What is an optical light microscope? How is it
constructed?
How does a microscope work? How can we
measure the size of a cell?
What is the advantage of studying
structures with a microscope?
What do we mean by the
resolution/resolving power of a microscope?
What is the formula for magnification? How do you do magnification
calculations?
What is the difference between a light
microscope and an electron microscope? Which is the most powerful?
What do we
use a microscope for in biology? and why is it such a useful investigative
tool?
A microscope is an important instrument for studying
cells e.g. the type of cell and the structure of cells.
Microscopes enable to see structures that we cannot
see with the unaided naked eye.
Plant and animal cells can be studied in greater detail
with a light microscope by magnifying the image.
Microscopes use a glass lens system to
magnify images - with a bigger image you see more detail.
You can increase the resolution of an
image by using more powerful and better quality lenses. Resolution means how
good a microscope is at distinguishing between two points that are close
together on an image. The higher the resolution the more clear is the image,
especially when looking for fine details e.g. in a cell.
Microscopes enable you to see objects (like
microorganisms) which you cannot see with the naked eye.
Microscopes using the visible part of the
electromagnetic spectrum (visible light) were invented in the 16th
century and the optical lens systems of light microscopes have been improved through the
following centuries even until today.
With these microscopes, by passing
light through a specimen up into a lens system, you can see
individual cells and smaller details such as nuclei and mitochondria in all
cells, and chloroplasts in plant cells.
Optical
light microscopes
Light microscopes using visible light and lenses to form
a magnified image of the object under investigation e.g. cells of plant or
animal tissue. With a light microscope you can see individual cells
and large subcellular structures like the nucleus, but not internal cell
structures such as ribosomes or plasmids. The best light microscopes can give a
magnification of 2000 times of a specimen's length.
The resulting image on a photographic plate, book or a
computer screen is called a light micrograph.
Very high magnification is not possible with optical
light microscopes. The limitation is due to the light gathering ability of
the microscope and the short working distance of the lenses. This limits the
total magnification of light microscope to about x 1500.
BUT, even with a high magnification, details may still
not be that clear. The microscope must have a high resolving power -
this is the resolution of the microscope. The resolving power is the
smallest distance between two points that can be clearly distinguished. For
optical light microscopes the best resolution is about 0.2
µm (200 nm).
However, unlike
electron microscopes (described below), light telescopes can be used t
observe living cells.
Electron microscopes - a means
of looking at cells in more detail
Changes in microscope technology have enabled us to see
cells with more clarity and detail than in the past, including simple
magnification calculations.
Over time the design and usefulness of microscopes has
improved, particularly using new technology in the 20th century and into the
21st century.
In the 20th century, with advances in atomic
physics, the electron microscope (EM) was invented in the 1930s which uses beams of
electrons instead of visible light photons. Electron microscopes use beams
of electrons instead of beams of visible light photons. They have a much
greater magnifying power and resolving power than optical light microscopes
- larger sharper images.
So, using an electron microscope, using electrons
instead of light photons, you can form images of very small subcellular
structures such as ribosomes, plasmids and the internal structure of
mitochondria (site of respiration) and chloroplasts (site of photosynthesis), because they have a much
higher resolving
power, but they are much more expensive!
Electron microscope images have a higher
resolution than light microscopes - a higher resolving power increases the distinction between
points on an image, i.e. you get a much sharper image of the fine detail
of cell structure.
Electron microscopes can produce much
greater magnified images (compared to light microscopes) of up to ten million (109) times the real length of the
specimen under investigation.
So electron microscopes are superior to optical
light microscopes in terms of both magnification and resolution.
The resulting image on a photographic plate or
a computer screen is called an electron micrograph.
Electrons do not form a colour spectrum,
so all images are in black and white.
Electron microscopes can't be used to look at
living cells - the electron beams would damage the function of
living cells.
The transmission electron microscope (TEM)
is particularly good at looking at very thin layers of biological
materials e.g. a layer of cells and investigate in great detail the
sub-cellular components of a cell e.g. plasmids and mitochondria and
also viruses.
A transmission electron microscope is a large instrument, not very portable and
very
expensive. To prepare specimens for examination is a complicated
process, and, unlike light microscopes, cannot be used to examine living
tissue.
In a TEM, as the electron beam passes through the
sample, some electrons are scattered and those that pass through
are focussed by electromagnetic coils (instead of lenses) to
produce an image on an electronic screen.
A TEM can examine very thin sections of
cells up to a magnification of 106 (million x) and
with a resolution of less than 1 nm (10-9 m). This is
200 x greater resolution than the best light microscopes.
The scanning electron microscope (SEM)
A scanning electron microscope works by
bouncing beams of electrons off the surface of a specimen. The
specimen must be first coated in an ultra-thin layer of a heavy
metal like gold. The scattered electrons are again focussed by
electromagnetic coils to produce an image on an electronic
screen.
A SEM is used to produce images
(micrographs) of the surface shape of structures of e.g. of
individual cells or small organisms.
This has enabled the magnification produced
by a microscope to be considerably increased to the point where you can see
even smaller structures such as the internal detailed structure of mitochondria,
chloroplasts and plasmids (hoops of DNA) so as to give a better understand
of their structure and how their role in cell behaviour - in other words
a powerful tool for better understanding how a cell works and the function
of sub-cellular structures.
e.g. in animal cells
or plant cells
where you
can observe the fine detailed complex internal structure of
important subcellular structures such as mitochondria (where
aerobic respiration takes place), chloroplasts (where
photosynthesis takes place), ribosomes (where protein synthesis
takes place) as well as the detailed structure of specialised
(differentiated) cells e.g. red blood cells (oxygen carriers) or
white blood cells (immune defence system). In other words,
electron microscopes allow more detailed studies of some pretty
important structures and their functions!
Mitochondria and ribosomes can
only be adequately viewed using an electron microscope and the 3D
structure of biological specimens requires the use of an scanning
electron microscope.
Extra note on microscopy
methods
There is a technique called 'super-resolved
fluorescence microscopy' which allows a much higher resolution
than normal light microscopy. Since this is based on light, it
means you can study living cells, which you can't do with
electron microscopes - electron beams kill cells!
e.g. the size and shape of cells
and subcellular
structures are important, they are also variable, and such differences can be important e.g.
The complexity of
mitochondria can indicate how active a cell is.
You can measure the ratio of
the area-volume of the cytoplasm to that of the cell's nucleus.
A high ratio of cytoplasmic area-volume : nucleus area-volume
can show a cell is about to divide. A low ratio can indicate a
cancer cell.
You need to be able to use the
following microscope formula:
For any microscope: magnification = length
of image / real length of object, and for light microscopes:
total magnifying power = magnification
of object lens x magnification of objective lens
and with a variety of units e.g. micro,
nano etc. as well as
expressing small numbers in standard form!
