameter.

STEP 2: CONVERT millimetres to micrometres.

1 000 µm = 1 mm
Therefore, 35 mm= 35 mm x 1 000= 35 000 µm.
STEP 3: CALCULATE MAGNIFICATION
SIZE OF IMAGE
MAGNIFICATION=

SIZE OF SPECIMEN

= 35 000 µm /7 µm
= X 5 000
QUESTIONS:

1. Calculate the magnification of the following drawing of an Amoeba as seen under

the light microscope, given the following information:

Actual length of specimen= 6.3 µm

Length of drawing= 70 mm

2. A student drew a Paramecium ( a protoctist) as seen under a light microscope. His

teacher measured the length of his drawing as 185 mm, and he stated his
magnification as X 608 without showing his calculations. What was the actual
length of the specimen (in micrometres) that he observed?
METHOD 2- USING A SCALE BAR
Magnification can be calculated using a SCALE BAR. This is a line drawn on the photograph
or drawing which has a label showing the actual length of the bar before being magnified.

LENGTH SCALE BAR

REPRESENTS

SCALE BAR
PHOTOMICROGRAPH OF SEA URCHIN EMBRYO SHOWING A SCALE BAR AND THE LENGTH
THE SCALE BAR REPRESENTS

STEP 1: MEASURE the LENGTH OF THE SCALE BAR.

Let’s say it is 21 mm.

STEP 2: CONVERT LENGTH OF SCALE BAR millimetres to micrometres.

1 000 µm = 1 mm
Therefore, 21 mm = 21 mm x 1 000 = 21 000 µm.

STEP 2: CALCULATE MAGNIFICATION

SIZE OF IMAGE
MAGNIFICATION=
SIZE OF SPECIMEN

LENGTH OF SCALE BAR

MAGNIFICATION=
LENGTH SCALE BAR RESPRESENTS
= 21 000 µm/50 µm
= X 420
QUESTION:
1. In the photomicrograph of an Amoeba shown below, the length that the scale bar
represents is 100 µm. The length of the scale bar is 20 mm.
Calculate the magnification of the image.

100 µm

IF WE WANT TO DETERMINE THE ACTUAL SIZE OF THE SPECIMEN:

a) Determine the magnification by using the steps outlined under the heading:
METHOD 2- USING A SCALE BAR

b) Measure the length of the image (in mm.)

c) Convert the length of the image from mm to µm.

d) Use the formula:

SIZE OF IMAGE

MAGNIFICATION=
SIZE OF SPECIMEN

Look at the photomicrograph of the sea urchin on the previous page again,
Let’s say it is 70 mm in diameter.
In µm = 70 x 1 000 = 70 000 µm
We had already calculated its nagnification as x 420.
Thus actual size of the sea urchin embryo =
SIZE OF IMAGE

MAGNIFICATION =
SIZE OF SPECIMEN

SIZE OF IMAGE
Therefore, SIZE OF SPECIMEN =

MAGNIFICATION

= 70 000
420

= 166.67 µm

QUESTION:
In the photomicrograph of red onion epidermal tissue shown below, the length of the scale
bar is 33 mm, and length of the image of the tissue is 90 mm.
Calculate the:

i) the magnification of the photomicrograph

ii) actual length of the tissue in micrometres.
 MEASURING THE LENGTH OF A SPECIMEN AS SEEN UNDER A LIGHT MICROSCOPE AND
CALCULATING ITS MAGNIFICATION

For a specimen which is large enough to be seen with the naked eye, its length can be
measured by using a ruler.
However, for measurements of length of a specimen under a light microscope, we use a
special type of ruler called an EYEPIECE GRATICULE (or RETICLE) (which is placed in the tube
containing the eyepiece lens).

LIGHT MICROSCOPE EYEPIECE GRATICULE (RETICLE)

Due to the fact that if the magnification of the objective lens is increased, the field of view
of the specimen decreases (that is, we see less of the specimen that we are viewing if we
increase the magnification of the objective lens). However, what we do actually see is a
magnified image with more detail of that part of the specimen that we are observing.

ONION EPIDERMAL TISSUE AS ONION EPIDERMAL TISSUE AS

OBSERVED UNDER THE X 4 OBSERED UNDER THE X 40
OBJECTIVE LENS (LOW OBJECTIVE LENS (HIGH
POWER/MAGNIFICATION) OF A POWER/MAGNIFICATION) OF A
LIGHT MICROSCOPE LIGHT MICROSCOPE

Therefore, we need to CALIBRATE the graticule for each magnification of the objective lens.
In doing this, the value of each division on the graticule will be different (but appropriate)
for each magnification of the objective lens.
We calibrate a graticule by using another special type of ruler called a STAGE MICROMETER
(which is placed on the stage of the microscope, hence its name). It is basically a microscope
slide with a tiny “ruler” at the centre of it.

VALUE OF EACH DIVISION ON STAGE

MICROMETER. IN THIS CASE, IT IS
0.01 mm or 10 µm

MAGNIFIED IMAGE OF THE

DIVISIONS OF A STAGE
MICROMETER

A STAGE MICROMETER
1 mm
 CALIBRATION OF AN EYEPIECE GRATICULE USING A STAGE MICROMETER

STEP 1: Place the stage micrometer on the stage of the light microscope.
STEP 2: Adjust the magnification of the objective lens that you want to use (e.g. X 10) so
that it faces towards the stage micrometer. Use the coarse and fine adjustment knobs to
bring the stage micrometer into focus.
STEP 3: Line up the stage micrometer scale and eyepiece graticule scale by turning the
eyepiece, and/or moving the stage micrometer on the stage.
ENSURE THAT TWO LARGE MARKINGS ON EACH SCALE ARE EXACTLY LINED UP WITH EACH
OTHER.
The final appearance of both scales should be as shown in the diagram below.

PERFECT
ALIGNMENT
HERE- POINT B

PERFECT
ALIGNMENT
HERE- POINT A

DIAGRAM SHOWING ALIGNMENT OF EYEPIECE GRATICULE SCALE AND STAGE MIRCOMETER

SCALE
 STEP 4: Observe two points where one eyepiece graticule division is perfectly aligned with a
stage micrometer division (from the left).
In the example above:
i) AT POINT A: the 50 mark of the stage micrometer is perfectly aligned with the
1.0 mark on the eyepiece graticule.
ii) AT POINT B: the 60 mark of the stage micrometer is perfectly aligned with the
5.5 mark on the eyepiece graticule .

STEP 5:
i) Count the number of GRATICULE divisions (UNITS) from point A to point B. on
the graticule.

In this example, there are 45 graticule divisions from point A to point B.

ii) Count the number of STAGE MICROMETER divisions from point A to point B.
In this example, there are 10 stage micrometer divisions from point A to point B.

Therefore, 45 graticule divisions = 10 stage micrometer divisions.

STEP 6: Calculate the total length corresponding to the stage micrometer divisions from
point A to point B.

If the value of each stage micrometer division is 0.01 mm (as stated on the stage
micrometer), then 10 micrometer divisions = 10 x 0.01 mm= 0.1 mm= 100 µm

STEP 7: Calculate the value of one graticule division.

VALUE OF ONE GRATICULE DIVISION (UNIT) = 100/45 = 2.22 µm.

REMEMBER: CONVERT ALL UNITS OF MEASUREMENT OF LENGTH TO µm WHEN DOING

CALCULATIONS!

WATCH NOW ON

https://www.youtube.com/watch?v=tv9GX0RZeBE
AS Biology - How to calibrate a microscope
QUESTION:

1.

EYEPIECE GRATICULE

STAGE MICROMETER

Calculate the value of one graticule division using the aligned eyepiece graticule and
stage micrometer above (each stage micrometer division = 0.01 mm)

2. A cell measures 84 graticule divisions.

When a stage micrometer is placed on the stage using the same objective lens, it
can be seen that 100 graticule divisions exactly line up with 8 stage micrometer
markings. The stage micrometer is marked off in 0.01 mm divisions. Calculate the
size of the cell.
 If you do not have a stage micrometer, or you are short on time to do calibrations, you
can use this table showing calibrated values of graticule divisions (in µm) for each
magnification of objective lens to do your microscopy work.

TABLE SHOWING THE VALUE OF EACH GRATICULE DIVISION (IN µm) AT EACH
OBJECTIVE LENS MAGNIFICATION

OBJECTIVE LENS
NUMERICAL VALUE OF EACH GRATICULE
MAGNIFICATION
DIVISION (IN MICROMETRES ( µm) )

X4 25
X 10 10
X 40 2.5

X 100 1

CALCULATION OF MAGNIFICATION FOR DRAWINGS OF

SPECIMENS SEEN UNDER THE LIGHT MICROSCOPE

The three cells below are observed under the x 10 objective lens of a light microscope.
They are found to occupy 98 graticule divisions under the x 10 objective lens .
2) In the table above, if a specimen is observed using the x 10 objective lens of a light
microscope, the numerical value of each division on the graticule is 10 micrometres (µm).

3) Now, since the three cells above occupy 98 graticule units/divisions, and we have
determined that each division under the x 10 objective lens is 10 µm, then:
length of specimen as observed under the x 10 objective lens= 98 x 10 µm=980 µm

4) Let us say that we had drawn the three cells as is, and when we measured it with a ruler,
the length of our drawing= 180 mm.

Convert this value to µm: 180 mm x 1 000= 180 000 µm (1 000 µm = 1 mm)

LENGTH OF DRAWING
5) MAGNIFICATION =
LENGTH OF SPECIMEN

= 180 000 µm/ 980 µm

= X 184

QUESTION:
You observed the cell under the light microscope under different magnifications of
objective lenses as shown below, and you made drawings of them.

The table below shows the magnification of the objective lens used and the corresponding
size of your drawing.

OBJECTIVE LENS MAGNIFICATION SIZE OF DRAWING (mm)

X4 30
X 10 25
X 40 21
X 100 18

Calculate the magnification for each drawing corresponding to each objective lens.
 RESOLUTION

The RESOLUTION of a microscope is the smallest distance between two

objects as seen under a microscope at which the objects appear distinct.

Light microscopes cannot show objects that are smaller than 200 nanometres across. They
may just appear as a blur.

LIMIT OF RESOLUTION = 0.45

Where = wavelength of light used.

In white light (visible light), blue light has the shortest wavelength ( = 450 nm).
Thus, the limit of resolution of the light microscope is: 0.45 x 450 nm = 200 nm.

TWO OBJECTS LESS THAN 200 nm TWO OBJECTS GREATER THAN OR EQUAL TO 200 nm
APART AS SEEN UNDER A LIGHT APART AS SEEN UNDER A LIGHT MICROSCOPE
MICROSCOPE

Look at the photomicrographs of the pollen grains as shown below. It can be clearly seen
that photomicrograph A was onserved under a light microscope with low resolution, and
photomicrograph B was onserved under a light microscope with high resolution.

PHOTOMICROGRAPH B OF POLLEN
PHOTOMICROGRAPH A OF POLLEN
GRAINS AS SEEN UNDER A LIGHT
GRAINS AS SEEN UNDER A LIGHT
MICROSCOPE WITH HIGH
MICROSCOPE WITH LOW
RESOLUTION
RESOLUTION
 THE ELECTRON MICROSCOPE
Due to the limits of magnification and resolution in using a light microscope, a more
powerful microscope is required to observe the structural details of cells (that is, their
ULTRASTRUCTURE).
A special type of microscope called an ELECTRON MICROSCOPE can be used for this
purpose.

HOW DOES AN ELECTRON MICROSCOPE WORK?

Electrons have a shorter wavelength than light (electrons have a wave-particle duality). It is
less than 1 nm.

Therefore, because of electrons’ shorter wavelength, the limit of resolution will be greater if
we use a microscope that uses a beam of electrons instead of a beam of light. Thus we will
be able to observe greater detail in our specimens.

Unlike light microscopes (which can distinguish between two objects which are 200 nm or
greater apart), an electron microscope has a limit of resolution of 0.5
nm. Thus objects which are 0.5 nm apart (or greater) can be distinguished as two separate
objects. We can thus magnify specimens much more than a light microscope and still obtain
a clear image.
Thus unlike a light microscope which we can magnify an image up to X 1 400 due to its low
limit of resolution, we
can magnify an image up to X 300 000 using an
electron microscope.

AN ELECTRON MICROSCOPE
The diagram below shows how an electron microscope works.

DIAGRAM SHOWING HOW AN ELECTRON MICROSCOPE WORKS

WATCH NOW ON

https://www.youtube.com/watch?v=pT-tnPiDjoo
Scanning electron microscopy
POINTS TO NOTE:
1. An electron gun and an anode produce a beam of electrons.
2. The lenses which focus the electron beam are ELECTROMAGNETS.
3. Specimens used must be dead since the beam of electrons has an
ionising effect and will kill the specimen inside of the microscope. They
must also be thin.
4. Stains used are heavy metal ions such as lead or osmium ions. These
are large and positively charged ions. Some parts of the cell take up the
the ions more than other parts.
The negatively charged electrons are attracted to those positive ion
stains. Thus, those electrons do not arrive on the screen. Therefore, the
screen remains dark in those regions. The structures in the specimen
which have taken up the stain appear dark.
5. Images are viewed on a screen or a photographic plate.
6. Images are in black and white. Colours can be added afterwards by a
computer program. Images produced by electron microscopes are called
ELECTRON MICROGRAPHS.
7. The internal setup is within a vacuum. This is because if electrons collide
with air molecules in the chamber, the air molecules will become ionised
and can cause damage.

TYPES OF ELECTRON MICROSCOPES

There are two types of electron microscopes: TRANSMISSION ELECTRON MICROSCOPES
(TEM) AND SCANNING ELECTRON MICROSCOPES (SEM).

TRANSMISSION ELECTRON MICROSCOPE (TEM)- This allows one to see internal structures
of the specimen. Details of structures inside of cells have been determined by using this
type of microscope.

TRANSMISSION ELECTRON MICROGRAPH OF A PLANT CELL

SCANNING ELECTRON MICROSCOPE (SEM)- This allows one to observe the details of the
SURFACE of a specimen.

I’M A MOSQUITO
AND I’M GONNA’
SUCK YOU DRY
SUCKA!!!!!

STRAND OF HUMAN HAIR

MOSQUITO

FROM LEFT TO RIGHT: RED BLOOD

CELL, PLATELET, T LYMPHOCYTE (A A POLLEN GRAIN
TYPE OF WHITE BLOOD CELL)

SCANNING ELECTRON MICROGRAPHS OF SOME BIOLOGICAL SPECIMENS

 COMPARISON OF LIGHT AND ELECTRON MICROSCOPY

FEATURE LIGHT MICROSCOPY ELECTRON

MICROSCOPY
Type of radiation Visible light: Electron beam λ<1 nm
used λ= 400 nm to 700 nm
Approximate 200 nm 0.5 nm
maximum
resolution
Approximate X 1400 X 300 000
maximum useful
magnification
Type of lens glass electromagnets
Specimen -Living cells; tissues; -Cells always dead.
preparation organisms. -TEM: Cells cut extremely
- Non-living specimens cut thinly.
thinly, mounted on -SEM: Surface prepared;
transparent medium on a specimen dehydrated and
slide. placed within a vacuum
within microscope.
Specimen staining Coloured stains which are Heavy metals which are
absorbed by different parts absorbed by different parts
of the cell or by different of cell or by different cell
cell types (e.g. methylene types (e.g. lead, osmium).
blue)
Image viewing Directly (eye, screen or Electrons fall on
monitor). fluorescent screen or fed to
a monitor to produce
image.
COMPARISON OF LIGHT AND ELECTRON MICROSCOPY
(CONTINUED)

FEATURE LIGHT MICROSCOPY ELECTRON

MICROSCOPY
Image appearance In colour (natural colour of -Black and white.
specimen or colour of -False colours can be
stains). added by using
computer imaging
software.
Distortion Material can be prepared Staining techniques
for viewing without a high and effect of vacuum
risk of distortion. increase distortion
risk.
Ease of use Cheap; little training Very expensive to
required to use. purchase and
maintain; thorough
training required.

Specimen Well-prepared slides can Specimens deteriorate

permanence last for years. during viewing. Cannot
be kept.