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Microscopy
1. Light microscopes
Most cells are very small, and their structures can only be seen
by using a microscope.
This is the oldest, simplest and most widely-used form of
microscopy.
Specimens are illuminated with light, which is focused using glass
lenses and viewed using the eye or photographic film.
Specimens can be living or dead, but often need to be coloured
with a coloured stain to make them visible. Many different stains
are available that stain specific parts of the cell such as DNA,
lipids, cytoskeleton, etc.
All light microscopes today are compound microscope, which
means they use several lenses to obtain high magnification.
Light microscopy has a resolution of about 200 nm, which is good
enough to see tissues and cells, but not the details of cell
organelles.
There has been a recent resurgence in the use of light
microscopy, partly due to technical improvements, which have
dramatically improved the resolution far beyond the theoretical
limit. For example fluorescence microscopy has a resolution of
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about 10 nm, while interference microscopy has a resolution of
about 1 n m .
Mechanism: light rays pass through the specimen on a
slide focused by an objective lens and an eyepiece lens. ---
> magnified image of the specimen on the retina of
your eye/screen/camera.
Microscopy Magnification
Magnification is how many times bigger the image of a
specimen observed is in compared to the actual (real-life) size
of the specimen.
A light microscope has two types of lenses:
o An eyepiece lens, which often has a magnification of x10
o A series of (usually 3) objective lenses, each with a
different magnification
To calculate the total magnification the magnification of
the eyepiece lens and the objective lens
are multiplied together:
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Total magnification =eyepiece lens magnification x objective lens
magnification
Drawing Magnification
The magnification of a microscope simply indicates how much
bigger the image is that the original object. To better
understand the images we produce using microscopes we need
to know the actual size of the specimen.
It is usually given as a magnification factor, e.g. x100. By using
more lenses microscopes can magnify by a larger amount, but
this doesn't always mean that more detail can be seen.
The size of cells is typically measured using
the micrometre (μm) scale, with cellular structures measured
in either micrometers (μm) or nanometers (nm)
When doing calculations all measurements must be in
the same units. It is best to use the smallest unit of
measurement shown in the question.
There are 1000 nanometers (nm) in a micrometre (µm)
There are 1000 micrometres (µm) in a millimetre (mm)
There are 1000 millimetres (mm) in a metre (m)
To convert units, multiply or divide depending if the units
are increasing or decreasing
Magnification does not have units
Magnification calculations
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Work out the real size of an object knowing the magnification:
a. This drawing of a mitochondrion has been magnified 100
000 times.
Use ruler to measure its length in mm (50 mm).
Convert this measurement to µm by multiplying by 1000.
50 x 1 000 = 50 000 µm
Substitute into the equation:
Step 1:
Check that units in magnification questions are the same
Remember that 1mm = 1000µm
2000 / 1000 = 2, so the actual thickness of the leaf is 2 mm
and the drawing thickness is 50 mm
Step 2: Calculate Magnification
Magnification = image size / actual size = 50 / 2 = 25
So the magnification is x 25
Measuring cells using a eyepiece graticule and stage micrometer
An eyepiece graticule and stage micrometer are used to measure
the size of the object when viewed under a microscope.
Eyepiece graticule is a little scale bar placed in the eyepiece of
light microscope. The graticule is marked off in 'graticule units’.
It is usually 100 divisions. Turn the eyepiece so that the graticule
scale lies over the object.
Calibration: the conversion of graticule units into real units (mm,
µm). For this special slide called a stage micrometer is placed
on the microscope stage and is brought into focus. This is
marked off in a tiny scale. The smallest markings are often 0.01
mm (10 µm) apart.
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Take the specimen off the stage or the microscope and replace it
with the stage micrometer. Use the same objective lens. Line up
the micrometer scale and the eyepiece graticule scale (by
turning the eyepiece and moving the micrometer on the stage).
Make sure that 2 large markings on each scale are lined up.
Eyepiece graticule scale (arbitrary
units)
Stage micrometer
(0.01mm & 0.1mm
divisions)
In the eyepiece graticule 100 units measure 0.25mm.the value of
each eyepiece unit is 0.25/100 = 0.0025mm
0.0025x1000 = 2.5μm
The diameter of the cell superimpose on the scale measure 20
eye piece units
The actual diameter is 20x2.5 μm= 50 μm
Resolution.
The amount of detail depends on the resolution of a microscope,
which is the smallest separation at which two separate objects
can be distinguished (or resolved). Resolution is therefore a
distance (usually in nm).
So, the resolution of a microscope is ultimately limited by the
wavelength of light (400-600nm for visible light).
As light passes through the specimen, it will be diffracted
The longer the wavelength of light, the more it is diffracted and
the more that this
diffraction will overlap as the points get closer together
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To improve the resolution a shorter wavelength of light is
needed, and sometimes microscopes have blue filters for this
purpose (because blue has the shortest wavelength of visible
light).
Problems with the light microscopy:
Less magnification(x1500) and resolution (can be used for
specimens above 200nm)
2. Electron microscopes
There are two kinds of electron microscope.
1. The Transmission Electron Microscope (TEM) works much like a
light microscope, transmitting a beam of electrons through a thin
specimen and then focusing the electrons to form an image on a
screen or on film. This is the most common form of electron
microscope and has the best resolution.
2. The scanning electron microscope (SEM) scans a fine beam of
electron onto a specimen and collects the electrons scattered by
the surface. This has poorer resolution, but gives excellent 3-
dimentional images of surfaces.
Mechanism
High voltage (50000V) is passed through a tungsten filament on
top of the column. Then the hot filament release steam of
electrons specimen very thin, placed in a vacuum to allow
electrons to pass through it. Electrons are focused onto
a fluorescent screen/photographic film ---> magnified image of the
specimen.
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Advantages of Electron Microscope
High magnification and High resolution (TEM-0.5 nm/SEM 5 to
20nm) compare to light microscopy Due to the higher
frequency of electron waves (a much shorter wavelength).
Larger samples SEM
Give 3D effects SEM
Surface structures can be seen
Problems with the electron microscopy:
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• The electron beam is scattered by air molecules, so to avoid this
there is a vacuum inside an
electron microscope, so it can't be used for living organisms.
• Specimens must be very thin, so are embedded in plastic for
support, and so can't be
manipulated under the microscope.
• Specimens can be damaged by the electron beam, so delicate
structures and molecules can be destroyed.
• Specimens are usually transparent to electrons, so must be
stained with an electron-dense chemical (usually heavy metals like
osmium, lead or gold).
• Initially there was a problem of artefacts (i.e. observed structures
that were due to the preparation process and were not real), but
improvements in technique have eliminated most of these.
Expensive to run and buy
Preparation of material is time consuming and require expert
training.
Comparison of Light and Electron Microscopes
Light Microscopes Electron Microscopes
illumination and
light from lamp electrons from hot wire
source
focusing glass lenses electromagnets
detection Eye or film phosphor screen or film
magnification X 1 500 -2000 X 500 000
resolution 200 nm 0.5 – 1 nm
tissues, cells and small cell organelles, microbes
used to observe
organisms and viruses
specimen living or dead dead
heavy metals and vacuum is
staining coloured dyes
needed
cost cheap to expensive very expensive
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