Exercises that will help you understand how your microscope works
1.
Calculating magnification
One of the most basic things you need to know is how much the microscope is magnifying the
specimen you are looking at. Each lens in the microscope enlarges the image and the total
magnification is given by this formulae:
Magnification of
eyepiece (ocular) lens
Magnification of
objective lens
X
=
Total
Magnification
Most school microscopes have an eyepiece lens that magnifies 10 times (x10). Objective lenses
are usually x4, x10 and x40. All microscope lenses will have magnifications etched into their metal
casings.
The table below gives the magnifications of the most common microscope configurations:
Eyepiece
(ocular) lens
magnification
x5
x10
(most
common)
x15
Objective lens magnification
Low power
x4
Medium
Power x10
High power
x40
x20
x50
x200
x40
x100
x400
Oil immersion
lenses x100
x500
x1000
Most common magnifications
x60
x150
x600
x1500
Check the microscope you are using and fill in the table below. You will need to know the
magnifications of your microscope when you examine specimens and draw diagrams.
All microscope observations and diagrams must be accompanied by an indication of the
magnification you used.
Eyepiece lens
magnification
X
Objective lens
magnifications
Low power
Total
magnifications
X
=
Medium
power
X
=
High power
X
=
Oil immersion
(if your
microscope
has one)
X
=
2.
“e” inversion
For this exercise you will need a small piece of paper ripped from a glossy magazine. Make sure
the piece has a letter ‘e’ on part of it. It is best to choose a portion where the font size is small or
you will not be able to see the entire letter in your ‘field-of-view’.
e
slide
cover slip
Place this on a microscope slide, there is no need for water but a cover slip will keep the paper flat.
Focus under Low Power. What do you notice about the orientation of your letter ‘e’?
________________________________________
Draw what you see here:
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Try moving the slide left/right, and up/down. What happens when you move the slide?
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Now use Medium Power and see if the same thing occurs. What about High Power?
(Of course you will probably not be able to see the entire letter).
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This exercise illustrates how difficult it can be when manipulating slides on the microscope stage. It
is particularly difficult when following moving organisms such as Paramecium because you must
move the slide in the opposite direction to the direction the organism moves.
Below are some pictures showing the type of images you should see:
3.
Indirect measurement under the microscope
In this exercise you will learn how to estimate the sizes of cells and other objects under the
microscope. This method of measurement is ‘indirect’ because the object itself is not measured,
size is estimated by knowing the diameter of the microscope’s ‘field-of-view’ (diam. f.o.v.).
The f.o.v. of the microscope is the circular area or image you can see when you focus on a
specimen.
All you will need for this exercise is a clear plastic ruler with millimetre marks on it. Place the ruler
on the stage so that the edge of the ruler cuts the f.o.v. in half (i.e. follows the diameter of the f.o.v.
focus under Low Power).
You will see something similar to this:
Field-of-view (lit area)
You will not be
able to see this area
mm marks of ruler
Adjust the ruler so that the left hand mm mark is half in and half out of your f.o.v. (see red arrow).
You can now simply measure the diameter of the f.o.v. by counting the mm marks. In the example
above the diam. f.o.v. is 4.5 mm
Millimetres however are not a small enough unit to use when measuring cells as they are very
small. Instead we use micrometers (usually called microns, symbol µm).
A micron is one millionth of a meter, remember that one millimetre is one thousandth of one meter.
This means that there are 1000 microns in one millimetre.
1 µm = 10-6 m ( 1/1,000,000th m)
or
1,000,000 µm = 1 m
1 mm = 10-3 m (1/1,000th m)
or
1,000 mm
= 1 m.
1 µm = 10-3 mm ( 1/1,000th mm)
or
1,000 µm
= 1 mm.
This means that the Low Power diameter f.o.v.
How to estimate the size of
cells:
Imagine a row of cells as shown
(right). There are about 14 cells
in one diameter of this L.P. f.o.v.
That means 14 cells in
4,500 µm (microns)
The average length of each cell
= 4,500 / 14
= 320 µm
= 4.5 mm
= 4,500 µm (microns)
Note that cells are not directly measured but their size is estimated indirectly.
It is now time to do the same exercise using Medium Power and if possible using High Power
This is similar to what you will see using Medium Power.
As before you should adjust the ruler so that the left hand mm mark is half in and half out of your
f.o.v. (see red arrow). Now simply measure the diameter of the f.o.v. by counting the mm marks. In
the example above the Medium Power diam. f.o.v. is 1.8 mm or 1,800 µm
It might not be possible to do this exercise using High Power as often there is insufficient room to
get the plastic ruler under the high power objective.
Your microscope:
Of course using different microscopes the measurements obtained in this exercise might be
different. Use the table below to add the values you obtained.
Objective
lens
Magnification
(eyepiece x
objective)
L.P.
x10 x4 = x40
M.P.
x10 x10 = x100
H.P.
x10 x40 = x400
Diameter f.o.v.
millimetre
micron
(mm)
(µm)
If you cannot estimate the H.P. diameter f.o.v. by observing the ruler you can calculate it using this
formulae:
H.P. diam f.o.v.
= L.P. diam f.o.v. X
L.P. magnification
H.P. magnification
For the microscope values used in this exercise:
H.P. diam f.o.v.
= 4,500 X
= 450 µm
40
400
(less than ½ of one mm)
4.
Understanding what the condenser and diaphragm do
The best way to understand how the condenser and diaphragm on the microscope you are using
can help you form an image is to experiment with it.
Condenser
The condenser is a lens under the stage that focuses light from the mirror or artificial light source
below the stage onto the specimen (on the slide on the stage). Normally the condenser is adjusted
to be as high as possible but be careful that it does not protrude above the stage and make contact
with the slide.
The condenser is usually adjusted in conjunction with the diaphragm. The diaphragm controls the
amount of light but also has an effect on the amount of contrast that the image has. Often this is its
most important function. You should always be prepared to adjust the condenser and diaphragm in
order to improve the image you see, especially when using High Power.
The 100FL microscope:
The 100FL has a simple condenser set up.
stage
In many self-illuminated microscopes (with a
built in light source) there is a lens above the
lamp that has the same function as the
condenser. It focuses light to a point at the
centre of the stage.
focussing
knobs
This type of condenser is non-adjustable.
condenser lens
lamp housing
switch
Microscopes that are more sophisticated have fully adjustable condensers and diaphragms.
The following images are of an Olympus microscope that many schools use.
Stage
Condenser
Diaphragm lever
Condenser focusing knob
Double-sided mirror
This microscope is not self-illuminated (does not have a build in light source) so you must use a
light bulb or use the microscope in a bright position.
Warning: never use a microscope in direct sunlight as it is possible to harm your eyes if you get
too much light from the sun.
The mirror is usually double sided:
 use the flat mirror is the microscope has a condenser
 use the concave mirror if there is no condenser. The mirror will help focus light in the same
way the condenser does.
The Condenser focusing knob moves the condenser up and down. Generally, it should be as
high as possible but you should experiment and see what happens to the image. Any effect will be
most noticeable when using high Power.
Diaphragm control
There are 2 types of diaphragm:

Rotating disc type as in the 100FL. The disc has 5 different sized holes which are
engaged by turning the disc. It ‘clicks’ into position. Look under the stage and see how it
works.

Iris diaphragm as in the Olympus microscope. This
diaphragm consists of a number of overlapping foils
and is adjusted by a small lever as in the 3
photographs at the right. Note that the position of
the lever changes in the 3 photographs.
The diaphragm helps to improve contrast. Stray light can
reduce contrast and the diaphragm only lets through the
light in the centre, which directly illuminates the specimen
being examined.
Lever
5.
Bright field and Dark field illumination
By far the majority of images you will look at will be ‘bright-field’ where the image is fully illuminated
from below.
Dark-field illumination however, uses light that is scattered by the specimen rather than being
transmitted through it. It works by introducing an opaque disc into the light beam.
Most microscopes are not designed for dark field illumination and it might only work under Low
Power. Despite this it is an interesting way of looking at some specimens and the images obtained
can give added detail that bright field illumination misses.
How is it done?
objective lens
slide with specimen and cover slip
opaque disc that interrupts the centre of the
light beam
lamp housing
bulb
The effect of the opaque disc is to interrupt the centre of the light beam. This means that light that
is normally transmitted through the specimen to the objective lens is blocked out. This gives the
dark background, hence the name ‘dark-field’ illumination.
The only light that passes to the objective lens is light that is scattered by particles on the slide.
Thus objects appear light against the dark background. Most objects are seen in unusual or false
colours but the effect does allow resolution of some objects that cannot be as easily seen with a
bright-field.
Comparison of Riccia leaf (an aquatic liverwort) under bright-field illumination (left) and dark-field
illumination (right).
In the images above, note the following:


There is an increase in contrast with dark-field illumination.
Many particles that are invisible under bright-field become visible under dark fields because
they scatter light. This is true of dust and any small objects
There are some easy ways in which you can quickly adapt a microscope to dark-field illumination
1.
Using a small opaque disc
mounted on a piece of glass. In
this case the disc is made of
black paper and the glass is
simply placed on top of the lamp
housing. (see photos right)
2.
An even simpler but not quite as
effective method is to place one’s
finger on top of the lamp housing
so that blocks off the centre of
the beam but allows most of the
light around the edge of the lamp
to pass to the specimen. Of
course this is done while you look
down the microscope so that you
can watch as you adjust. (see
photo below and diagram right)
3.
Small coins can also be used. In
this case, an old NZ 1 cent coin is
about the correct size. Place it on
a slide and balance this on top of
the lamp housing (photo right).
lamp housing
most of light beam
passes around finger
finger positioned
so that it blocks
centre of beam
6.
Depth of field exercise
To understand this concept you need to make up a slide with three overlapping pieces of thread. It
is easier to do this exercise if you:
 use threads of three distinctive colours and
 use small pieces of cello-tape to secure each thread as you mount it on the slide.
It is not necessary to use a cover slip for this exercise.
slide
three overlapping threads
Small pieces of cello-tape
Focus under Low Power. What do you notice about the three threads as you focus up and down?
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The term ‘depth of field’ is used to describe the range of distances objects can be focussed by a
lens. Microscope lenses have very shallow depths of field. Because the three threads mounted on
your slide overlap they will be at different heights and thus different distances from the objective
lens of the microscope. While one is in focus the other two will not be in focus because the
objective lens’s depth of field is too shallow to focus on all three threads at the same time.
Focused on lowest, green
thread
Microscope objectives
have a very narrow
‘depth-of-field’, This means
that any part of the specimen
above or below this zone
will be out of focus.
focal distance of lens
narrow depth-of-field
Focused on middle, yellow
thread
objective
lens
Focused on top, red thread
In practice this means that it is best to ‘focus
up and down’ while observing any specimen
under the microscope. As you focus up and
down different parts of the specimen will come
in and out of focus and you will see much more
than if you maintain a constant focus.
This will be especially true if you have a thick
specimen or a unicellular organism that is
swimming at different depths in the fluid
between the slide and the cover slip..
7.
Resolution exercise
For this exercise you will need a small piece of paper ripped from a glossy magazine that includes
part of a coloured illustration.
slide
cover slip
piece of coloured picture
Place this on a microscope slide, there is no need for water but a cover slip will keep the paper flat.
Focus under low power. What do you notice about the picture?
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Below are some pictures showing the type of image you should see:
Low power x40
Medium power x100
Low power x40
Low power x40
Can you see what this is a picture of?
It is in fact someone’s eye and eyebrow.
What this exercise illustrates is the important difference between magnification and resolution.
Magnification simply means enlarging the image so that it appears bigger than the specimen.
Resolution refers to the increased detail you are able to see. It is the ability to distinguish two
points or objects that are very close together.
In the example above our eyes are not able to resolve the dots of ink that make up the coloured
picture in a glossy magazine. However the microscope not only enlarges the image it also
increases resolution so that we can now resolve them.