Leeuwenhoek microscope of about 1670 (left) and

Zeiss microscope of 2020 (right)

• It is difficult to obtain the magnifications of more than 400x with a light
microscope. It will be impossible to observe two separate dots if they are
very close to each other.
• A hand lens or microscope allows smaller details to be distinguished but
there is a limit because of distortions caused by the wavelength of light.
• Electron microscopes use beams of electrons instead of light. The
wavelength of electrons is much shorter than the wavelength of visible light.
• The first electron microscopes were designed and constructed in Germany
during the 1930s. They came into use in research laboratories in the 1940s
and 50s.
• Some electron microscopes can give magnifications up to 1,000,000x
• Making the separate parts of an object distinguishable by eye is called
resolution.
• Because electron microscopes have better resolution, they can give much higher
magnification. This means much smaller structures can be seen than with a light
microscope.
• Electron microscopes have allowed scientists to investigate the detailed structure
(ultrastructure) of cells.
• Electron microscopes do have some disadvantages. They can only give black and white
images, so any colour in electron micrographs has to be added artificially.
• The methods used to prepare material for the electron microscope always kill the
cells. In any case, cells would die inside an electron microscope because there is a
vacuum and the beams of electrons are very destructive.
• In contrast, light microscopes can be used to examine living material and produce
images in colour. Therefore, both types of microscopes are very useful in research and
continue to be widely used.
1. Fluorescent stains and immunofluorescence
• Most of the chemicals in cells are white or colourless so they are difficult to distinguish unless stained.
• Stains used in microscopy are coloured substances that bind to some chemicals but not others.
• For example, methylene blue binds to DNA and RNA so it stains the nucleus dark blue and cytoplasm a lighter
blue.
• Fluorescence is when a substance absorbs light and then re-emits it at a longer wavelength. Fluorescent stains
have been used for over 100 years. Some absorb ultraviolet light and re-emit it as blue light, for example.

The fluorescent stain (yellow)

may be linked directly to the
antibody that binds to the
target antigen (green).

Alternatively, it may be linked

indirectly by an antibody that
binds to the primary antibody
Special fluorescence microscopes have been designed and built with intense light sources such as high-
power LEDs or lasers that emit a single wavelength. This light is absorbed and re-emitted by the sample,
generating particularly bright images.
Immunofluorescence is a development of fluorescent staining. Antibodies that bind to particular chemicals
(antigens) in the cell are produced. Then fluorescent markers of different colours are linked to these
antibodies. A multicoloured fluorescent image can then be produced showing where the chemicals are
located. There are many research applications of this technique; for example, it can be used to find out if
one specific type of protein is being produced in a cell.

In this image produced by

immunofluorescence, DNA in the
nuclei is stained cyan. Microtubule
proteins of the cytoskeleton, which are
normally invisible, are stained
magenta. This image was produced
using a Nikon RTS2000MP custom laser
scanning microscope
2. Freeze-fracture electron microscopy
• This technique is used to produce images of surfaces within cells. A sample is plunged into liquified propane at
–190°C so it rapidly freezes.
• A steel blade is then used to fracture the frozen sample. The fracture goes through the weakest points of the cells.
• Some of the ice at the fractured surface is removed by vaporization, to enhance the texture of the surface. This is
called etching.
• Then a vapour of platinum or carbon is fired onto the fracture surface at an angle of about 35° to form a coating.
This creates a replica of the fracture surface.
• The replica is removed from the frozen sample and can be examined
using an electron microscope.
• It is about 2 nanometres thick on average but the thickness varies
because of the angle at which the coating is applied.
• This gives the impression of a 3D image through shadowing. The
weakest point in cells is usually the middle of membranes, between
the two layers of phospholipid.
• The freeze-fracture process gives a unique image of this part of cells.
When these images were First produced, they led to a fundamental
change in theories about
Freeze-fracture image of a yeast cell, showing a
large vacuole, smaller vesicles (unlabeled),
plasma membrane (PM), cell wall (CW) and a
lipid droplet (LD). The vacuole, vesicles and
plasma membrane appear convex or concave
because the fracture followed the centre of the
membrane, which was curved. The lipid droplet
is cross-fractured because it is not surrounded
by a membrane
Cryogenic electron microscopy
• This technique is often called cryo-EM.
• It is mainly used for researching the structure of proteins.
• A thin layer of a pure protein solution is applied to a grid. The solution is flash-frozen,
to create smooth vitreous ice (like glass) and prevent the information of water
crystals.
• Liquid ethane just above its melting point of –182.6°C is usually used as the coolant.
• The grid with the frozen protein solution is placed in an electron microscope and
detectors record the pattern of electrons transmitted by individual protein
molecules.
• Because the protein molecules are randomly orientated in the layer of frozen
solution, many different patterns are produced. Using computational algorithms,
these patterns are combined to produce a 3D image of the protein molecules.
• Previous methods for analysing protein structure only produced images of a protein in
its most stable form. However, cryo-EM analyses proteins at the instant in time when
the water around them froze. This allows scientists to research proteins that change
from one form to another as they carry out their function.
• Since 2010, cryo-EM techniques have improved rapidly. They can now give resolutions of
0.12 nanometres. This allows the generation of images of individual atoms in a protein
or other molecule. Over 10,000 protein structures have now been shared in the Electron
Microscopy Data Bank (EMDB).
• The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank
and Richard Henderson for their work in developing cryo-EM.
▸Figure 24 Pyocin is a
protein produced by
bacteria to kill other
bacteria by bursting their
cell wall and membrane.
The cryo-EM image
(a) shows pyocin pre-
contraction and post-
contraction. The scale bar
represents 30 nm. Two
computer-generated
coloured images of pyocin
(b and c) show components
of the protein before and
after it contracts
This sequence of diagrams shows how pyocin binds to and then pierces its target
A2.2.4 Structures common to cells in all living organisms
Cells vary considerably in size, shape and structure, but they share some common
features:

1. Plasma membrane: This is the outer boundary of the cell and encloses all of its
contents. The plasma membrane controls the entry and exit of substances. It can
pump substances in, even if the concentration outside the cell is very low. It is also
very effective at preventing entry of unwanted or even toxic substances. It allows a
cell to maintain concentrations of substances that are very different from those in the
surrounding environment. The permeability of the plasma membrane relies on a
structure based on lipids. Occasionally the plasma membrane of a cell bursts. This is
known as lysis and can be caused by excess pressure or by viruses. It can even be
carried out by the cell itself (autolysis). Lysis always leads to the death of the cell; this
shows that the plasma membrane is a vital structure.
2. Cytoplasm: Water is the main component of cytoplasm and there are many
substances dissolved or suspended in this water. Enzymes in the cytoplasm catalyze
hundreds or even thousands of different chemical reactions. These reactions are the
metabolism of the cell. Metabolism provides a cell with energy and produces all the
proteins and other substances that make up the structure of a cell. Proteins are quite
easily damaged, so even when a cell is not growing the cytoplasm must continuously
break down and replace its proteins.

3. DNA: Genes, made of DNA, contain the information needed for a cell to carry out all
its functions. Many genes hold the instructions for making a protein. Some proteins are
structural so are needed for growth and repair. Others act as enzymes, without which a
cell cannot control chemical reactions and does not have a functioning metabolism.