Download CHAPTER 2 - MICROSCOPY

Chapter 2
- Microscopy
CHAPTER 2 - MICROSCOPY
Cells are studied by a combination of methods
One of the most important tools used to study cell structures has been the
microscope. In fact, cells were not described until 1665, when Robert Hooke
examined a piece of cork using a microscope he had made. In his book Micrographia,
published in 1665, Hooke drew what he saw and described many objects that he
viewed through his microscope. Hooke did not actually see cells in the cork; he saw
the walls of dead cork cells. Not until much later was it realized that the interior
enclosed by the walls is the important part of living cells.
2.1. Light microscopes
A few years after Hooke described dead cork cells, the Dutch naturalist Anto
van Leeuwenhoek viewed living cells with small lenses that he made. However, he
did not share his lens making techniques, and more than a century passed before
biologists realized the importance of microscopes and what they could reveal. It was
not until the early 19th century that microscopes were sufficiently developed for
biologists to begin their study of cells.
Most cells are too small to be seen. Even if you use a magnifying glass or a
hand lens, a cell would only appear as big as a full stop. You need a microscope to
see the detailed structure of cells. The most common type of microscope used in
schools and colleges is called a light microscope. The name ‘light microscope’ means
that light is passed through the specimen that is being studied.
Light rays are focused on to a transparent specimen by a condenser lens. The
rays pass through the specimen and are focused again by two more lenses – the
objective lens and the eyepiece lens. These two lenses produce a magnified image.
As many biological specimens are colourless and nearly transparent, stains
are often used to make different parts show up clearly. Stains usually colour just a
particular part of a cell; iodine solution, for example, colours starch grains blueblack. Some stains, such as methylene blue or iodine solution, can be added to living
cells. In other cases, the specimen is ‘fixed’ by adding a chemical such as acetic acid
or alcohol, known as a fixative. These chemicals react with substance in the cell,
making them insoluble and so anchoring them in position. The cells are killed when
the fixative is added. Stains may be added either before or after the fixing process.
So long as the specimen is thin enough to allow light to pass through, there
are no limitations on what you can look at using a light microscope. Living, moving
organisms such as protoctists can be watched, or you can look at a permanent
stained preparation of a thin section through a piece of a human tissue.
2.2 The function of the parts of the microscope
Tube – the microscope image is viewed through the tube with the aid of an eyepiece
(one in a monocular microscope and two in a binocular microscope)
Eyepiece – the image projected by the objective is further magnified when viewed
through this lens (Fig 2.1)
Arm – the central element integrating all the mechanical and optical components to
form the complete microscope. It supports the body tube and is the part you can
grasp to carry the microscope.
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Objective lenses – form a magnified image of the objective in the intermediate
image plane. There are three objective lenses to choose from: low power objective,
medium power objective and high power objective.
Stage – supports the slide which is clamped into position by means of clips. This has
a hole in it that allows light to shine up through the specimen.
Coarse adjustment – moves the tube and lenses up and down to approximately the
right position so that the specimen is in focus. This knob is used only with the low
power objective lens.
Fine adjustment – moves the tube and lenses up and down to put the specimen at
the right position so that the specimen is perfectly focused. It is used to achieve fine
focus with the high power and medium power objectives.
Fig 2.1
Light source – a bulb which supplies light, it is situated inside the base of the
microscope.
Iris diaphragm - a hole under the stage that regulates the amount of light that
goes through a specimen on the stage, it collects light and illuminates the specimen.
Mirror – has a flat surface on one side and concave surface on the other side that is
used to reflect light up through the specimen on the stage.
The microscope is used to see tiny objects that are invisible to the naked
eyes. It uses lenses to magnify the object to focus (display) it in greater details. A
very good light microscope can magnify about 1500 times, and can show many of
the important structures in animal and plant cells. The discovery and the introduction
of the microscope have significantly contributed (more than any other instrument) to
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the development and understanding of biology as a science. There are two types of
microscope named after the source of illumination: light microscope and electron
microscope. The light microscope is the one that you are going to use in your biology
laboratory.
2.3. Electron microscopes
The principle is the same as that of a light microscope except that beams of
electrons are used instead of beams of light. They are focused using electromagnets
rather than glass lenses. As electrons are easily stopped by air molecules, the space
inside an electron microscope must be a vacuum. As our eyes do not respond to
electrons, the electrons are allowed to hit a fluorescent screen which emits visible
light where the electrons hit (Fig 2.2 and 2.4).
Electrons cannot penetrate materials as well as light rays can, so specimens
for viewing in an electron microscope must be much thinner than those used in a
light microscope. This and the fact that the specimen has to be placed in a vacuum,
places great limitations on what can be viewed using an electron microscope. In
particular, it is not usually possible to look at living material.
As in a light microscope, specimens are stained. Heavy metal ions, such as
lead or osmium, are added to the specimen and are taken up by particular parts of
the cells. Atoms of these metals have large, positively charged nuclei which scatter
electrons rather than letting them pass straight through. These electrons therefore
do not arrive on the screen, so leaving a dark area in the image. The structures in a
cell which have taken up these heavy metal stains therefore appear dark.
Fig 2.2
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2.4 Magnification and resolution
Magnification is the number of times larger an image is than the specimen.
For example, if a cell is 10µm in diameter, and a microscope produces an image of it
which is 1mm (1000 µm) in diameter, then the microscope has magnified the
specimen 100 times.
Size of image
Magnification = -----------------------Size of specimen
The magnification produced by a light microscope depends on the strengths of
the objective lens and the eyepiece lens. If you are using a x40 objective lens and a
x10 eyepiece lens, then your specimen is being magnified 400 times.
There is no limit to the amount a light microscope can magnify. By putting in
stronger lenses, or more lenses, you could produce a huge image several metres
across. But the image would not be at all clear, and you would not be able to see any
more detail than before. This is because the resolution of a light microscope is
limited. Indeed, at higher magnification, the image would become blurrier. Light
microscopes can effectively magnify objects only about 1000 times
Resolution is the degree of detail which can be seen (Fig 2.5). The limit of
resolution of a microscope is the minimum distance by which two points can be
separated and still be seen as two separate points and not one fuzzy one. Newspaper
photography’s, for example, have a rather poor resolution, being made up of quite
large dots which you can see with the naked eye. This limits the amount of detail
which you can see. Compare this with a good quality photograph with a higher
resolution (smaller dots)
The limit of resolution depends on the wavelength of light. The resolution limit
is about 0.45 times the wavelength. Shorter wavelengths give the best (smallest)
resolution. The shortest wavelength light, which we can see in blue light, has a
wavelength of about 450nm. This gives a resolution of about 0.45x 450nm, which is
close to 200nm. Any objects smaller than this, or any points less than 200nm apart,
will either be invisible or appear as blurs.
Electron beams, however, have a much shorter wavelength than light; so
much better resolutions can be resolution around 400 times better than that of light
microscopes, being able to separate objects as light as 0.5 nm apart.
Resolution is the capacity to distinguish fine detail in an image. This is
defined as the minimum distance between two points at which they can both be seen
separately rather than a single, blurred point.
Resolution is the ability of an optical instrument to show two close objects as
separate. For e.g, what looks to your unaided eye like a single star in the sky may be
resolved as two separate stars with the help of a telescope?
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Fig 2.3
2.5 Comparison of advantages and disadvantages of the light and electron
microscopes (Fig 2.3)
LIGHT MICROSCOPE
ELECTRON MICROSCOPE
Advantages
Disadvantages
Cheap to purchase and operate
Small and portable-can be used almost
anywhere
Unaffected by magnetic fields
Preparation of material is relatively
quick and simple, requiring only a little
expertise
Material rarely distorted by preparation
Natural colour of the material can be
observed
Disadvantages
Expensive to purchase and operate
Very large and must be operated in
special rooms
Affected by magnetic field
Preparation of material is lengthy and
requires considerable expertise and
sometimes complex equipment
Preparation of material may distort it
All images are in black and white
Magnifies objects up to 2000*
The depth of field is restricted
Magnifies objects over 500000*
It is possible to investigate a greater
depth of field
Advantages
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Fig 2.4
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Fig 2.5
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