Microscopy

The light or optical microscope was invented in the 17th century. It has been refined in many ways over
the years but it is essentially still the same. The light microscope works as follows; light rays from a
source beneath the stage are passed through the sample to glass lenses in series. The two lenses are
called the objective lens and the ocular (eyepiece) lens. Most light microscopes have three or four
objective lenses on a rotating turret. These lenses magnify the image by 4x to 100x. The light then
passes up the body tube to an ocular lens that magnifies the image another 10x to 15x.

There is a limit to the amount of detail the light microscope can show, which is determined by the
resolving power (or resolution). The resolution is defined as the minimum distance by which two points
must be separated in order for them to be perceived as two separate points, rather than a single fused
image. For the light microscope this distance is approximately 0.2µm. So in theory it might seem
possible to magnify an object indefinitely by means of glass lenses in series. This has been put into
practice and has only produced a larger and fuzzier picture; so the resolution is not improved and no
more detail is visible. The resolution of the light microscope is imposed by the wavelength of visible
light, and means that little is gained by magnifying an object more than 1500 times. This limits the
amount of structural detail that can be seen within a cell.

Higher magnification with good resolution can be obtained by using a special objective lens. This lens is
called an Oil Immersion lens; this is a lens with fluid (oil) between the sample and the objective, but
even with this lens it not possible to achieve effective magnification above 2000 times.

Principle of immersion microscopy.

Path of rays with immersion medium (yellow) (left half) and
without (right half). Rays (black) coming from the object (red)
at a certain angle and going through the coverslip (orange, as
the slide at the bottom) can enter the objective (dark blue)
only when immersion is used. Otherwise, the refraction at the
coverslip - air interface causes the ray to miss the objective
and its information is lost.

Various stains and dyes are frequently used in microscopy to highlight structures in biological tissues for
viewing. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers
or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within
individual cells.
The light microscope opened up a new world of structural detail for biologists, revealing the variety of
cell forms making up new organisms. However, the limitations in resolution prevented them from seeing
the fine detail within a cell. This problem was overcome by using radiation with a wavelength less than
that of light.

In 1933 the Electron microscope was developed. The electron microscope works on the same principles
as the light microscope but instead of light rays, with wavelengths in the order of 500nm, a beam of
electrons of wavelengths 0.005nm is used. This means that the electron microscope can magnify up to
500,000x without loss of resolution, compared to the best light microscopes which magnify only around
2000x.

The light microscope uses glass lenses to focus the light rays, whereas the electron beam of the electron
microscope is focused by powerful electromagnets. The image produced by the electron microscope
cannot be detected directly by the naked eye. The beam of electrons is directed on to a screen from
which black and white photographs, called Photoelectron micrographs, can be taken.

There are two main types of electron microscope. These are called the Transmission electron
microscope (TEM), and the Scanning electron microscope (SEM).

In the TEM, a beam of electrons is passed through thin, specially prepared slices of material. Details in
light microscope samples can be enhanced by stains that absorb light; similarly TEM samples of
biological tissues can utilize high atomic number stains to enhance contrast (high atomic number = large
nucleus of atom = greater chance of absorbing/scattering an electron). The stain absorbs electrons or
scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of
heavy metals such as osmium, lead, or uranium may be used prior to TEM observation to selectively
deposit electron dense atoms in or on the sample in desired cellular or protein regions, requiring an
understanding of how heavy metals bind to biological tissues.

Therefore, parts of the sample allow the electrons through while other parts absorb/scatter the
electrons. As air molecules would also absorb/scatter the electrons, a vacuum has to be created within
the instrument.

Where electrons are absorbed by the material, and therefore do not reach the screen, the image is dark.
Such areas are said to be electron dense. Where electrons penetrate, the screen appears bright. These
areas are termed electron transparent. As electrons have a very small mass, they do not easily penetrate
materials and so the sections need to be exceedingly thin (at most hundreds of nm thick). This
sectioning creates a flat image and the natural contouring of a specimen cannot be seen.

To overcome this, the SEM was developed. In the SEM a fine beam of electrons is passed to and fro
across the specimen, beginning at one end and working across to the other. At each point on the
specimen the electron beam loses some energy, and that lost energy is converted into other forms, such
as heat, emission of low-energy secondary electrons, light emission (cathodoluminescence) or x-ray
emission. The display of the SEM maps the varying intensity of any of these signals into the image in a
position corresponding to the position of the beam on the specimen when the signal was generated.
Most commonly, low energy secondary electrons are amplified and transmitted to a screen. The
resultant image shows holes and depressions as dark areas, ridges, and extensions of the surface as
bright areas. In this way the natural contouring of the material may be observed.

Biological (and other non-conductive) specimens tend to gather a charge when scanned by the electron
beam, which causes scanning faults and other image artifacts. They are therefore usually coated with an
ultrathin coating of electrically-conducting material, commonly gold, deposited on the sample either by
low vacuum sputter coating or by high vacuum evaporation. Conductive materials in current use for
specimen coating include gold, gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium
and graphite. Coating prevents the accumulation of static electric charge on the specimen during
electron irradiation.

A spider coated in gold, having been prepared for viewing with a scanning electron microscope.
However there are problems with both these forms of electron microscope, in that they require
expensive and complex preparation techniques, coupled with the need for a high vacuum. This means
that the material being observed must be dehydrated, therefore dead and is frequently considerably
distorted; what you see may be very different from the original material.

In response to this a new generation of electron microscopes has been developed - the Environment
Scanning Electron Microscope (ESEM). These microscopes allow the material on view to be kept at a low
or partial vacuum, while the region around the electron gun is at high vacuum. This is achieved by
separating the microscope column into a series of chambers. Each of these chambers has its own
pressure. There is only a minute hole between chambers; the hole is wide enough to allow the tiny
electron beam through. A special low voltage detector which can operate at low vacuum is used to
detect the scattered electrons, secondary electrons and X-rays which provide the image.

Overleaf is a table of comparison, of the advantages and disadvantages of the light and electron
microscope.

Light/Optical Microscope Electron Microscope

Cheap to purchase and operate Expensive to purchase and operate

Small and portable- can be used almost Very large and must be operated in special
anywhere rooms

Unaffected by magnetic fields Affected by magnetic fields

Preparation of material is relatively quick and Preparation of material is lengthy and

simple, requiring only a little expertise requires considerable expertise and
sometimes complex equipment

Material rarely distorted by preparation Preparation of material may distort it

Natural colour of the material can be observed All images are black and white

Magnifies objects up to 2000x Magnifies objects over 500,000x

The depth of the field is restricted It is possible to investigate a greater depth of

field