Introduction: Microscope
From Thrilling Toy to Important Tool
Like no other invention, the microscope has unveiled the secrets of
nature. The human eye has a resolution in the order of 100 um (10-4
m), which is about the thickness of a hair. With the microscope, whole
worlds become available, filled with knowledge that can serve as
inspiration to our fantasy. The exploration of microcosmos has led to
numerous discoveries, without which we would be left with the limited
knowledge our eyes give us.
The development of the conventional microscope at the end of the 16th
century would lead to a great step forward for science, particularly in
biology and medicine. In the beginning though, the microscope was
mainly a toy in rich homes. But many important discoveries followed.
The first scientific results based on microscopy dealt with the
circulating blood system and changed our view of the human body.
Scientists have also discovered and explored life's own building block –
the cell. Different types of bacteria and the following struggle against
diseases, as well as studies of different materials and their qualities
are other valuable results.
Through ingenious inventions, the limit of what scientists could reveal
from the hidden expanded continuously during the seventeenth and
eighteenth centuries. Finally, at the end of the nineteenth century
physical limits in the form of the wavelength of light stopped the quest
to see further into the microcosmos. With the theories of quantum
physics, new possibilities appeared – the electron with its extremely
short wavelength could be used as "light-source" in microscopes with
unprecedented resolution. The first prototype of the electron
microscope was constructed around 1930. In the following decades,
smaller and smaller things could be studied. Viruses were identified
and with magnifications up to one million, even atoms finally became
visible.
Since photography has developed hand in hand with different
techniques of microscopy, the public has been able to follow close in
the footsteps of scientists. Pictures of cell division, nerves that make
up the brain and single atoms have changed our view of the human
body and nature itself. Even today our ability to lurk into nature
increases further, owing to new techniques of microscopy for studying
delicate processes within the cell or the building of materials atom by
atom with nanotechnology.
1) Time Line
14th century – The art of grinding lenses is
developed in Italy and spectacles are made to
improve eyesight.
1590 – Dutch lens grinders Hans and Zacharias
Janssen make the first microscope by placing
two lenses in a tube.
1667 – Robert Hooke studies various object
with his microscope and publishes his results in
Micrographia. Among his work were a
description of cork and its ability to float in
water.
1675 – Anton van Leeuwenhoek uses a simple
microscope with only one lens to look at blood,
insects and many other objects. He was first to
describe cells and bacteria, seen through his
very small microscopes with, for his time,
extremely good lenses.
18th century – Several technical innovations
make microscopes better and easier to handle,
which leads to microscopy becoming more and
more popular among scientists. An important
discovery is that lenses combining two types of
glass could reduce the chromatic effect, with its
disturbing halos resulting from differences in
refraction of light.
1830 – Joseph Jackson Lister reduces the
problem with spherical aberration by showing
that several weak lenses used together at
certain distances gave good magnification
without blurring the image.
1878 – Ernst Abbe formulates a mathematical
theory correlating resolution to the wavelength
of light. Abbes formula make calculations of
maximum resolution in microscopes possible.
1903 – Richard Zsigmondy develops the
ultramicroscope and is able to study objects
below the wavelength of light.
The Nobel Prize in Chemistry 1925 »
1932 – Frits Zernike invents the phase-contrast
microscope that allows the study of colorless
and transparent biological materials.
The Nobel Prize in Physics 1953 »
1938 – Ernst Ruska develops the electron
microscope. The ability to use electrons in
microscopy greatly improves the resolution and
greatly expands the borders of exploration.
The Nobel Prize in Physics 1986 »
1981 – Gerd Binnig and Heinrich Rohrer invent
the scanning tunneling microscope that gives
three-dimensional images of objects down to
the atomic level.
2) Resolving Power Line
What can you see with the different types of microscopes? The
human eye is capable of distinguishing objects down to a
fraction of a millimeter. With the use of light and electron
microscopes it is possible to see down to an angstrom and
study everything from different cells and bacteria to single
molecules or even atoms.
* Light microscope includes phase contrast and fluorescence microscopes. Electron microscope includes
transmisson electron microscope.
3) Development Milestones
3.1) The Phase Contrast Microscope
The phase contrast microscope is widely used for examining such
specimens as biological tissues. It is a type of light microscopy that
enhances contrasts of transparent and colorless objects by influencing
the optical path of light. The phase contrast microscope is able to show
components in a cell or bacteria, which would be very difficult to see in
an ordinary light microscope.
Altering the Light Waves
The phase contrast microscope uses the fact that the light passing
trough a transparent part of the specimen travels slower and, due to
this is shifted compared to the uninfluenced light. This difference in
phase is not visible to the human eye. However, the change in phase
can be increased to half a wavelength by a transparent phase-plate in
the microscope and thereby causing a difference in brightness. This
makes the transparent object shine out in contrast to its surroundings.
The Invisible Can Be Seen
The phase contrast microscope is a vital instrument in biological and
medical research. When dealing with transparent and colorless
components in a cell, dyeing is an alternative but at the same time
stops all processes in it. The phase contrast microscope has made it
possible to study living cells, and cell division is an example of a
process that has been examined in detail with it. The phase contrast
microscope was awarded with the Nobel Prize in Physics, 1953.
The phase-plate increases the phase
difference to half a wavelength. Destructive
interference between the two sorts of light
when the image is projected results in the
specimen appearing as a dark object.
3.2) The Fluorescence Microscope
In fluorescence microscopy, the sample you want to study is itself the
light source. The technique is used to study specimens, which can be
made to fluoresce. The fluorescence microscope is based on the
phenomenon that certain material emits energy detectable as visible
light when irradiated with the light of a specific wavelength. The
sample can either be fluorescing in its natural form like chlorophyll and
some minerals, or treated with fluorescing chemicals.
The Sample Gets Excited
The basic task of the fluorescence microscope
is to let excitation light radiate the specimen
and then sort out the much weaker emitted
light to make up the image. First, the
microscope has a filter that only lets through
radiation with the desired wavelength that
matches your fluorescing material. The
radiation collides with the atoms in your
specimen and electrons are excited to a
higher energy level. When they relax to a
lower level, they emit light.
To become visible, the emitted light is
separated from the much brighter excitation
light in a second filter. Here, the fact that the
emitted light is of lower energy and has a longer wavelength is used.
The fluorescing areas can be observed in the microscope and shine out
against a dark background with high contrast.
Specific Details are Marked
Fluorescence microscopy is a rapid expanding technique, both in the
medical and biological sciences. The technique has made it possible to
identify cells and cellular components with a high degree of specificity.
For example, certain antibodies and disease conditions or impurities in
inorganic material can be studied with the fluorescence microscopy.
Principle of Fluorescence
1. Energy is absorbed by the atom which becomes excited.
2. The electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, emitting a
photon (or a packet of light) - the atom is fluorescing.
3.3) The Transmission Electron Microscope
The transmission electron microscope (TEM) operates on the same
basic principles as the light microscope but uses electrons instead of
light. What you can see with a light microscope is limited by the
wavelength of light. TEMs use electrons as "light source" and their
much lower wavelength makes it possible to get a resolution a
thousand times better than with a light microscope.
You can see objects to the order of a few angstrom (10-10 m). For
example, you can study small details in the cell or different materials
down to near atomic levels. The possibility for high magnifications has
made the TEM a valuable tool in both
medical, biological and materials research.
Magnetic Lenses Guide the Electrons
A "light source" at the top of the microscope
emits the electrons that travel through
vacuum in the column of the microscope.
Instead of glass lenses focusing the light in
the light microscope, the TEM uses
electromagnetic lenses to focus the
electrons into a very thin beam. The
electron beam then travels through the
specimen you want to study. Depending on
the density of the material present, some of the electrons are
scattered and disappear from the beam. At the bottom of the
microscope the unscattered electrons hit a fluorescent screen, which
gives rise to a "shadow image" of the specimen with its different parts
displayed in varied darkness according to their density. The image can
be studied directly by the operator or photographed with a camera.
3.4) The Scanning Tunneling Microscope
The scanning tunneling microscope (STM) is a type of electron
microscope that shows three-dimensional images of a sample. In the
STM, the structure of a surface is studied using a stylus that scans the
surface at a fixed distance from it.
Currents Control the Surface
An extremely fine conducting probe is held close to the sample.
Electrons tunnel between the surface and the stylus, producing an
electrical signal. The stylus is extremely
sharp, the tip being formed by one single
atom. It slowly scans across the surface
at a distance of only an atom's diameter.
The stylus is raised and lowered in order
to keep the signal constant and maintain
the distance. This enables it to follow
even the smallest details of the surface it
is scanning. Recording the vertical
movement of the stylus makes it possible
to study the structure of the surface atom
by atom. A profile of the surface is
created, and from that a computergenerated contour map of the surface is produced.
Important in Many Sciences
The study of surfaces is an important part of physics, with particular
applications in semiconductor physics and microelectronics. In
chemistry, surface reactions also play an important part, for example
in catalysis. The STM works best with conducting materials, but it is
also possible to fix organic molecules on a surface and study their
structures. For example, this technique has been used in the study of
DNA molecules.