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 microscope’s 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 hasn’t. 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,

This creates an enlarged image of the subject. Compound optical microscopes can produce a magnified image of a specimen up to 1000× and. at its simplest. 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. 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. It is this real image that is viewed by the eyepiece lens that provides further enlargement. By carefully focusing a brightly lit specimen. 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. Some microscopes have a fourth objective lens. The objective lens is. and the objective lens being used. a very high powered magnifying glass. allowing the final image to become focused precisely on the plane of the retina. there are three objective lenses: a scanning lens (4×). To use this lens. at high magnifications. for example. are used to study thin specimens as they have a very limited depth of field. the relative distance between the specimen and the objective lens changes. When the focusing knob of the light microscope is turned. called an oil immersion lens.the basic optical principles of a microscope are quite simple. a highly enlarged image can be seen. the eyepiece is a compound lens. An oil immersion lens usually has a magnification of 50 to 100×. 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). On a typical compound optical microscope. low power lens (10×) and high power lens (ranging from 20 to 100×). a drop of immersion oil is placed on top of the cover slip. . a lens with a very short focal length. with one component lens near the front and one near the back of the eyepiece tube. usually about 10×. the virtual image comes to a focus between the two lenses of the eyepiece. 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. The total magnification attained by the microscope is the product of the magnifications produced by the objective lens and the ocular lens. This forms an air-separated couplet. The actual power or magnification of an optical microscope is the product of the powers of the ocular (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 many designs. In most microscopes.

however. At its simplest. the laser scanning confocal microscope. so we will see a little information briefly about the bright field and phase-contrast microscopy. typically a large knurled wheel to adjust coarse focus. Bright field microscopy is used to view stained or naturally pigmented organelles in the cell.The stage is a platform below the objective which supports the specimen being viewed. have their own controllable light source that is focused through an optical device called a condenser. 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. about 3 µm. The phase-contrast microscope makes highly transparent objects more visible. light is provided and controlled in a variety of ways. are one of the smallest organelles to be seen through microscopy. The arm is usually able to pivot on its joint with the foot to allow the viewing angle to be adjusted. In the center of the stage is a hole through which light passes to illuminate the specimen. and phase-contrast microscope. fluorescence microscope. is usually the shortest wavelength used in light microscopy. The fluorescence microscope and confocal microscope will be explained in detail later. The stage usually has arms to hold slides (rectangular glass plates with typical dimensions of 25 mm by 75 mm. Further image enhancement is accomplished by using a specific monochromatic light source. Most microscopes. We have already known that under the division of light microscope. We can distinguish different parts of an object because they affect light differently from one another. together with a smaller knurled wheel to control fine focus. 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. Therefore. with diaphragms and filters available to manage the quality and intensity of the light. it must be large enough to be able to perturb the wave motion of the light rays that strike it. In order to see a specific structure of the cell. Blue light. The easiest way to enhance the contrast of the relatively transparent cells is to stain them. Mounted on the arm are controls for focusing. The illumination source is below the stage. 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. A common filter is the one that allows only blue light to pass through. comes a few other types of microscope such as the bright field microscope. One way to achieve this is by using a filter. daylight is directed via a mirror. about 450 nm. mitochondria. on which the specimen is mounted). .

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. the phase-contrast microscope converts differences in refractive index into differences in intensity (relative brightness and darkness). As a consequence. delivers an image that has an apparent three dimensional quality. the edges of structures. For example. Phase-contrast microscopes are more useful for examining intracellular components of living cells at relatively high resolution. mitotic chromosomes. Contrast in DIC microscopy depends on the rate of change of refractive index across a specimen. However. the dynamic motility of mitochondria.One basis for such differences is refractive index. lipid. dead cells. 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. Regions of different composition are likely to have different refractive indexes. are seen with especially good contrast. termed differential interference contrast (DIC). or sometimes Nomarski interference after its developer. Another type of interference system. Cell organelles are made up of different proportions of various molecules: DNA. . The phase-contrast microscope has optical handicaps that results in loss of resolution. and water. and vacuoles can be followed and filmed with this optics. salts. Normally such differences cannot be detected by our eyes. carbohydrate. and the image suffers from interfering halos and shading produced were sharp changes in refractive index occur. 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. 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. RNA. The phase-contrast microscope is a type of interference microscope. which are visible to the eye. protein. where the refractive index varies markedly over a relatively small distance.

These advances in the live cell imaging have been made possible to a large extent by innovations in fluorescence microscopy. The specimen is illuminated with light of a specific wavelength or wavelengths which is absorbed by the fluorophores. The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the . a component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore such as green fluorescent protein (GFP). In most cases. the excitation filter. the dichroic mirror or dichromatic beamsplitter. causing them to emit longer wavelengths of light of a different color than the absorbed light. and the emission filter. The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Fluorescence microscope Over the past few decades. fluorescein or DyLight 488. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor lamp). 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.Figure 1: Paramecium sp. viewed under a light microscope.

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

The excitatory light is passed from above or. yellow (YFP). for inverted microscopes. When a muscle of one of these mice was exposed surgically. in which researchers generated strains of mice whose neurons contained differently colored fluorescent proteins. In the latter case the transmitted excitatory light reaches the objective together with light emitted from the specimen. A filter between the objective and the detector filters out the excitation light from fluorescent light.robust fluorophores. 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. which then synthesize a chimeric protein containing fluorescent GFP fused to the protein under study. Variants of GFP that fluoresce in shades of blue (BFP). have also been generated by mutagenesis experiments. for example. which fluoresce in a variety of distinguishable colors. In addition. Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences. the labeled proteins participate in the normal activities of the cell. only reflected excitatory light reaches the objective together with the emitted light and this method therefore gives an improved signal to noise ratio. In recent years. through the objective and then onto the specimen instead of passing it first through the specimen. Since most of the excitatory light is transmitted through the specimen. a distantly related red fluorescent tetrameric protein (DsRed) has been isolated from a sea anemone. Studies can often be made more informative by the simultaneous use of GFP variants that exhibit different spectral properties. by minimizing illumination. 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. In all of these strategies. and cyan (CFP) have been generated through directed mutagenesis of the GFP gene. The type of information that can be obtained using GFP variants is illustrated by the study. the investigators could observe the dynamic interactions between the variously colored neurons and the neuromuscular junctions being innervated. or by introducing a scavenger system to reduce the rate of photobleaching. 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. They watched. as branches from a CFP-colored neuron . Monomeric variants of DsRed. This recombinant DNA is used to transfect cells. and their location can be followed microscopically to reveal the dynamic activities in which the protein participates. from below.

for synaptic contact with the muscle tissue. In the absence of bound cGMP. which can measure distances between fluorochromes in the nanoscale range. GFP variants have also been useful in a technique. all of the ‘winning’ branches belong to one of the neurons. FRET can be used to study such changes as they occur in vitro or within a living cell. FRET can also be used to follow many other processes including protein folding or the association and dissociation of components within a membrane. 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). 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. 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. called fluorescence resonance energy transfer (FRET). . In each case they found that. 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. FRET is typically employed to measure changes in distance between two parts of a protein or between two separate proteins within a large structure. As a result. Binding of cGMP induces a conformational change in the protein that brings the two fluorochromes into close enough proximity fro FRET to occur. the two fluorochromes are too far apart for energy transfer to occur. while all the ‘losing’ branches belong to the other neuron. when two neurons compete for innervations of different muscle fibers. This transfer of energy reduces the fluorescence intensity of the donor and increases the fluorescence intensity of the acceptor.

the entire specimen is flooded in light from a light source. 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. microtubules are marked green by an antibody bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC.Figure 2: Endothelial cells under the fluorescence microscope. Nuclei are stained blue with DAPI. In a conventional fluorescence microscope. This technique has gained popularity in the scientific and industrial . The principle of confocal imaging was patented by Marvin Minsky in 1957 and aims to overcome some limitations of traditional wide-field fluorescence microscopes. 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. Confocal microscope The development of a new type of light microscope with imaging techniques. Bovine pulmonary artery endothelial (BPAE) cells.

Light emitted from the specimen is brought to focus at a site within the microscope that contains a pinhole aperture. and you are throwing out useful light. so the entire sample is fluorescing at the same time. due to diffraction. but nonetheless. Because the focal point of the objective lens of the microscope forms an image where the pinhole is. The pinhole is conjugate to the focal point of the lens. thus illuminating only a thin plane within the specimen. the aperture and the illuminated plane in the specimen are confocal. In contrast. thus it is a confocal pinhole. The advantage of fluorescence for microscopy is that you can often attach fluorescent dye molecules to specific parts of your sample. the highest intensity of the excitation light is at the focal point of the lens. the sample plane and the pinhole/screen are conjugate planes. In this type of microscope. you can cause one type of dye to fluoresce. Confocal microscopes are typically used with fluorescence optics. the sample is completely illuminated by the excitation light. out of focus points in the specimen become invinsible. Adding a pinhole/screen combination solves this problem. As described earlier. Normally. short wavelength incident light is absorbed by the fluorochromes in a specimen and reemitted at longer wavelength. This contributes to a background haze in the resulting image. Any smaller. The image of a point source of light isn't actually a point. a confocal microscope uses point illumination and a pinhole in an . and then another. the specimen is illuminated by a finely focused laser beam that rapidly scans across the specimen at a single depth. As a result. and you see more out of focus light. Thus. whereas any light rays that might emanate from above or below this plane are prevented from participating in image formation. By changing the excitation light. to distinguish two different parts of your sample. these two points are known as "conjugate points" or alternatively. so that only those parts are the ones seen in the microscope. 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. Of course. the observer views the specimen at different depths by changing the position of the objective lens by rotating the focusing knob. You can also use more than one type of dye. When a whole cell or a section of an organ is examined under a standard light microscope. Any larger. it's actually an Airy disk.communities and typical applications are in life sciences and semiconductor inspection. the other parts of the sample do get some of this light and they do fluoresce. The size of the confocal pinhole needs to be matched to the size of the Airy disk. Light rays emitted from the illuminated plane of the specimen can pass through the aperture.

The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging of samples. From there. for example. The detector is attached to a computer which builds up the image. The thickness of the focal plane is defined mostly by the inverse of the square of the numerical aperture of the objective lens. a similar effect happens with points of light in the focal plane. one pixel at a time. The light that passes through the pinhole is measured by a detector.optically conjugate plane in front of the detector to eliminate out of focus informations. The practical effect of this is that your image comes from a thin section of your sample. so. So a confocal microscope has slightly better resolution horizontally. a photomultiplier tube. As only one point in the sample is illuminated at a time. The limitation is in the scanning mirrors. only one point of the sample is observed. Also. Three types of confocal microscopes are commercially available: 1) Confocal laser scanning microscopes. the laser hits two mirrors which are mounted on motors. these mirrors scan the laser across the sample. particularly in the sample depth direction. as an example. and also by the optical properties of the specimen and the ambient index of refraction. but not at the focal point emitted light from these areas is blocked by the pinhole screen. 2D or 3D imaging requires scanning over a regular raster. As only light produced by fluorescence very close to the focal plane can be detected the image resolution. So. 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. the microscope is really efficient at rejecting out of focus fluorescent light. Dye in the sample fluoresces. a rectangular pattern of parallel scanning lines in the specimen. and the emitted light gets descanned by the same mirrors that are used to scan the excitation light from the laser. By scanning many thin sections through your sample. A laser is used to provide the excitation light (in order to get very high intensities). 3) Programmable Array . is much better than that of wide-field microscopes. you can build up a very clean three-dimensional image of the sample. By having a confocal pinhole. the name "confocal" comes from this configuration. The emitted light passes through the dichroic and is focused onto the pinhole. 2) Spinning-disk (Nipkow disk) confocal microscopes. The laser light reflects off a dichroic mirror. as well as vertically. there never is a complete image of the sample at any given instant.

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.Microscopes (PAM). The . 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. Spinning-disk confocal microscopes can achieve video rate imaging a desirable feature for dynamic observations such as live cell imaging but at lower resolution. The construction and operation of the scanning electron microscopes are very different from that of transmission electron microscopes. Confocal laser scanning microscopes generally yield better image quality than Nipkow and PAM but imaging frame rates are typically very slow. 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. Figure 3: β-tubulin in Tetrahymena (a ciliated protozoan) viewed under a confocal microscope.

which typically has an energy ranging from a few hundred eV to 40 keV. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering. Because water contains such a high percentage of the weight of the living cells and is present in association with virtually every macromolecule. 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. its removal can have a very destructive effect on cell structure. which can be used in a standard tungsten filament SEM if the vacuum system is upgraded and field emission guns (FEG). which extends from less than 100 nm to around 5 µm into the surface. and the resulting image is therefore a distribution map of the intensity of the signal . typically in the final lens. Other types of electron emitters include lanthanum hexaboride (LaB6) cathodes. an electron beam is thermionically emitted from an electron gun fitted with a tungsten filament cathode. thereby allowing it to be heated for electron emission. emission of secondary electrons by inelastic scattering and the emission of electromagnetic radiation. but is devoid of fluid. The raster scanning of the CRT display is synchronised with that of the beam on the specimen in the microscope. The electron beam. Tungsten is normally used in thermionic electron guns because it has the highest melting point and lowest vapour pressure of all metals. composition and other properties such as electrical conductivity. The size of the interaction volume depends on the electron's landing energy. When the primary electron beam interacts with the sample. In a typical SEM. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current.4 nm to 5 nm in diameter.electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography. the atomic number of the specimen and the specimen's density. which may be of the cold-cathode type using tungsten single crystal emitters or the thermally-assisted Schottky type. is focused by one or two condenser lenses to a spot about 0. and because of its low cost. the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume. Electronic amplifiers of various types are used to amplify the signals which are displayed as variations in brightness on a cathode ray tube. 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. each of which can be detected by specialized detectors. as required for observing the specimen under vacuum. using emitters of zirconium oxide. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column.

Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude from about 10 to 500. Resolving power of an SEM is related to the diameter of the electron beam. and not to image the specimen. 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. In a SEM. extensions. SEM can provide a great range of magnification. At the cellular level. Assuming that the display screen has a fixed size. another electron beam synchronously scans the face of a cathode-ray tube.000 times. or the voltage supplied to the x. The result is an image on the screen that reflects the surface topology of the specimen because it is this topology (the crevices. and extracellular materials that interact with the environment. but their function is to focus the beam to a spot. and vice versa. The image may be captured by photography from a high resolution cathode ray tube. hills. magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. SEMs may have condenser and objective lenses. 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. higher magnification results from reducing the size of the raster on the specimen. The SEM also provides remarkable depths of focus. although it might not be very versatile or achieve very high resolution. Provided the electron gun can generate a beam with sufficiently small diameter. approximately 500 times that of the light microscope at a corresponding magnification. as in scanning probe microscopy. and not by objective lens power. The more electrons collected from the specimen at a given spot. y deflector plates. the stronger the signal to the tube and the greater the intensity of the beam on the screen at the corresponding spot. a SEM could in principle work entirely without condenser or objective lenses. Magnification is therefore controlled by the current supplied to the x. Unlike optical and transmission electron microscopes. . y scanning coils. This property gives SEM images their three-dimensional quality. image magnification in the SEM is not a function of the power of the objective lens. the SEM allows the observer to appreciate the structure of the outer cell surface and all of the various processes.being emitted from the scanned area of the specimen. In addition to the beam that scans the surface of the specimen. and pits) that determines the number of electrons collected from the various parts of the surface.

example wet biological samples or oil-bearing rock need to be either dried or cryogenically frozen. ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. as can uncoated biological samples. The resolution is also limited by the size of the interaction volume. Processes involving phase transitions. Coating is thus unnecessary. liquid transport. The pressure of gas in the chamber can be controlled. such as the drying of adhesives or melting of alloys. so carbon coatings are routinely used in conventional . which in turn depends on both the wavelength of the electrons and the electron-optical system which produces the scanning beam. Positively charged ions generated by beam interactions with the gas help to neutralize the negative charge on the specimen surface. may conceal small features on the surface of the sample and may reduce the value of the results obtained. Uncoated plastics and elastomers can be routinely examined. or the extent to which the material interacts with the electron beam. so the resolution of the SEM is not high enough to image individual atoms. Coating can be difficult to reverse. The spot size and the interaction volume are both large compared to the distances between atoms. the ability to image bulk materials not just thin films or foils. and the type of gas used can be varied according to need. Conventional SEM requires samples to be imaged under vacuum. chemical reactions. 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. including the ability to image a comparatively large area of the specimen. though. samples that produce a significant amount of vapor. solid-air-gas systems and living organisms in general cannot be observed. as is possible in the shorter wavelength such as higher energy transmission electron microscope (TEM). and X-ray analysis unhindered. and the variety of analytical modes available for measuring the composition and properties of the specimen. because a gas atmosphere rapidly spreads and attenuates electron beams. 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. Consequently. The SEM has compensating advantages. 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. X-ray analysis is difficult with a coating of a heavy metal.The spatial resolution of the SEM depends on the size of the electron spot.

2) photometric stereo (use of 4 images from backscattered electron detector). 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. and thus appear brighter in the image. but ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive specimens. . corrosion measurement and height step measurement. where forensic analysis may need to be repeated by several different experts.SEMs. 3D data can be measured in the SEM with different methods: 1) photogrametry (2 or 3 images from tilted specimen). Possible applications are roughness measurement. Since heavy elements (high atomic number) backscatter electrons more strongly than light elements (low atomic number). BSE are used to detect contrast between areas with different chemical compositions. ESEM may be the preferred for electron microscopy of unique samples from criminal or civil actions. measurement of fractal dimenstion. Figure 4: These pollen grains taken on an SEM show the characteristic depth of field of SEM micrographs.

A little water is to be added to the edge of the coverslip if there are dry parts. a drop of water is added to the slide and the specimen is added to it but sometimes it can be done vice versa. 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. 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. To prepare a section. methylene blue. 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. The goal of fixation is to maintain cellular structure as close as possible to the native state. A slide is placed flat on the surface. Iodine. The most common fixatives for the light microscope are solutions of formaldehyde. 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). or crystal violet may be added to specimens in order to increase contrast. water is required. the cells are first killed by immersing the tissue in a chemical solution. first the slide is placed on a flat surface.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. Air bubbles are prevented from being captured under the coverslip. 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 whole mount is an intact object. either living or dead. usually used for inanimate objects that do not require water to live. used to prepare that holds living organisms either mobile or not. thereby compromising the intensity of your immunostaining. specimen is laid on the slide (the specimen should be as thin as possible. called a fixative. 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. and the coverslip is placed on the specimen as flat as possible. one cell layer thick is best). The procedures are. the coverslip is lowered slowly so that it spreads the water out. excess water can be dabbed of with a paper towel. In a wet mount. A dry mount doesn’t need water. Fixation can damage or mask antigenic sites.

alcohol. or adding antioxidants. Microwave fixation is useful for penetrating thick tissue samples. the rest of the cell. a coverslip is mounted over the tissue using a mounting medium that has the same refractive index as the glass slide and coverslip. Mountants often have scavengers which soak-up free radicals and reduce photobleaching. After fixation. The goal of fixation is to maintain cellular structure as close as possible to the native state. Sectioning produces very thin slices for mounting. Embedding supports the tissue in wax or resin so that it can be cut into thin sections. these sometimes reduce the initial brightness of the samples. indeed. 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. Fluorescence microscope The sample preparation for simultaneous measurements is similar to that used for Atomic Force Microscope imaging. and they are let to dry by lying flat before imaging. Sections are cut with a microtome . Slides and coverslips are the simplest things that are usually used to view samples. Slides are sealed after letting them dry if they are not going to be imaged immediately. or acetic acid. Mounting medium helps preserve samples and raises the refractive index to give good performance with oil objectives. However. the tissue is dehydrated by transfer through a series of alcohols and then usually embedded in paraffin (wax). light signals can be faint. you can go through the rest of the protocol without losing the protein of interest and. which dissolves the wax. where it can be stained or treated with antibodies or other agents. in fluorescence microscopy. After staining. Mounting medium helps preserve your sample and raises the refractive index to give good performance with oil objectives. Paraffin is used as an embedding medium because it is readily dissolved by organic solvents. leaving the thin slice of tissue attached to the slide. to increase the efficiency of the fluorescence. Water removed from the specimen using ethanol. which provides mechanical support during sectioning. often in conjunction with chemical fixatives. Slides containing adherent paraffin sections are immersed in toluene. Particularly important for electron microscopy because water molecules deflect the electron beam which blurs the image. Just put a spot of any mounting medium on the sample before putting the coverslip on the slide. Once fixed. using a hydrophobic medium and replacing water with glycerol.

Some substances emit light when stimulated. These methods typically require that a coverslip be placed on top of the sample to protect and preserve it. Ideally one would compare a fixed sample to a living specimen so we know what artifacts we have induced by fixation. this step in sample preparation must be omitted. Tissue needs to be submerged in the fixative often following perfusion and gently agitated. Mountants often have scavengers which soak-up free radicals and reduce photobleaching these sometimes reduce the initial brightness of the samples. Note. however. Confocal microscope Good tissue preservation is generally a trade-off with preserving antigenicity. Cells and tissue remain osmotically active after fixation in . 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. Since such a coverslip prevents the AFM probe from interacting with the sample. temperature. Instead. The filter used in the system permits only short wave blue rays to fall on the object. pH. preparation must be tailored to each sample and the amount of fluorescent dye has to be adapted to the new imaging conditions. time and method (perfusion or immersion) can all affect the outcome. osmolarity. All processing induces artifacts but we can only try to minimize them. The most common fixative is buffered 4% paraformaldehyde used at 4oC. or Flurocin isothiocynate. Fixation must adequately stabilize antigenic sites. The source of light in fluorescence microscope is UV lamp or a carbonic arc. while iodine in KI solution is used for plant tissues to stain in other light microscopes. Several factors must be considered when deciding on a fixation protocol: composition of the fixative. If fixation is inadequate. Methylene blue is often used for animal cells. and a compromise may need to be found. Commercial formalin contains methanol (6-15%) and other impurities that may affect fixation. Fluorescence dyes are usually used to stain the objects which are not able to emit color (autofluorescent). for subsequent treatment.or an ulramicrotome to make them a few micrometres thick. that since this may result in a loss of binding specificity of the dye. Paraformaldehyde is prepared fresh from powder or purchased in vials sealed with inert gas. and other tissue components. Different stains are used for different types of tissues. For example. Examples of fluorescence dyes are Rhodamine. the fluorescence in our sample can diffuse and disappear over a possibly very short time. Auramine.

Cells fixed with solvents do not require additional permeabilizing as the solvent has already extracted enough of the membrane. There are several ways to do this. They precipitate proteins. so it is necessary to know that the molecules you are interested in have not been washed away in the permeabilization step. The mountant used under the coverslip is important for several reasons such as. the use of a fixative that does not destroy in vivo structure and organization must be found.aldehydes. 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. If a component of the cytoplasm or nucleus is to be labeled. To provide the ability to take full advantage of the three-dimensional reconstruction capability of the confocal microscope. Tissue is fixed for a minimum of one to four hours or longer. and they depend on the fixation method chosen. Paraformaldehyde penetrates rapidly but fixes slowly. the plasma membrane must be permeabilized. Cell cultures are generally fixed for a minimum of 30 minutes. The degree of shrinkage may be almost insignificant for monolayers of cells. Different detergents solubilize different molecules within the membrane. the laser lines available dictate which fluorophores can be used. Samples fixed with glutaraldehyde must subsequently be treated with sodium borohydride. the mountant must have . 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. Fluorescence detection is not the only way to use a confocal microscope. Permeabilization is another area. Tissue will be shrunken and distorted.5. but will distort tissue samples dramatically. Cells fixed with cross-linking aldehydes need to have the plasma membrane integrity breached by the use of chemical agents. Methanol and acetone are not good fixatives for preserving most structures. In general. Fixation times vary with the type of specimen and the accessibility of the epitope. Glutaraldehyde induces autofluorescence in the tissue. Therefore solvent fixation is doubly efficient for this reason. It is important to remember that different specimens may require a different fixation method. Fluorescein is sensitive to pH absorption or emission where it’s maximal is at pH 8. The concentration of detergent is another detail that should be adjusted carefully. Commonly used reagents include like X-100 saponin or deoxycholin. Checking for excessive swelling or shrinkage in the tissue is possible. Testing and optimizing for each new sample type will insure that the best balance between preservation and labeling is obtained. like fixation where fine tuning is necessary.

The wells in depression slides are usually too deep to allow focusing above 10X or 20X. Two important reasons for coating. Nonconductive specimens tend to charge when scanned by the electron beam. commonly gold. They are therefore usually coated with an ultrathin coating of electrically-conducting material. 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. Cells cultured on coverslips are ideal since they can be flipped over. In most cases. rubber cement. Coverslips are too thick to be used as spacers for most samples. To prevent compression. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. A slide is usually sealed around the coverslip with nail polish. this causes scanning faults and other image artifacts. even when there is more than enough specimen conductivity to prevent charging. For the same reason. are to maximise signal . 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. it is better not to use too much mountant under the coverslip. deposited on the sample either by low vacuum sputter coating or by high vacuum evaporation. A coverslip placed directly on cells or tissue will compress the sample. 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. The working distance of a lens is the space between the front element of the objective and the top of the coverslip. elevate the coverslip with a spacer as close as possible to the size of the tissue. and especially in secondary electron imaging mode. do not use an aqueous mountant with fixed tissue. This prevents evaporation of the mountant and stabilizes the coverslip for use with immersion lenses.an additive to prevent photobleaching. Thickness of the coverslip is important for optimal image quality. Scanning electron microscope For conventional imaging in the SEM. The coverslip thickness an objective lens is designed for is engraved on the lens. at least at the surface. and electrically grounded to prevent the accumulation of electrostatic charge at the surface. The refractive index of the mountant should be as close to that of the fixed tissue as possible. Pap pens. specimens must be electrically conductive. mounted on the slide and the thickness of the spacer is immaterial.

For SEM. This requires that the primary electron beam's potential and current be tuned to the characteristics of the sample specimen. such as glutaraldehyde. especially for backscattered electron imaging. dried insects or shells can be examined with little further treatment. but living cells and tissues and whole. a specimen is normally required to be completely dry. the electron beam can penetrate several micrometres below the surface. 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. since the specimen chamber is at high vacuum. low-Z material.and improve spatial resolution. and optionally followed by postfixation with osmium tetroxide. dry materials such as wood. this is commonly achieved by critical point drying. feathers. Coating with a high-Z material such as gold maximises secondary electron yield from within a surface layer a few nm thick. 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. Fixation is usually performed by incubation in a solution of a buffered chemical fixative. The fixed tissue is then dehydrated. . 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. that is. 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. material with low atomic number such as carbon. signal increases with atomic number. soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure. 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. The improvement in resolution arises because in low-Z materials. generating signals from an interaction volume much larger than the beam diameter and reducing spatial resolution. sometimes in combination with formaldehyde and other fixatives. 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. Hard. The high pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal. bone. Because air-drying causes collapse and shrinkage. Broadly.

Another technique is negative staining. ribosomes. cryofixation may be used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens. The preparation method reveals the proteins embedded in the lipid bilayer. The technique provides excellent contrast and produces a three dimensional effect.which involves replacement of water in the cells with organic solvents such as ethanol or acetone. Freeze-fracturing. such as viruses. so that no gas-liquid interface is present within the sample during drying. 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. cytoskeletal elements and so on. As a result. 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. drop method and spray method which is applied only when very small volumes of specimen are available. and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide at high pressure. Shadow casting is another technique to visualize isolated particles by casting their shadows. The carbon dioxide is finally removed while in a supercritical state. as SEM is also well suited for examining very small particulate materials. the specimen stands out by its relative brightness on the viewing screen. . 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. The grids containing the specimens are placed in a sealed chamber which is then evacuated by vacuum pump. The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically- conductive double-sided adhesive tape. freeze-etch or freeze-and- break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. Some methods of the negative staining are float method.

lead or gold. the images are seen in a electronic device such as the computer or some other equipments often. the natural colors of the specimens are preserved and maintained but only black and white images are shown in the electron microscopes. The specimens are rarely distorted during sample preparation for light microscopes whereas preparations distort materials in electron microscopy. 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. 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. Stains in the specimens are required sometimes to make the cells visible in the light microscope but in electron microscopes.Differences in all these microscopes Between the light microscope and the electron microscope. 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. During the imaging process of the samples prepared in the light microscopes. there are many procedures which are complicating and it takes quite a long time to prepare the sample specimens. Secondly. The light microscopes are usually small and portable whereas the electron microscopes are large and needs special rooms to be kept. that means the differences between Scanning electron microscope and the others which are the light microscope. many differences can be given. fluorescence microscope and the confocal microscope. 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. the electron beams can damage the specimens so they must be stained with an electron-dense chemical usually heavy-metals like osmium. The sample preparation in the matter is simple and short time duration needed for light microscopes but for electron microscopes. 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. which shows 2 dimensional images of the . The main difference is the resolution of the microscopes where electron microscopes have a much higher resolution than light microscopes. 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.

specimen. a pinhole is present for imaging purpose but this is not present in the fluorescence microscope. Confocal microscopes are larger than fluorescence microscopes as the imaging system will be attached to it normally. Confocal microscopes provide high contrast images of specimens without the image artifacts normally present with conventional contrasting techniques. 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. monochromatic light. an example is for the confocal microscope. Other differences of these microscopes are seen in the structures and the light sources and pointing of the microscopes. well focused. which easily illuminates individual points within a specimen. . that can be assembled in an imaging system to construct a 3 dimensional image but not as natural as under the fluorescence microscope. 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. 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 .

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