1.0 CELL STRUCTURE AND FUNCTION
1.1 MICROSCOPY
Objectives
By the end of the subtopic, learners should be able to:
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Calibrate the eyepiece graticule of a microscope.
Draw and determine linear dimensions of specimens.
Distinguish between magnification and resolution.
Prepare and mount temporary slides on microscopes
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Microscopy is the use of microscopes to view objects and areas of objects that cannot be
seen with the naked eye.
There are three branches of microscopy are, namely optical, electron and scanning probe
microscopy.
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a. Light microscope
o The diagram below shows the light microscope.
Fig.1.1.1: The light microscope
o The eyepiece lens magnifies and focuses the image from the objective onto the eye.
o The objective lens collects light passing through the specimen and produces a magnified
image.
o Condenser lenses focus light onto the specimen that is on the stage, held between the
cover slip and the slide.
o They are particularly helpful when coupled with the highest objective lens.
o The light source produces the light that is focused onto the specimen.
o Usually, there are three or four objective lenses on a microscope, consisting of 4x, 10x,
40x and 100x magnification powers.
o To obtain the total magnification of an image, one needs to multiply the eyepiece lens
power by the objective lens power.
o If one couples a 10x eyepiece lens with a 40x objective lens, the total magnification
would be 10 x 40 = 400 times.
o The stage is a flat platform that supports the slide being analysed.
o The diaphragm controls the intensity and size of the cone light projected on the
specimen.
o The more transparent the specimen, the less the light required.
o Coarse adjustment knobs move the stage up or down, in order to coarse adjust the
focus.
o Fine adjustment knobs are used to fine adjust the focus.
b. The electron microscope
o There are two types which are the scanning electron microscope and the transmission
electron microscope.
o It uses electrons to create an image of the specimen being examined.
o A scanning electron microscope scans a focused electron beam over a surface to create
an image.
o The electrons in the beam interact with the sample, producing various signals that can
be used to obtain information about the surface topography and composition.
o The diagram below shows an electron microscope.
Fig.1.1.2: The scanning electron microscope
o The Scanning Electron Microscope can produce three-dimensional images.
o Thus great detail of many tissue or cellular arrangements can be shown, unlike on a
light microscope.
o The resolution is 0.2nm, which is 1000x more than a light microscope.
o Because of this resolution, electron microscopes can produce very detailed images of
organelles inside the cells, which could otherwise not be seen.
o Electron microscopes are very expensive, and to use and prepare samples for the
microscopes is also very expensive.
o One must be expertly trained and able to work with a high degree of skill.
o Electron beams are deflected by molecules in the air, requiring the sample to be placed
in a vacuum.
o Images produced are always in black and white and so must be falsely coloured.
Resolution and magnification
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Resolution is the ability to distinguish between two separate points.
Inability to resolve means the two points are seen as one.
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It refers to the amount of detail that can be seen, that is, the greater the resolution, the
greater the detail that can be seen.
For a light microscope, the maximum resolution is 200nm, meaning that any two objects
with a distance of less than 200nm between them cannot be distinguished as separate.
For an electron microscope, the resolution is about 0.2nm, which is 1000 times that of the
light microscope.
Magnification is the enlargement of an object. It is the number of times that an image is
greater than the actual object.
Magnification can be calculated using the formula:
𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 = 𝑖𝑚𝑎𝑔𝑒 𝑠𝑖𝑧𝑒 ÷ 𝑎𝑐𝑡𝑢𝑎𝑙 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑏𝑗𝑒𝑐𝑡
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The magnification of a light microscope is formed using a mixture of the powers of the
eyepiece and the objective lens.
The eyepiece produces a power of 10x and the objective lens can produce various different
powers, so if it were to produce a power of 100x, the final magnification would be 1000x
(10 x 100).
The image would appear 1000 times larger than it actually is.
Light microscopes generally have three different objective lenses to allow the slide to be
viewed in three separate manners.
Such microscopes are known as compound light microscopes.
Measuring specimens
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Measurement of length is based on a comparison of the object under scrutiny with another
of known dimensions, or with a standardized, calibrated scale, for example, a ruler.
When measuring the specimen, a transparent scale called an eyepiece graticule can be
used.
The diagram below shows an eyepiece gratitude.
The eyepiece scale is divided into 100 small divisions.
Fig.1.1.3: An eyepiece graticule
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It is placed in the microscope eyepiece so that it can be seen at the same time as the object
to be measured.
To know the actual size of the eyepiece units, the eyepiece graticule scale should be
calibrated.
This is done by using a transparent ruler, called a stage micrometre scale.
It is placed on the microscope stage and brought into focus.
The stage micrometre scale is 1 mm divided into 100 divisions so each division is equivalent
to 10µm.
The images of the two scales are superimposed as shown on the diagram below.
Fig. 1.1.4: The eyepiece and the stage micrometre superimposed
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Count the number of divisions on the eyepiece graticule that corresponds to the total
length of the stage micrometre (Z).
Calculate the actual length (A) represented by one division of the eyepiece graticule.
To find the actual length represented by the whole length of the eyepiece graticule (W):
𝑊 =𝐴×𝑍
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Fig.1.1.5 below shows how to calibrate the eyepiece graticule.
Fig.1.1.5: How to calibrate the eyepiece graticule
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This can now be used to measure the length of any specimen that is viewed under the
microscope.
Preparing microscope slides
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The specimen is placed on a slide, in a drop of water.
A cover slip is carefully lowered onto the slide to cover the specimen at an angle of 45
degrees.
This can be done using a mounted needle.
To improve visibility, the specimen can be stained by irrigating with, for example, iodine
solution or potassium iodide solution when observing cells.
Staining improves the contrast between different structures.
This stains the contents of the cell yellow with the nucleus strongly stained than the
cytoplasm.
The vacuole and the cell wall are not stained.
Different types of stains can be used to stain the cell contents differently for easy visibility.
If there are any air bubbles, they are removed by gently pressing on top of cover slip to
move them to the edge.
Excess water is dabbed off using paper towels or filter paper.
Fig 1.1.6 shows the stages of preparing a slide.
Fig.1.1.6: Preparing a microscope slide
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In light microscopy, the oil immersion technique is used to increase the resolving power of a
microscope.
This is achieved by immersing both the objective lens and the specimen in a transparent oil
of high refractive index, thereby increasing the numerical aperture of the objective lens.
Many condensers also give optimal resolution when the condenser lens is immersed in oil.
Oil immersion objectives are used only at very large magnifications that require high
resolving power.
Samples for electron microscope need to be sectioned into thin slices to allow electrons to
pass through them.
The first step in the preparation process is fixation.
In this step, living tissue is chemically treated to stabilise it.
This also kills the tissue sample at the same time.
This should be done as quickly as possible because, as soon as tissue is removed from its
natural environment, it starts to change.
The samples are first fixed and dehydrated and then embedded in hard resin so that they
can be easily cut.
To be visualised by an electron microscope, biological samples need to be:
o fixed so the electron beam does not destroy them.
o dried thoroughly so the vacuum does not affect them.