Cell biologists often need to examine the structure of cells
and their components. The microscope is an indispensable tool for this purpose
because most cellular structures are too small to be seen by an unaided eye.

In fact, the beginnings of cell biology can be traced to the
invention of the light microscope, which made it possible for scientists to see
enlarged images of cells for the first time. The first generally useful light
microscope was developed in 1590 by Z Janssen and his nephew, H Janssen. Many
important microscopic observations were reported during the next century,
notably those of Robert Hooke, who observed the first cells, and Antonie van
Leeuwenhoek, whose improved microscopes provided our first glimpses of internal
cell structure. Since then, the light microscope has undergone numerous
improvements and modifications, right up to the present time.

Just as the invention of the light microscope heralded a
wave of scientific achievement by allowing us to see cells for the first time,
the development of the electron microscope in the 1930s revolutionised our
ability to explore cell structure and function. Because it is at least a
hundred times better at visualising objects than the light microscope, the
electron microscope ushered in a new era in cell biology, opening our eyes to
an exquisite sub-cellular architecture never before seen and changing the way
we think about cells forever.

But despite its inferior resolving power, the light
microscope has not fallen into disuse. To the contrary, light microscopy has
experienced a renaissance in recent years as the development of specialised new
techniques has allowed researchers to explore aspects of cell structure and
behaviour that cannot be readily studied by electron microscopy. These advances
have involved the merging of technologies from physics, engineering, chemistry,
and molecular biology, and they have greatly expanded our ability to study
cells using the light microscope.

Although light and electron microscopes differ in many ways,
they make use of similar optical principles to form images. Regardless of the
kind of microscope being used, three elements are always needed to form an
image — a source of illumination, a specimen to be examined, and a system of
lenses that focuses the illumination on the specimen and forms the image. In a
light microscope, the source of illumination is visible light, and the lens
system consists of a series of glass lenses. The image can either be viewed
directly through an eyepiece or focused on a detector, such as photographic
film or an electronic camera. In an electron microscope, the illumination source
is a beam of electrons emitted by a heated tungsten filament, and the lens
system consists of a series of electromagnets. The electron beam is focused
either on a fluorescent screen or on photographic film or is digitally imaged
using a detector.

Despite these differences in illumination source and
instrument design, both types of microscopes depend on the same principles of
optics and form images in a similar manner. When a specimen is placed in the
path of a light or electron beam, physical characteristics of the beam are
changed in a way that creates an image, which can be interpreted by the human
eye or recorded on a photographic detector. To understand this interaction
between the illumination source and the specimen, we need to understand the concept
of wavelength.

If two people hold onto opposite ends of a slack rope and
wave the rope with a rhythmic up-and-down motion they will generate a long,
regular pattern of movement in the rope called a wave form. The distance from
the crest of one wave to the crest of the next is called the wavelength. If
someone standing to one side of the rope tosses a large object such as a beach
ball toward the rope, the ball may interfere with, or perturb, the wave form of
the rope’s motion. However, if a small object such as a softball is tossed
toward the rope, the movement of the rope will probably not be affected at all.
If the holders move the rope more rapidly, the motion of the rope will still
have a wave form but the wavelength will be shorter. In this case, a softball
is tossed towards the rope and it is quite likely to perturb the rope’s
movement.

This simple analogy illustrates an important principle — the
ability of an object to perturb a wave motion depends crucially on the size of
the object in relation to the wavelength of the motion. This principle is of
great importance in microscopy, because it means that the wavelength of the
illumination source sets a limit on how small an object can be seen.

To understand this relationship, recognise that the moving
rope is analogous to the beam of light (photons) or electrons that is used as
an illumination source in a light or electron microscope, respectively — in
other words, both light and electrons behave as waves. When a beam of light or
electrons encounters a specimen, the specimen alters the physical
characteristics of the illuminating beam, just as the beach ball or softball
alters the motion of the rope. And because an object can be detected only by
its effect on the wave, the wavelength must be comparable in size to the object
that is to be detected.

By this relationship between wavelength and object size, we
can readily appreciate why very small objects can be seen only by electron
microscopy — the wavelengths of electrons are much shorter than those of
photons. Thus objects such as viruses and ribosomes are too small to perturb a
wave of photons but they can readily interact with a wave of electrons.

By Tapan Kumar Maitra

(The writer is associate professor, Head, Department of
Botany, Ananda Mohan College, Kolkata, and also fellow, Botanical Society of
Bengal, and can be contacted at [email protected])