Microscopes and
Cells
2.1.4 Comparison of relative sizes of
molecules, cell membrane thickness, viruses,
bacteria, organelles and cells, using the
appropriate SI unit.
Size relationships
of biological and chemical
levels of organization are
compared
-Notice the diversity and
how the power of 10 is used
-Although sizes are expressed
in length and diameter, cells
and organisms are 3-D
SIZE OF VARIOUS CELLS AND STRUCTURES:
Molecules: 1 nm
Membranes (on organelles): 10 nm
Viruses: 100 nm
Bacteria: 1 um
Organelles: up to 10 um
Most cells: up to 100 um
Measurements above in 2D, remember all structures have 3D
shape.
The Metric System
Know how to convert from one unit to
another.
Kilo1000
Units
Hecto100
Dekunits
10
units
Divide
Multiply
Basic
Unit
Deci0.1
units
Centi0.01
units
Milli0.001
units
What units are used to measure cells?
1 mm = 1000 micrometers (um)
1 mm = 1,000,000 nanometers (nm)
Or…
A micrometer is 1 x 10-3 mm (0. 001)
A nanometer is 1 x 10-6 mm (0.000001 mm)
Magnification and Scale bars
Specimen size how large the specimen actually is
Image size how large the specimen appears in a
drawing or photograph
Magnification how much larger the image is than
the actual size
Formula used for these calculations:
Magnification = size of image
size of specimen
Microscopy Calculations Youtube video
Formulas
Magnification =
Size of image =
Size of specimen =
Calculating Linear Magnification
What is the actual size
of this specimen in um?
60mm/5 = 12mm
12mm x 1000 um =
Magnification x5
12,000 um
60 mm
Measuring
picture
2.1.5 Calculate the linear magnification of
drawings and the actual size of specimens in
images of known magnification
Magnification could be stated (for example, ×250) or
indicated by means of a scale bar
Scale bar
1um
Calculating image size using Scale Bar
Scale bar
0.1mm
If we want to see a cell…
We have to
magnify it
Magnification:
making something
that is small appear
larger
A cell from the
inside of your
cheek
Body tube
Revolving nosepiece
Objective lens
Eyepiece
Arm
Stage
Stage clips
Diaphragm
Light source
Coarse
adjustment
knob
Fine adjustment
Base
Light Microscopy
Advantages
Can view living
specimens
Inexpensive and
easy to use
Light Microscopy
Disadvantages
Resolution is limited.
Resolution: the ability to form
separate images of objects that
are close together
Resolving power: the minimum
distance two points can be
separated and still be individually
distinguished as two separate
points.
The smaller the resolving power,
the better the resolution.
Light Microscopy
Disadvantages
Can only magnify a
limited number of
times (ours go up to
1000x; best light
microscopes magnify
up to 4000x)
Limited by focal length
of lens
Electron Microscopes
To magnify an image a
large number of times,
you must use an
electron microscope.
Specimen has a beam
of electrons passed
through it
Electron Microscopes
There are different types of
electron microscopes
In a transmission electron
microscope (TEM), an electron
beam passes through a very thin
section of material
An image is formed because the
electrons pass through some
parts of the section but not
others
In a scanning electron
microscope (SEM), a narrow
beam of electrons is scanned in
a series of lines across the
surface of the specimen
The electrons that are reflected
or emitted from the surface are
collected by a detector and
converted into an electrical
signal, which is used to build a 3D image, line by line, on a TV
screen
18
Electron Microscope Advantages
Images can be
magnified thousands
of times (up to
250,000x)
A lot of detail can be
seen
Electron Microscopy
Advantages
Can magnify
1000’s of
times
Details are
easily visible
HIV, magnified
24,000x
Electron Microscopy
Disadvantages
Expensive ($$$$$)
Must use heavy
metal dyes, which
kill organisms
Transmission vs.
Scanning EM
Transmission EM’s view
cross-sections
SEM’s view surfaces only
Comparison of Light and Electron Microscopes
Light Microscopes
Electron Microscopes
Material can be prepared easily for examination.
Often, a sample can simply by placed on a slide
with a few drops of water and a cover slip. An
image can be obtained within seconds
Preparation of material for examination always
involves a long series of procedures. These take
several days to complete and often involve the
use of toxic chemicals
Living material can be examined, so specimens
do not always have to be killed. There is less
danger of artificial structures appearing and
causing confusion if the specimen is still alive
Living material cannot survive in the vacuum
inside electron microscopes. Tissues therefore
have to be killed as the first stage in the
preparation of them for examination
Movement can be observed if living material is
examined, including the flow of blood, streaming
of cytoplasm inside cells and the locomotion of
microscopic organisms
No movement can be observed as the material is
always dead. Movement can only be deduced
indirectly by complex experiments, often
involving radioactive tracers
Colors can be seen – both natural colors and
artificial colors caused by staining
Only monochrome images are produced, with
black, white, and shades of grey
The field of view (the area which can be
observed at once) is relatively large ~2mm
across at low power with typical microscopes
Only a small field if view can be examined at
once – in a TEM the max uninterrupted view is
about 100mm across
The resolution of light microscopes is relatively
poor – about .25mm so the max useful
magnification is only about x600. Many
structures within cells cannot be seen clearly
The short wavelength of electrons gives very
good resolution – about .25nm. This allows
magnification of up to x500,000. Very small
objects therefore become visible including many
23
of the details of cell structure
To calculate total
magnification:
Multiply the
magnification on the
objective by the
magnification found
on the eyepiece
You will need this
for every specimen
you draw under the
scope!
Field of View (FOV)
Field of View: Sometimes abbreviated "FOV", it is
the diameter of the circle of light that you see when
looking into a microscope.
As the power gets greater, the field of view gets
smaller.
You can measure this by placing a clear metric ruler
on the stage and counting the millimeters from one
side to the other. Typically you will see about 4.5mm
at 40X, 1.8mm at 100X, 0.45mm at 400X and
0.18mm at 1000X.
http://www.microscope-microscope.org/basic/microscope-glossary.htm
Calculating FOV
1.
2.
3.
4.
5.
6.
Measuring the microscope field of view on lowest
power
Place a clear plastic ruler with mm markings on top of
the stage of your microscope.
Looking through the lowest power objective, focus your
image.
Count how many divisions of the ruler fit across the
diameter of the field of view.
Multiply the number of divisions by 1000 to obtain the
field of view in micrometers (µm).
Record this in µm (1mm = 1000 µm ).
Magnified at 40X, the lines of the ruler are clearly
visible.
http://www.saskschools.ca/curr_content/biology20/unit1/UNIT1MODULE2LESSON2.htm
FOV Mathematical Calculation
Total Magnification Low Power =
Total Magnification at Other Power
FOV at Other Power
FOV at Low Power
Practice calculating FOV
Example:
If a 5x FOV is 3 mm, what is the 40x FOV of that
microscope?
Total Magnification Low Power = FOV at Other Power
Total Magnification at Other Power FOV at Low Power
5
40
=
FOV at Other Power
3mm
(3)(5) = (FOV of higher power)(40)
=0.375 mm FOV of higher power