Introduction

Microscopes are instruments that produce an enlarged image of an object. Robert Hooke and
Antonie van Leeuwenhoek used glass lenses to magnify small cells and cause them to appear
larger than the 100-micrometer limit imposed by the human eye. The glass lens adds additional
focusing power. Because the glass lens makes the object appear closer, the image on the back
of the eye is bigger than it would be without the lens. Van Leeuwenhoek's home-made
microscopes were very small simple instruments, with a single, yet strong lens. They were
awkward in use, but enabled van Leeuwenhoek to see detailed images. It took about 150 years
of optical development before the compound microscope was able to provide the same quality
image as van Leeuwenhoek's simple microscopes, due to timely difficulties of configuring
multiple lenses. Still, despite widespread claims, van Leeuwenhoek is not the inventor of the
microscope. It is difficult to say who invented the compound microscope. Dutch spectacle-
makers Hans Janssen and his son Zacharias Janssen are often said to have invented the first
compound microscope in 1590, but this was a declaration made by Zacharias Janssen himself
during the mid 1600s. The date is unlikely, as it has been shown that Zacharias Janssen
actually was born around 1590. Another favorite for the title of 'inventor of the microscope' was
Galileo Galilei. He developed an occhiolino or compound microscope with a convex and a
concave lens in 1609. Galileo's microscope was celebrated in the Accademia dei Lincei in 1624
and was the first such device to be given the name "microscope" a year latter by fellow Lincean
Giovanni Faber. Christiaan Huygens, another Dutchman, developed a simple 2-lens ocular
system in the late 1600s that was achromatically corrected, and therefore a huge step forward
in microscope development. The Huygens ocular is still being produced to this day, but suffers
from a small field size, and other minor problems.

A light source, which may be external to the microscope or built into its base, illuminates the
specimen. Some common microscopes that can be used in the study of cells are: a) light
(optical) microscopes, b) phase contrast microscopes, c) transmission electron microscopes, d)
scanning electron microscopes. Under the division of light microscopes, come the fluorescence
microscope and a new type of microscope which can be used in a wide range of cytological
studies including those of cancerous cell, and the microscope namely confocal microscope (laser
scanning confocal microscope) which will be discussed later. Under the division of electron
microscopes, comes the scanning electron microscope and the transmission electron
microscope, but we are only about to discuss about the scanning electron microscope. Light
microscopes, even the compound ones, are not powerful enough to resolve many of the
structures within the cells. For example, a membrane is only 5 nanometers thick. Why not just
add another magnifying stage to the microscope and so increase its resolving power? Because
when two objects are closer than a few hundred nanometers, the light beams reflecting from
two images start to overlap. The only way two light beams can get closer together and still be
resolved is if their wavelengths are shorter. One way to avoid overlap is by using a beam of
electrons rather than a beam of light. Electrons have a much shorter wavelength, and a
microscope employing electron beams has 1000 times the resolving power of a light
microscope.

Resolution is the ability to ability to distinguish between two separate points. The limit of
resolution of a microscope is the minimum distance between two points at which they are still
distinguished as two separate points. If the two points cannot be resolved, they will be seen as
one point. A microscope with a high resolution power will enable two small objects close
together to be seen as two separate objects. A microscope with a low resolution power will
cause the two small objects to be seen as one object. A general rule is that if an object is
smaller than half the wavelength of the radiation used to view the object, it cannot be seen
separately from nearby objects.
Principals of how the microscope works

Light microscope

All optical microscopes share the same basic components. A light source, which may be external
to the microscope or internally built into its base, illuminates the specimen. The sub-stage
condenser lens gathers the diffuse rays from the light source and illuminates the specimen with
a small cone of bright light that allows very small parts of the specimen to be seen after
magnification. The eyepiece is a cylinder containing two or more lenses to bring the image to
focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are
interchangeable and many different eyepieces can be inserted with different degrees of
magnification. Typical magnification values for eyepieces include 5x, 10x and 2x. In some high
performance microscopes, the optical configuration of the objective lens and eyepiece are
matched to give the best possible optical performance. This occurs most commonly with
apochromatic objectives. The objective lens is a cylinder containing one or more lenses,
typically made of glass, to collect light from the sample. At the lower end of the microscope
tube one or more objective lenses are screwed into a circular nose piece which may be rotated
to select the required objective lens. Typical magnification values of objective lenses are 4x, 5x,
10x, 20x, 40x, 50x and 100x. Some high performance objective lenses may require matched
eyepieces to deliver the best optical performance. The light rays focused on the specimen by
the condenser lens are then collected by the microscopes objective lens. From this point, we
need to consider two sets of light rays that enter the objective lens: those that the specimen
has altered and those that it hasnt. The latter group consists of light from the condenser that
passes directly into the objective lens, forming the background light of the visual field. The
former group of the light rays emanates from the many parts of the specimen. These light rays
are brought to focus by the objective lens to form a real, enlarged image of the object within
the column of the microscope. The image formed by the objective lens is used as an object by a
second lens system, the ocular lens, to form an enlarged and virtual image. A third lens system
located in the front part of the eye uses the virtual image produced by the ocular lens as an
object to produce a real image on the retina.

The optical components of a modern microscope are very complex and for a microscope to
work well, the whole optical path has to be very accurately set up and controlled. Despite this,
the basic optical principles of a microscope are quite simple. The objective lens is, at its
simplest, a very high powered magnifying glass, for example, a lens with a very short focal
length. This is brought very close to the specimen being examined so that the light from the
specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged
image of the subject. This image is inverted and can be seen by removing the eyepiece and
placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit
specimen, a highly enlarged image can be seen. It is this real image that is viewed by the
eyepiece lens that provides further enlargement. In most microscopes, the eyepiece is a
compound lens, with one component lens near the front and one near the back of the eyepiece
tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus
between the two lenses of the eyepiece, the first lens bringing the real image to a focus and
the second lens enabling the eye to focus on the virtual image. In all microscopes the image is
viewed with the eyes focused at infinity (mind that the position of the eye in the above figure is
determined by the eye's focus). Headaches and tired eyes after using a microscope are usually
signs that the eye is being forced to focus at a close distance rather than at infinity.

On a typical compound optical microscope, there are three objective lenses: a scanning lens
(4), low power lens (10) and high power lens (ranging from 20 to 100). Some microscopes
have a fourth objective lens, called an oil immersion lens. To use this lens, a drop of immersion
oil is placed on top of the cover slip, and the lens is very carefully lowered until the front
objective element is immersed in the oil film. Such immersion lenses are designed so that the
refractive indexes of the oil and of the cover slip are closely matched so that the light is
transmitted from the specimen to the outer face of the objective lens with minimal refraction.
An oil immersion lens usually has a magnification of 50 to 100. The actual power or
magnification of an optical microscope is the product of the powers of the ocular (eyepiece),
usually about 10, and the objective lens being used. Compound optical microscopes can
produce a magnified image of a specimen up to 1000 and, at high magnifications, are used to
study thin specimens as they have a very limited depth of field. When the focusing knob of the
light microscope is turned, the relative distance between the specimen and the objective lens
changes, allowing the final image to become focused precisely on the plane of the retina. The
total magnification attained by the microscope is the product of the magnifications produced by
the objective lens and the ocular lens.
The stage is a platform below the objective which supports the specimen being viewed. In the
center of the stage is a hole through which light passes to illuminate the specimen. The stage
usually has arms to hold slides (rectangular glass plates with typical dimensions of 25 mm by 75
mm, on which the specimen is mounted). The illumination source is below the stage, light is
provided and controlled in a variety of ways. At its simplest, daylight is directed via a mirror.
Most microscopes, however, have their own controllable light source that is focused through an
optical device called a condenser, with diaphragms and filters available to manage the quality
and intensity of the light. The whole of the optical assembly is attached to a rigid arm which in
turn is attached to a robust U shaped foot to provide the necessary rigidity. The arm is usually
able to pivot on its joint with the foot to allow the viewing angle to be adjusted. Mounted on
the arm are controls for focusing, typically a large knurled wheel to adjust coarse focus,
together with a smaller knurled wheel to control fine focus.

We have already known that under the division of light microscope, comes a few other types of
microscope such as the bright field microscope; fluorescence microscope, the laser scanning
confocal microscope, and phase-contrast microscope. The fluorescence microscope and
confocal microscope will be explained in detail later, so we will see a little information briefly
about the bright field and phase-contrast microscopy. Bright field microscopy is used to view
stained or naturally pigmented organelles in the cell, and it involves the use of color detection.
The stains or pigments absorb and alter the light that passes through the sample and is seen as
a color that is distinctly different from the background or direct light not passing through the
sample. The easiest way to enhance the contrast of the relatively transparent cells is to stain
them. Further image enhancement is accomplished by using a specific monochromatic light
source. One way to achieve this is by using a filter. A common filter is the one that allows only
blue light to pass through. Blue light, about 450 nm, is usually the shortest wavelength used in
light microscopy. In order to see a specific structure of the cell, it must be large enough to be
able to perturb the wave motion of the light rays that strike it. Therefore, mitochondria, about 3
m, are one of the smallest organelles to be seen through microscopy. Organelles smaller than
mitochondria cannot be resolved because they are small compared to the wavelengths of visible
light and are not able to perturb the wave motion of light travelling o our eyes.

The phase-contrast microscope makes highly transparent objects more visible. We can
distinguish different parts of an object because they affect light differently from one another.
One basis for such differences is refractive index. Cell organelles are made up of different
proportions of various molecules: DNA, RNA, protein, lipid, carbohydrate, salts, and water.
Regions of different composition are likely to have different refractive indexes. Normally such
differences cannot be detected by our eyes. However, the phase-contrast microscope converts
differences in refractive index into differences in intensity (relative brightness and darkness),
which are visible to the eye. Phase-contrast microscopes accomplish this result by separating
the direct light that enters the objective lens from the diffracted light emanating from the
specimen and causing light rays from these two sources to interfere with one another. The
relative brightness or darkness of each part of the image reflects the way in which the light
from that part of the specimen interferes with the direct light. Phase-contrast microscopes are
more useful for examining intracellular components of living cells at relatively high resolution.
For example, the dynamic motility of mitochondria, mitotic chromosomes, and vacuoles can be
followed and filmed with this optics. Simply watching the way tiny particles and vacuoles of cells
are bumped around in a random manner in a living cell conveys an excitement about life that is
unattainable by observing stained, dead cells. The phase-contrast microscope has optical
handicaps that results in loss of resolution, and the image suffers from interfering halos and
shading produced were sharp changes in refractive index occur. The phase-contrast microscope
is a type of interference microscope. Other types of interference microscopes minimize these
optical artifacts by achieving a complete separation of direct and diffracted beams using
complex light paths and prisms. Another type of interference system, termed differential
interference contrast (DIC), or sometimes Nomarski interference after its developer, delivers an
image that has an apparent three dimensional quality. Contrast in DIC microscopy depends on
the rate of change of refractive index across a specimen. As a consequence, the edges of
structures, where the refractive index varies markedly over a relatively small distance, are seen
with especially good contrast.
Figure 1: Paramecium sp. viewed under a light microscope.

Fluorescence microscope

Over the past few decades, the light microscope has been transformed from an instrument
designed primarily to examine sections of fixed tissues to one capable of observing the dynamic
events occurring at the molecular level in the living cells. These advances in the live cell
imaging have been made possible to a large extent by innovations in fluorescence microscopy.
In most cases, a component of interest in the specimen is specifically labeled with a fluorescent
molecule called a fluorophore such as green fluorescent protein (GFP), fluorescein or DyLight
488. The specimen is illuminated with light of a specific wavelength or wavelengths which is
absorbed by the fluorophores, causing them to emit longer wavelengths of light of a different
color than the absorbed light. The illumination light is separated from the much weaker emitted
fluorescence through the use of an emission filter. Typical components of a fluorescence
microscope are the light source (xenon arc lamp or mercury-vapor lamp), the excitation filter,
the dichroic mirror or dichromatic beamsplitter, and the emission filter. The filters and the
dichroic are chosen to match the spectral excitation and emission characteristics of the
fluorophore used to label the specimen. In this manner, a single fluorophore is imaged at a
time. Multi-color images of several fluorophores must be composed by combining several
single-color images.

The fluorescence microscopy allows viewers to observe the location of certain compounds called
fluorochromes or fluorophores. Fluorochromes absorb invinsible, ultraviolet radiation and
release a portion of the energy in the longer, visible wavelengths, a phenomenon called
fluorescence. The light source in the fluorescence microscope produces a beam of ultraviolet
light that travels through a filter, which blocks all wavelengths except that which is capable of
exciting the fluorochrome. The beam of monochromatic light is focused on the specimen
containing the fluorochrome, which becomes excited and emits the light of a visible wavelength
that is focused by the objective lens into an image that can be seen by the viewer. Because the
light source produces only ultrsviolet (black) light, objects stained with a fluorochrome appear
brightly colored against a black background, providing very high contrast. There are many
different ways that fluorescence compounds can be used in cell and molecular biology. In one
of its most common applications, a fluorochrome (such as rhodamine or fluorescein) is
covalently linked (conjugated) to an antibody to produce a fluorescent antibody that can be
used to determine the location of a specific protein within the cell. This technique is called
immunofluorescence. Fluorochromes can also be used to locate DNA or RNA molecules that
contain specific nucleotide sequences. In other examples, fluorochromes have been used to
study the sizes of molecules that can pass between cells, as indicators of transmembrane
potentials, or as probes to determine the free Ca 2+ concentration in the cytosol. Fluorescently
labeled proteins can also be used to study dynamic processes as they occur in a living cell. For
example, a specific fluorochrome can be linked to a cellular protein, such as actin or tubulin,
and the fluorescently labeled protein injected into a living cell. Most fluorescence microscopes in
use are epifluorescence microscopes, for example, excitation and observation of the
fluorescence are from above the specimen). These microscopes have become an important part
in the field of biology, opening the doors for more advanced microscope designs, such as the
confocal microscope and the total internal reflection fluorescence microscope (TIRF). The
Vertico SMI combining localisation microscopy with spatially modulated illumination uses
standard fluorescence dyes and reaches an optical resolution below 10 nanometers.
Fluorophores lose their ability to fluoresce as they are illuminated in a process called
photobleaching. Special care must be taken to prevent photobleaching through the use of more
robust fluorophores, by minimizing illumination, or by introducing a scavenger system to reduce
the rate of photobleaching.

Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life

sciences. The excitatory light is passed from above or, for inverted microscopes, from below,
through the objective and then onto the specimen instead of passing it first through the
specimen. In the latter case the transmitted excitatory light reaches the objective together with
light emitted from the specimen. The fluorescence in the specimen gives rise to emitted light
which is focused to the detector by the same objective that is used for the excitation. A filter
between the objective and the detector filters out the excitation light from fluorescent light.
Since most of the excitatory light is transmitted through the specimen, only reflected excitatory
light reaches the objective together with the emitted light and this method therefore gives an
improved signal to noise ratio. A common use in biology is to apply fluorescent or fluorochrome
stains to the specimen in order to image a protein or other molecule of interest.

In recent years, a noninvasive approach has been widely employed that utilizes a fluorescent
protein called Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria from where
a recombinant DNA is constructed in which the coding region of the GFP is joined with the
coding region of the protein under study. This recombinant DNA is used to transfect cells, which
then synthesize a chimeric protein containing fluorescent GFP fused to the protein under study.
In all of these strategies, the labeled proteins participate in the normal activities of the cell, and
their location can be followed microscopically to reveal the dynamic activities in which the
protein participates. Studies can often be made more informative by the simultaneous use of
GFP variants that exhibit different spectral properties. Variants of GFP that fluoresce in shades
of blue (BFP), yellow (YFP), and cyan (CFP) have been generated through directed mutagenesis
of the GFP gene. In addition, a distantly related red fluorescent tetrameric protein (DsRed) has
been isolated from a sea anemone. Monomeric variants of DsRed, which fluoresce in a variety
of distinguishable colors, have also been generated by mutagenesis experiments. The type of
information that can be obtained using GFP variants is illustrated by the study, in which
researchers generated strains of mice whose neurons contained differently colored fluorescent
proteins. When a muscle of one of these mice was exposed surgically, the investigators could
observe the dynamic interactions between the variously colored neurons and the neuromuscular
junctions being innervated. They watched, for example, as branches from a CFP-colored neuron
for synaptic contact with the muscle tissue. In each case they found that, when two neurons
compete for innervations of different muscle fibers, all of the winning branches belong to one
of the neurons, while all the losing branches belong to the other neuron. GFP variants have
also been useful in a technique, called fluorescence resonance energy transfer (FRET), which
can measure distances between fluorochromes in the nanoscale range. FRET is typically
employed to measure changes in distance between two parts of a protein or between two
separate proteins within a large structure. FRET can be used to study such changes as they
occur in vitro or within a living cell. FRET is based on the fact that excitation energy can be
transferred from one fluorescent group to another fluorescent group as long as the two groups
are in very close proximity (1-10 nm). This transfer of energy reduces the fluorescence intensity
of the donor and increases the fluorescence intensity of the acceptor. The efficiency of transfer
between two fluorescent groups that are bound to sites on a protein decreases sharply as the
distance between the two groups increases. As a result, determination of changes in
fluorescence of the donor and acceptor groups that occur during a process provides a measure
of changes in the distance between them at various stages in the process. In the absence of
bound cGMP, the two fluorochromes are too far apart for energy transfer to occur. Binding of
cGMP induces a conformational change in the protein that brings the two fluorochromes into
close enough proximity fro FRET to occur. FRET can also be used to follow many other
processes including protein folding or the association and dissociation of components within a
membrane. The separation of the cytoplasmic tails of integrin subunits following activation by
talin is an example of an event that has been studied by FRET.
Figure 2: Endothelial cells under the fluorescence microscope. Nuclei are stained blue with
DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are
labelled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial (BPAE) cells.

Confocal microscope

The development of a new type of light microscope with imaging techniques. Confocal
microscopy is an optical imaging technique used to increase micrograph contrast and to
reconstruct three-dimensional images by using a spatial pinhole to eliminate out of focus light in
specimens that are thicker than the focal plane. The principle of confocal imaging was patented
by Marvin Minsky in 1957 and aims to overcome some limitations of traditional wide-field
fluorescence microscopes. In a conventional fluorescence microscope, the entire specimen is
flooded in light from a light source. All parts of the specimen in the optical path are excited and
the resulting fluorescence is detected by the microscope photodetector or camera as
background signal. This technique has gained popularity in the scientific and industrial
communities and typical applications are in life sciences and semiconductor inspection. When a
whole cell or a section of an organ is examined under a standard light microscope, the observer
views the specimen at different depths by changing the position of the objective lens by
rotating the focusing knob. But the fact that the specimen contains different levels of focus
reduces the ability to form a crisp image because those parts of the specimen above and below
the plane of focus interfere with the light rays from that part that is in the plane of focus. In
this type of microscope, the specimen is illuminated by a finely focused laser beam that rapidly
scans across the specimen at a single depth, thus illuminating only a thin plane within the
specimen. Confocal microscopes are typically used with fluorescence optics. As described
earlier, short wavelength incident light is absorbed by the fluorochromes in a specimen and
reemitted at longer wavelength. The advantage of fluorescence for microscopy is that you can
often attach fluorescent dye molecules to specific parts of your sample, so that only those parts
are the ones seen in the microscope. You can also use more than one type of dye. By changing
the excitation light, you can cause one type of dye to fluoresce, and then another, to distinguish
two different parts of your sample. Light emitted from the specimen is brought to focus at a site
within the microscope that contains a pinhole aperture. Thus, the aperture and the illuminated
plane in the specimen are confocal. Normally, the sample is completely illuminated by the
excitation light, so the entire sample is fluorescing at the same time. Of course, the highest
intensity of the excitation light is at the focal point of the lens, but nonetheless, the other parts
of the sample do get some of this light and they do fluoresce. This contributes to a background
haze in the resulting image. Adding a pinhole/screen combination solves this problem. Because
the focal point of the objective lens of the microscope forms an image where the pinhole is,
these two points are known as "conjugate points" or alternatively, the sample plane and the
pinhole/screen are conjugate planes. The pinhole is conjugate to the focal point of the lens,
thus it is a confocal pinhole. The image of a point source of light isn't actually a point; due to
diffraction, it's actually an Airy disk. The size of the confocal pinhole needs to be matched to the
size of the Airy disk. Any smaller, and you are throwing out useful light. Any larger, and you see
more out of focus light.

Light rays emitted from the illuminated plane of the specimen can pass through the aperture,
whereas any light rays that might emanate from above or below this plane are prevented from
participating in image formation. As a result, out of focus points in the specimen become
invinsible. In contrast, a confocal microscope uses point illumination and a pinhole in an
optically conjugate plane in front of the detector to eliminate out of focus informations, so, the
name "confocal" comes from this configuration. As only light produced by fluorescence very
close to the focal plane can be detected the image resolution, particularly in the sample depth
direction, is much better than that of wide-field microscopes. However as much of the light
from sample fluorescence is blocked at the pinhole this increased resolution is at at the cost of
decreased signal intensity so long exposures are often required. As only one point in the sample
is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster, as an
example, a rectangular pattern of parallel scanning lines in the specimen. The thickness of the
focal plane is defined mostly by the inverse of the square of the numerical aperture of the
objective lens, and also by the optical properties of the specimen and the ambient index of
refraction. The thin optical sectioning possible makes these types of microscopes particularly
good at 3D imaging of samples.

A laser is used to provide the excitation light (in order to get very high intensities). The laser
light reflects off a dichroic mirror. From there, the laser hits two mirrors which are mounted on
motors; these mirrors scan the laser across the sample. Dye in the sample fluoresces, and the
emitted light gets descanned by the same mirrors that are used to scan the excitation light from
the laser. The emitted light passes through the dichroic and is focused onto the pinhole. The
light that passes through the pinhole is measured by a detector, for example, a photomultiplier
tube. So, there never is a complete image of the sample at any given instant, only one point of
the sample is observed. The detector is attached to a computer which builds up the image, one
pixel at a time. The limitation is in the scanning mirrors.

By having a confocal pinhole, the microscope is really efficient at rejecting out of focus
fluorescent light. The practical effect of this is that your image comes from a thin section of
your sample. By scanning many thin sections through your sample, you can build up a very
clean three-dimensional image of the sample. Also, a similar effect happens with points of light
in the focal plane, but not at the focal point emitted light from these areas is blocked by the
pinhole screen. So a confocal microscope has slightly better resolution horizontally, as well as
vertically.

Three types of confocal microscopes are commercially available: 1) Confocal laser scanning
microscopes, 2) Spinning-disk (Nipkow disk) confocal microscopes, 3) Programmable Array
Microscopes (PAM). Each of these classes of confocal microscope has particular advantages and
disadvantages, most systems are either optimised for resolution or high sensitivity for video
capture. Confocal laser scanning microscopes generally yield better image quality than Nipkow
and PAM but imaging frame rates are typically very slow. Spinning-disk confocal microscopes
can achieve video rate imaging a desirable feature for dynamic observations such as live cell
imaging but at lower resolution.

Figure 3: -tubulin in Tetrahymena (a ciliated protozoan) viewed under a confocal microscope.

Scanning electron microscope

The scanning electron microscope is utilized primarily to examine the surfaces of objects
ranging in size from a virus to an animal head. The construction and operation of the scanning
electron microscopes are very different from that of transmission electron microscopes. The
scanning electron microscope (SEM) is a type of electron microscope that images the sample
surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The
electrons interact with the atoms that make up the sample producing signals that contain
information about the sample's surface topography, composition and other properties such as
electrical conductivity. The goal of specimen preparation for scanning electron microscope is to
produce an object that has the same shape and surface properties as the living state, but is
devoid of fluid, as required for observing the specimen under vacuum. Because water contains
such a high percentage of the weight of the living cells and is present in association with
virtually every macromolecule, its removal can have a very destructive effect on cell structure.

In a typical SEM, an electron beam is thermionically emitted from an electron gun fitted with a
tungsten filament cathode. Tungsten is normally used in thermionic electron guns because it
has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be
heated for electron emission, and because of its low cost. Other types of electron emitters
include lanthanum hexaboride (LaB6) cathodes, which can be used in a standard tungsten
filament SEM if the vacuum system is upgraded and field emission guns (FEG), which may be of
the cold-cathode type using tungsten single crystal emitters or the thermally-assisted Schottky
type, using emitters of zirconium oxide. The electron beam, which typically has an energy
ranging from a few hundred eV to 40 keV, is focused by one or two condenser lenses to a spot
about 0.4 nm to 5 nm in diameter. The beam passes through pairs of scanning coils or pairs of
deflector plates in the electron column, typically in the final lens, which deflect the beam in the
x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface.
When the primary electron beam interacts with the sample, the electrons lose energy by
repeated random scattering and absorption within a teardrop-shaped volume of the specimen
known as the interaction volume, which extends from less than 100 nm to around 5 m into the
surface. The size of the interaction volume depends on the electron's landing energy, the
atomic number of the specimen and the specimen's density. The energy exchange between the
electron beam and the sample results in the reflection of high-energy electrons by elastic
scattering, emission of secondary electrons by inelastic scattering and the emission of
electromagnetic radiation, each of which can be detected by specialized detectors. The beam
current absorbed by the specimen can also be detected and used to create images of the
distribution of specimen current. Electronic amplifiers of various types are used to amplify the
signals which are displayed as variations in brightness on a cathode ray tube. The raster
scanning of the CRT display is synchronised with that of the beam on the specimen in the
microscope, and the resulting image is therefore a distribution map of the intensity of the signal
being emitted from the scanned area of the specimen. The image may be captured by
photography from a high resolution cathode ray tube, but in modern machines is digitally
captured and displayed on a computer monitor and saved to a computer's hard disc. Image
formation in the SEM is indirect. In addition to the beam that scans the surface of the
specimen, another electron beam synchronously scans the face of a cathode-ray tube,
producing an image similar to that seen on a television screen. The electrons that bounce off
the specimen and reach the detector control the strength of the beam in the cathode-ray tube.
The more electrons collected from the specimen at a given spot, the stronger the signal to the
tube and the greater the intensity of the beam on the screen at the corresponding spot. The
result is an image on the screen that reflects the surface topology of the specimen because it is
this topology (the crevices, hills, and pits) that determines the number of electrons collected
from the various parts of the surface. SEM can provide a great range of magnification.
Resolving power of an SEM is related to the diameter of the electron beam. The SEM also
provides remarkable depths of focus, approximately 500 times that of the light microscope at a
corresponding magnification. This property gives SEM images their three-dimensional quality. At
the cellular level, the SEM allows the observer to appreciate the structure of the outer cell
surface and all of the various processes, extensions, and extracellular materials that interact
with the environment.

Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude from

about 10 to 500,000 times. Unlike optical and transmission electron microscopes, image
magnification in the SEM is not a function of the power of the objective lens. SEMs may have
condenser and objective lenses, but their function is to focus the beam to a spot, and not to
image the specimen. Provided the electron gun can generate a beam with sufficiently small
diameter, a SEM could in principle work entirely without condenser or objective lenses, although
it might not be very versatile or achieve very high resolution. In a SEM, as in scanning probe
microscopy, magnification results from the ratio of the dimensions of the raster on the
specimen and the raster on the display device. Assuming that the display screen has a fixed
size, higher magnification results from reducing the size of the raster on the specimen, and vice
versa. Magnification is therefore controlled by the current supplied to the x, y scanning coils, or
the voltage supplied to the x, y deflector plates, and not by objective lens power.
The spatial resolution of the SEM depends on the size of the electron spot, which in turn
depends on both the wavelength of the electrons and the electron-optical system which
produces the scanning beam. The resolution is also limited by the size of the interaction
volume, or the extent to which the material interacts with the electron beam. The spot size and
the interaction volume are both large compared to the distances between atoms, so the
resolution of the SEM is not high enough to image individual atoms, as is possible in the shorter
wavelength such as higher energy transmission electron microscope (TEM). The SEM has
compensating advantages, though, including the ability to image a comparatively large area of
the specimen; the ability to image bulk materials not just thin films or foils; and the variety of
analytical modes available for measuring the composition and properties of the specimen.

Conventional SEM requires samples to be imaged under vacuum, because a gas atmosphere
rapidly spreads and attenuates electron beams. Consequently, samples that produce a
significant amount of vapor, example wet biological samples or oil-bearing rock need to be
either dried or cryogenically frozen. Processes involving phase transitions, such as the drying of
adhesives or melting of alloys, liquid transport, chemical reactions, solid-air-gas systems and
living organisms in general cannot be observed. The first commercial development of the
Environmental SEM (ESEM) in the late 1980s allowed samples to be observed in low-pressure
gaseous environment and high relative humidity. The accumulation of electric charge on the
surfaces of non-metallic specimens can be avoided by using environmental SEM in which the
specimen is placed in an internal chamber at higher pressure than the vacuum in the electron
optical column. Positively charged ions generated by beam interactions with the gas help to
neutralize the negative charge on the specimen surface. The pressure of gas in the chamber
can be controlled, and the type of gas used can be varied according to need. Coating is thus
unnecessary, and X-ray analysis unhindered. This was made possible by the development of a
secondary-electron detector capable of operating in the presence of water vapour and by the
use of pressure-limiting apertures with differential pumping in the path of the electron beam to
separate the vacuum region around the gun and lenses from the sample chamber. ESEM is
especially useful for non-metallic and biological materials because coating with carbon or gold is
unnecessary. Uncoated plastics and elastomers can be routinely examined, as can uncoated
biological samples. Coating can be difficult to reverse, may conceal small features on the
surface of the sample and may reduce the value of the results obtained. X-ray analysis is
difficult with a coating of a heavy metal, so carbon coatings are routinely used in conventional
SEMs, but ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive
specimens. ESEM may be the preferred for electron microscopy of unique samples from criminal
or civil actions, where forensic analysis may need to be repeated by several different experts.
Backscattered electrons (BSE) consist of high-energy electrons originating in the electron beam
that are reflected or back-scattered out of the specimen interaction volume by elastic scattering
interactions with specimen atoms. Since heavy elements (high atomic number) backscatter
electrons more strongly than light elements (low atomic number), and thus appear brighter in
the image, BSE are used to detect contrast between areas with different chemical compositions.

3D data can be measured in the SEM with different methods: 1) photogrametry (2 or 3 images
from tilted specimen), 2) photometric stereo (use of 4 images from backscattered electron
detector). Possible applications are roughness measurement, measurement of fractal
dimenstion, corrosion measurement and height step measurement.

Figure 4: These pollen grains taken on an SEM show the characteristic depth of field of SEM
micrographs.
Sample preparation for each microscope

Light microscope

Specimens to be observed with the light microscope are broadly divided into two categories:
whole mounts and sections. A whole mount is an intact object, either living or dead, and can
consist of an entire microscopic organism such as a protozoan or a small part of a larger
organism. Most tissues of plants and animals are much too opaque for microscopic analysis
unless examined as a very thin slice or section. A dry mount doesnt need water; usually used
for inanimate objects that do not require water to live. A slide is placed flat on the surface,
specimen is laid on the slide (the specimen should be as thin as possible, one cell layer thick is
best), and the coverslip is placed on the specimen as flat as possible. In a wet mount, water is
required; used to prepare that holds living organisms either mobile or not. The procedures are,
first the slide is placed on a flat surface, a drop of water is added to the slide and the specimen
is added to it but sometimes it can be done vice versa, the coverslips are held by its sides and
its bottom edge placed on the side of the specimen (holding the coverslips at angle 45 o helps),
the coverslip is lowered slowly so that it spreads the water out. Air bubbles are prevented from
being captured under the coverslip. A little water is to be added to the edge of the coverslip if
there are dry parts, excess water can be dabbed of with a paper towel. Iodine, methylene blue,
or crystal violet may be added to specimens in order to increase contrast. The stain can be
directly added to the water when first preparing the slide or it can be added later after first
viewing the specimen without the stain. A drop of the stain is added along one edge of the
coverslip. Placing a piece of paper towel along the opposite edge of the coverslip will help draw
the stain under the coverslip.

To prepare a section, the cells are first killed by immersing the tissue in a chemical solution,
called a fixative. The goal of fixation is to maintain cellular structure as close as possible to the
native state. A good fixative rapidly penetrates the cell membrane and immobilizes all of its
macromolecular material so that the structure of the cell is maintained as close as possible to
that of the living state. Fixation can damage or mask antigenic sites, thereby compromising the
intensity of your immunostaining. You may find the need to test several fixatives before you
find the one that produces the right balance of antibody binding and structural integrity of the
sample. The most common fixatives for the light microscope are solutions of formaldehyde,
alcohol, or acetic acid. Microwave fixation is useful for penetrating thick tissue samples, often in
conjunction with chemical fixatives. After fixation, the tissue is dehydrated by transfer through a
series of alcohols and then usually embedded in paraffin (wax), which provides mechanical
support during sectioning. Paraffin is used as an embedding medium because it is readily
dissolved by organic solvents. Slides containing adherent paraffin sections are immersed in
toluene, which dissolves the wax, leaving the thin slice of tissue attached to the slide, where it
can be stained or treated with antibodies or other agents. Slides and coverslips are the simplest
things that are usually used to view samples. After staining, a coverslip is mounted over the
tissue using a mounting medium that has the same refractive index as the glass slide and
coverslip. Mounting medium helps preserve samples and raises the refractive index to give good
performance with oil objectives. Mountants often have scavengers which soak-up free radicals
and reduce photobleaching, these sometimes reduce the initial brightness of the samples. Just
put a spot of any mounting medium on the sample before putting the coverslip on the slide,
and they are let to dry by lying flat before imaging. Slides are sealed after letting them dry if
they are not going to be imaged immediately.

Fluorescence microscope

The sample preparation for simultaneous measurements is similar to that used for Atomic Force
Microscope imaging. However, in fluorescence microscopy, light signals can be faint. Some
common methods of increasing the efficiency of fluorescent emissions cannot be used in the
MFP-3D because it would prevent the AFM probe from coming into contact with the sample
for example, using a hydrophobic medium and replacing water with glycerol, or adding
antioxidants, to increase the efficiency of the fluorescence. The goal of fixation is to maintain
cellular structure as close as possible to the native state. Once fixed, you can go through the
rest of the protocol without losing the protein of interest and, indeed, the rest of the cell.
Mounting medium helps preserve your sample and raises the refractive index to give good
performance with oil objectives. Water removed from the specimen using ethanol. Particularly
important for electron microscopy because water molecules deflect the electron beam which
blurs the image. Embedding supports the tissue in wax or resin so that it can be cut into thin
sections. Sectioning produces very thin slices for mounting. Sections are cut with a microtome
or an ulramicrotome to make them a few micrometres thick. Different stains are used for
different types of tissues. Some substances emit light when stimulated. The source of light in
fluorescence microscope is UV lamp or a carbonic arc. The filter used in the system permits only
short wave blue rays to fall on the object. Methylene blue is often used for animal cells, while
iodine in KI solution is used for plant tissues to stain in other light microscopes. Fluorescence
dyes are usually used to stain the objects which are not able to emit color (autofluorescent).
Examples of fluorescence dyes are Rhodamine, Auramine, or Flurocin isothiocynate. Mountants
often have scavengers which soak-up free radicals and reduce photobleaching these sometimes
reduce the initial brightness of the samples. These methods typically require that a coverslip be
placed on top of the sample to protect and preserve it. Since such a coverslip prevents the AFM
probe from interacting with the sample, this step in sample preparation must be omitted.
Instead, preparation must be tailored to each sample and the amount of fluorescent dye has to
be adapted to the new imaging conditions. For example, it might be necessary to increase the
amount of fluorochrome that is going to bind to the specimen to obtain a larger signal for the
FM measurement. Note, however, that since this may result in a loss of binding specificity of
the dye, and a compromise may need to be found.

Confocal microscope

Good tissue preservation is generally a trade-off with preserving antigenicity. Fixation must
adequately stabilize antigenic sites, and other tissue components, for subsequent treatment. If
fixation is inadequate, the fluorescence in our sample can diffuse and disappear over a possibly
very short time. Ideally one would compare a fixed sample to a living specimen so we know
what artifacts we have induced by fixation. All processing induces artifacts but we can only try
to minimize them. Several factors must be considered when deciding on a fixation protocol:
composition of the fixative, pH, osmolarity, temperature, time and method (perfusion or
immersion) can all affect the outcome. The most common fixative is buffered 4%
paraformaldehyde used at 4oC. Paraformaldehyde is prepared fresh from powder or purchased
in vials sealed with inert gas. Commercial formalin contains methanol (6-15%) and other
impurities that may affect fixation. Tissue needs to be submerged in the fixative often following
perfusion and gently agitated. Cells and tissue remain osmotically active after fixation in
aldehydes. Checking for excessive swelling or shrinkage in the tissue is possible. Fixation times
vary with the type of specimen and the accessibility of the epitope. Cell cultures are generally
fixed for a minimum of 30 minutes. Tissue is fixed for a minimum of one to four hours or
longer. Paraformaldehyde penetrates rapidly but fixes slowly. Glutaraldehyde induces
autofluorescence in the tissue. Samples fixed with glutaraldehyde must subsequently be treated
with sodium borohydride. Methanol and acetone are not good fixatives for preserving most
structures. They precipitate proteins. Tissue will be shrunken and distorted. The degree of
shrinkage may be almost insignificant for monolayers of cells, but will distort tissue samples
dramatically. To provide the ability to take full advantage of the three-dimensional
reconstruction capability of the confocal microscope, the use of a fixative that does not destroy
in vivo structure and organization must be found. It is important to remember that different
specimens may require a different fixation method. Testing and optimizing for each new sample
type will insure that the best balance between preservation and labeling is obtained.

If a component of the cytoplasm or nucleus is to be labeled, the plasma membrane must be

permeabilized. There are several ways to do this, and they depend on the fixation method
chosen. Cells fixed with solvents do not require additional permeabilizing as the solvent has
already extracted enough of the membrane. Therefore solvent fixation is doubly efficient for
this reason. Cells fixed with cross-linking aldehydes need to have the plasma membrane
integrity breached by the use of chemical agents. Commonly used reagents include like X-100
saponin or deoxycholin. Permeabilization is another area, like fixation where fine tuning is
necessary. Different detergents solubilize different molecules within the membrane, so it is
necessary to know that the molecules you are interested in have not been washed away in the
permeabilization step. The concentration of detergent is another detail that should be adjusted
carefully. The idea is to selectively remove plasma membrane constituents to allow access to
the cytoplasm without altering either the antigenicity or morphology of the sample. The choice
label depends upon the available equipment such as lasers or filters and the availability of
certain fluorochromes conjugated to required antibodies for use in multiple labeling schemes. In
general, the laser lines available dictate which fluorophores can be used. Fluorescence detection
is not the only way to use a confocal microscope.

The mountant used under the coverslip is important for several reasons such as, Fluorescein is
sensitive to pH absorption or emission where its maximal is at pH 8.5, the mountant must have
an additive to prevent photobleaching. The refractive index of the mountant should be as close
to that of the fixed tissue as possible. In most cases, do not use an aqueous mountant with
fixed tissue. The working distance of a lens is the space between the front element of the
objective and the top of the coverslip. Any space between the coverslip and the tissue adds to
the working distance since a manufacturers figure for the working distance of a lens presumes
that the tissue is just under the coverslip. Pap pens, rubber cement, or any other substance
used to create a well for immunostaining must be completely removed before coverslipping as
any remnants left on the slide can elevate the coverslip. The resulting increase in the distance
between the front element of the objective lens and the tissue will prevent focusing within the
sample if this distance exceeds the working distance of the lens. For the same reason, it is
better not to use too much mountant under the coverslip. The wells in depression slides are
usually too deep to allow focusing above 10X or 20X. A coverslip placed directly on cells or
tissue will compress the sample. To prevent compression, elevate the coverslip with a spacer as
close as possible to the size of the tissue. Coverslips are too thick to be used as spacers for
most samples. Cells cultured on coverslips are ideal since they can be flipped over, mounted on
the slide and the thickness of the spacer is immaterial. Thickness of the coverslip is important
for optimal image quality. The coverslip thickness an objective lens is designed for is engraved
on the lens. A slide is usually sealed around the coverslip with nail polish. This prevents
evaporation of the mountant and stabilizes the coverslip for use with immersion lenses.

Scanning electron microscope

For conventional imaging in the SEM, specimens must be electrically conductive, at least at the
surface, and electrically grounded to prevent the accumulation of electrostatic charge at the
surface. Metal objects require little special preparation for SEM except for cleaning and
mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the
electron beam, and especially in secondary electron imaging mode, this 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. Two important reasons for coating, even when
there is more than enough specimen conductivity to prevent charging, are to maximise signal
and improve spatial resolution. Broadly, signal increases with atomic number, especially for
backscattered electron imaging. The improvement in resolution arises because in low-Z
materials, that is, material with low atomic number such as carbon, the electron beam can
penetrate several micrometres below the surface, generating signals from an interaction volume
much larger than the beam diameter and reducing spatial resolution. Coating with a high-Z
material such as gold maximises secondary electron yield from within a surface layer a few nm
thick, and suppresses secondary electrons generated at greater depths, so that the signal is
predominantly derived from locations closer to the beam and closer to the specimen surface
than would be the case in an uncoated, low-Z material.

Nonconducting specimens may be imaged uncoated using specialized SEM instrumentation such
as the Environmental SEM (ESEM) or field emission gun (FEG) SEMs operated at low voltage.
Environmental SEM instruments place the specimen in a relatively high pressure chamber where
the working distance is short and the electron optical column is differentially pumped to keep
vacuum adequately low at the electron gun. The high pressure region around the sample in the
ESEM neutralizes charge and provides an amplification of the secondary electron signal. Low
voltage SEM of non-conducting specimens can be operationally difficult to accomplish in a
conventional SEM and is typically a research application for specimens that are sensitive to the
process of applying conductive coatings. Low-voltage SEM is typically conducted in an FEG-SEM
because the FEG is capable of producing high primary electron brightness even at low
accelerating potentials. Operating conditions must be adjusted such that the local space charge
is at or near neutral with adequate low voltage secondary electrons being available to neutralize
any positively charged surface sites. This requires that the primary electron beam's potential
and current be tuned to the characteristics of the sample specimen.

For SEM, a specimen is normally required to be completely dry, since the specimen chamber is
at high vacuum. Hard, dry materials such as wood, bone, feathers, dried insects or shells can
be examined with little further treatment, but living cells and tissues and whole, soft-bodied
organisms usually require chemical fixation to preserve and stabilize their structure. Fixation is
usually performed by incubation in a solution of a buffered chemical fixative, such as
glutaraldehyde, sometimes in combination with formaldehyde and other fixatives, and optionally
followed by postfixation with osmium tetroxide. The fixed tissue is then dehydrated. Because
air-drying causes collapse and shrinkage, this is commonly achieved by critical point drying,
which involves replacement of water in the cells with organic solvents such as ethanol or
acetone, and replacement of these solvents in turn with a transitional fluid such as liquid carbon
dioxide at high pressure. The carbon dioxide is finally removed while in a supercritical state, so
that no gas-liquid interface is present within the sample during drying. The dry specimen is
usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically-
conductive double-sided adhesive tape, and sputter coated with gold or gold or palladium alloy
before examination in the microscope. If the SEM is equipped with a cold stage for cryo-
microscopy, cryofixation may be used and low-temperature scanning electron microscopy
performed on the cryogenically fixed specimens. Freeze-fracturing, freeze-etch or freeze-and-
break is a preparation method particularly useful for examining lipid membranes and their
incorporated proteins in "face on" view. The preparation method reveals the proteins embedded
in the lipid bilayer. Another technique is negative staining, as SEM is also well suited for
examining very small particulate materials, such as viruses, ribosomes, cytoskeletal elements
and so on. The shapes of individual proteins and nucleic acids can also be resolved as long as
they are made to have sufficient contrast from their surroundings. One of the best ways to
make such substances visible is through this method in which heavy-metal deposits are
collected everywhere on a specimen grid except where the particles are present. As a result,
the specimen stands out by its relative brightness on the viewing screen. Some methods of the
negative staining are float method, drop method and spray method which is applied only when
very small volumes of specimen are available. Shadow casting is another technique to visualize
isolated particles by casting their shadows. The grids containing the specimens are placed in a
sealed chamber which is then evacuated by vacuum pump. The technique provides excellent
contrast and produces a three dimensional effect.
Differences in all these microscopes

Between the light microscope and the electron microscope, many differences can be given, that
means the differences between Scanning electron microscope and the others which are the
light microscope, fluorescence microscope and the confocal microscope. The main difference is
the resolution of the microscopes where electron microscopes have a much higher resolution
than light microscopes. Secondly, the price where light microscopes are much cheaper than the
electron microscope because it is cheaper to operate the light microscope but the production of
electron beam is quite expensive. The light microscopes are usually small and portable whereas
the electron microscopes are large and needs special rooms to be kept. The sample preparation
in the matter is simple and short time duration needed for light microscopes but for electron
microscopes, there are many procedures which are complicating and it takes quite a long time
to prepare the sample specimens. The specimens are rarely distorted during sample preparation
for light microscopes whereas preparations distort materials in electron microscopy. A vacuum
space is needed in the electron microscope for a purpose but it is not required in the light
microscope which is a one of the reasons why light microscopes are smaller in size. During the
imaging process of the samples prepared in the light microscopes, the natural colors of the
specimens are preserved and maintained but only black and white images are shown in the
electron microscopes. The advantage of the electron microscopes is that in can magnify an
image over than 500 000 times whereas a light microscope only can magnify objects up to 2000
times. Specimens viewed in the light microscopes can be either living ones or dead ones but the
specimens viewed in the electron microscope can only be dead ones as they must be fixed in
plastic and viewed in a vacuum. Stains in the specimens are required sometimes to make the
cells visible in the light microscope but in electron microscopes, the electron beams can damage
the specimens so they must be stained with an electron-dense chemical usually heavy-metals
like osmium, lead or gold.

The fluorescence microscope and confocal microscope has a few similarities but the confocal
microscope is much newer and the image found in the confocal microscope looks much clearer
and realistic through 2-D imaging. The images that can be seen through the fluorescence
microscope are clearer with their colors and some specimens are alive where even their motility
can be observed but in confocal microscopy, the images are seen in a electronic device such as
the computer or some other equipments often, which shows 2 dimensional images of the
specimen, that can be assembled in an imaging system to construct a 3 dimensional image but
not as natural as under the fluorescence microscope. Confocal microscopes are larger than
fluorescence microscopes as the imaging system will be attached to it normally. A fluorescence
microscope will have filters that permit only certain wavelength of lights to pass through the
specimen (usually blue light only) but confocal microscopes uses laser beam as a light source
because laser provides high intensity, well focused, monochromatic light, which easily
illuminates individual points within a specimen. Other differences of these microscopes are seen
in the structures and the light sources and pointing of the microscopes, an example is for the
confocal microscope, a pinhole is present for imaging purpose but this is not present in the
fluorescence microscope. The usage of confocal microscopes are costly than the fluorescence
microscopes as well as they could view relatively thick specimens and is well suited for
fluorescence applications. Confocal microscopes provide high contrast images of specimens
without the image artifacts normally present with conventional contrasting techniques. There
are high possibilities for the confocal microscope to damage the specimen from the laser
illumination but this does not happen in fluorescence microscopes.
Appendices

Figure 5: A light microscope

Figure 6: A fluorescence microscope

Figure 7: A confocal microscope

Figure 8: A scanning electron microscope

References

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3) Murad H. and M.U.A. Atiqui, 1991, Biological Techniques in Electron Microscopy, Satish
Kumar Jain for CBS Publishers & Distributors, New Delhi, India.

4) Raven P. H., Johnson G. B., 2005, Biology: Seventh edition, McGraw-Hill Companies
Inc., New York.

5) V.H. Talib, 1999, A handbook of Medical Laboratory Technology: 5 th Edition, McGraw-Hill

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