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Introduction Cells are small and in almost all situations a microscope is needed to observe them and their subcellular components. In fact the invention of the microscope led to the discovery and description of cells by Hooke in 1655. The microscope is still an extremely important tool in biological research. The light microscope has a limited capability in regards to the size of a particle that can be examined. The electron microscope provides additional resolution that allows for the examination of subcellular structures and even molecules.
History The first person to publish extensive, accurate observations of microorganisms was the amateur Microscopist Antony van Leeuwenhoek (1632–1723) of Delft, The Netherlands . Leeuwenhoek earned his living as a draper and haberdasher (a dealer in men’s clothing and accessories), His microscopes could magnify around 50 to 300 times, and he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45° angle to the specimen plane. Beginning in 1673, Leeuwenhoek sent detailed letters describing his discoveries to the Royal Society of London. It is clear from his descriptions that he saw both bacteria and protozoa. In 1893 August Köhlerdeveloped a key principle of sample illumination, Köhler illumination, which is central to achieving the theoretical limits of resolution for the light microscope. Further developments in sample illumination came from the discovery of phase contrast by Frits Zernikein 1953, and differential contrast microscope illumination by Georges Nomarskiin 1955; both of which allow imaging of unstained, transparent samples.
Simple microscopy A simple microscope is used to obtain small magnifications. It is usually used for study of microscopic algae, fungi and biological specimen. Light from a light source (mirror) passes through a thin transparent object . A biconvex lens magnifies the size of the object to get an enlarged virtual image. The image is viewed from the other side.
Light Microscopy Microbiologists currently employ a variety of light microscopes in their work; bright-field, dark-field, phase-contrast, and fluorescence microscopes are most commonly used. Modern microscopes are all compound microscopes. That is, the magnified image formed by the objective lens is further enlarged by one or more additional lenses.
Light Microscopy • Resolution of 0.2µm • Magnification – objective and projection lens • Resolution • D = 0.61λ/N sin α Resolution is improved by using shorter wavelengths or increasing either N or α.
Application • All branches of biology uses Microscopes especially in Molecular Biology and Histology ( study of cells ) , Microscopes are the backbone of studying biology , The biologists use it to view the details that cannot be seen by the naked eye such as the small parasites and small organisms which is important for the disease control research. • Microscopes are used in viewing the specimens that are relatively very small in size , they are used to view the cellular structures of organs , germs and bacteria , They play a very important role in laboratory for the tissues and organisms which are too small to be seen clearly with the naked eye . • Microscopes has opened up a whole new dimension in science , By using Microscopes scientists were able to discover the existence of the microorganisms , study the structure ofcells , and see the smallest parts of plants , animals , and fungi .
Limitation • Light is a major limitation of a light microscope. More specifically, the wavelength of the light waves that illuminate the specimen limits the resolution. The wavelength of visible light ranges from about 400 to 700 nanometres. The best compound microscopes cannot resolve parts of a specimen that are closer together than about 200 nanometres.
Dark-field microscopy • The Dark-field microscope allows a viewer to observe living, unstained cells and organisms by simply changing the way in which they are illuminated. • A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image . • The field surrounding a specimen appears black, while the object itself is brightly illuminated . • It also is used to identify certain bacteria like the thin and distinctively shaped Treponema pallidumthe causative agent of syphilis.
Volvox and Spirogyra: dark-field microscopy Treponema pallidum : dark-field microscopy
Simple stains . Crystal violet stain of Escherichia coli Methylene blue stain of Corynebacterium
Differential staining Gram stain Purple cells are gram positive. Red cells are gram negative Acid-fast stain Red cells are acid-fast. Blue cells are non-acid-fast.
Electron Microscopy • An instrument that uses a beam of electrons rather than light to generate an image. The German physicist, Ernst Ruska, working with electrical engineer Max Knoll, developed the first prototype electron microscope in 1931 • The transmission electron microscope works on similar principles to an optical microscope but uses electrons in the place of light and electromagnets in the place of glass lenses. Use of electrons, instead of light, allows for much higher resolution. • Development of the transmission electron microscope was quickly followed in 1935 by the development of the scanning electron microscope by Max Knoll. • Transmission electron microscopes became popular following the Second World War. Ernst Ruska, working at Siemens, developed the first commercial transmission electron microscope and, in the 1950s, • The first commercial scanning electron microscope was developed by Professor Sir Charles Oatleyand his postgraduate student Gary Stewart, and marketed by the Cambridge Instrument Company as the "Stereo scan".
Transmission EM • theoretically 0.005 nm; practically 0.1 nm –1 nm (2000x better than LM) • High – velocity electron beam passes through the sample • 50-100 nm thick sections • 2-D sectional image – surface details are revelaed • Subcellular organelles • Scanning EM • Resolution about 10 nm • Secondary electrons released from the metal coated unsectioned specimen • 3-D surface image
The microscope’s inventors, Gerd Binnig and Heinrich Rohrer, shared the 1986 Nobel Prize in Physics for their work, together with Ernst Ruska, the designer of the first transmission electron microscope. • It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail • Fixation with chemicals like glutaraldehyde or osmium tetroxide to stabilize cell structure, the specimen is dehydrated with organic solvents (e.g., acetone or ethanol). • Complete dehydration is essential because most plastics used for embedding are not water soluble. Next the specimen is soaked in unpolymerized, liquid epoxy plastic until it is completely permeated, and then the plastic is hardened to form a solid block. Thin sections are cut from this block with a glass or diamond knife using a special instrument called an ultramicrotome.
Transmission electron microscope • In negative staining, the specimen is spread out in a thin film with either phosphotungstic acid or uranyl acetate. The background dark, whereas the specimen appears bright in photographs. • Negative staining is an excellent way to study the structure of viruses, bacterial gas vacuoles, and other similar objects • In shadowing, a specimen is coated with a thin film of platinum or other heavy metal by evaporation at an angle of about 45° from horizontal so that the metal strikes the microorganism on only one side. In one commonly used imaging method, the area coated with metal appears dark in photographs, whereas the uncoated side and the shadow region created by the object is light . • This technique is particularly useful in studying virus morphology, prokaryotic flagella, and DNA.
Scanning electron microscope • Microorganisms must first be fixed, dehydrated, and dried to preserve surface structure and prevent collapse of the cells when they are exposed to the SEM’s high vacuum. • Before viewing, dried samples are mounted and coated with a thin layer of metal to prevent the build up of an electrical charge on the surface and to give a better image. • To create an image, the SEM scans a narrow, tapered electron beam back and forth over the specimen . When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary electrons. • Secondary electrons entering the detector strike a scintillator causing it to emit light flashes that a photomultiplier converts to an electrical current and amplifies.
Visualize unstained living cells • Phase Contrast microscopy • Thin layers of cells but not thick tissues • Differential Interference contrast • Suited for extremely small details and thick objects • Thin optical section through the object
Phase contrast microscopy • Unpigmented living cells are not clearly visible in the bright field microscope because there is little difference in contrast between the cells and water. • One solution to this problem is to kill and stain cells before observation to increase contrast and create variations in colour between cell structures. A phase-contrast microscope converts slight differences in refractive index and cell density into easily detected variations in light intensity and is an excellent way to observe living cells. • Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial components such as endospores and inclusion bodies that contain poly--hydroxyalkanoates (e.g., poly-hydroxybutyrate), polymetaphosphate, sulphur, or other substances. • Phase contrast microscopes also are widely used in studying eukaryotic cells.
Differential-interference-contrast • Differential-interference-contrast allow objects that differ slightly in refractive index or thickness to be distinguished within unstained or living cells. • Differences in the thickness or refractive index within the specimen result in a differential retardation of light which shifts the phase or deviates the direction of the light. • The interference effects between the incident and diffracted light enhance small differences in the refractive index or thickness of the specimen and leads to an increased resolution without staining.
Fluorescence Microscopy • An object also can be seen because it actually emits light, and this is the basis of fluorescence microscopy. • When some molecules absorb radiant energy, they become excited and later release much of their trapped energy as light. Any light emitted by an excited molecule will have a longer wavelength (or be of lower energy) than the radiation originally absorbed. • Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state. • The fluorescence microscope exposes a specimen to ultraviolet, violet, or blue light and forms an image of the object with the resulting fluorescent light. The most commonly used fluorescence microscopy is epifluorescence microscopy, also called incident light or reflected light fluorescence microscopy.
The most commonly used fluorescence microscopy is epifluorescence microscopy, also called incident light or reflected light fluorescence microscopy • Major Function: Localization of specific cellular molecules – example proteins • Major Advantages: • Sensitivity: "glow” against dark background • Specificity: immunofluorescence • Cells may be fixed or living • Fluorescent dyes or proteins (Flurochromes) • flurochromes may be indirectly or directly associated with the cellular molecule • Multiple flurochromes may be used simultaneously
Absorb light at one wavelength and emit light at a specific and longer wavelength DAPI-4,6diamidino-2-phenylindole GFP-green fluorescent protein FITC-fluorescein isothiocyanate CY3- cyanine dye3 ALEXA568 CY5-cyanine dye 5
Immunofluorescence Microscopy and Specific Proteins • Fluorescently tagged primary anti body • Fluorescently tagged secondary antibody • Fluorescently labeled antibody to tagged proteins such as Myc Myc is a regulator gene that codes for a transcription factor. The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and celluar transformation.
CONFOCAL MICROSCOPY • The rise of fluorescence microscopy drove the development of a major modern microscope design, the confocal microscope. • The principle was patented in 1957 by Marvin Minsky, although laser technology limited practical application of the technique. It was not until 1978 • when Thomas and Christoph Cremer developed the first practical confocal laser scanning microscope and the technique rapidly gained popularity through the 1980s. • This overcomes the limitations of Fluorescence microscopy • Blurred images • Thick specimens
Confocal Microscopy • Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. • It enables the reconstruction of three-dimensional structures from sets of images obtained at different depths (a process known as optical sectioning) within a thick object. • This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science. • Aconfocal microscope only "sees" images one depth level at a time. In effect, the CLSM achieves a controlled and highly limited depth of focus.
Scanning Probe Microscopy • scanning probe microscopes, measure surface features by moving a sharp probe over the object’s surface. • The scanning tunnelling microscope, invented in 1980, is an excellent example of a scanning probe microscope. It can achieve magnifications of 100 million and allow scientists to view atoms on the surface of a solid. • The electrons surrounding surface atoms tunnel or project out from the surface boundary a very short distance. The scanning tunnelling microscope has a needle like probe with a point so sharp that often there is only one atom at its tip. • The probe is lowered toward the specimen surface until its electron cloud just touches that of the surface atoms. If a small voltage is applied between the tip and specimen, electrons flow through a narrow channel in the electron clouds. • This tunnelling current, as it is called, is extraordinarily sensitive to distance and will decrease about a thousand fold if the probe is moved away from the surface by a distance equivalent to the diameter of an atom.
Freeze fracture technique • In the freeze fracturing process, a specimen is frozen rapidly and cracked on a plane through the tissue. This fracture occurs along weak portions of the tissue such as membranes or surfaces of organelles. After cleaving, both surfaces are shadowed with a platinum film. • This coating produces a replica of the surfaces. The replica is then coated with carbon and is then imaged in the transmission electron microscope. • While there are several uses for freeze fracture, one of the most common is to determine the prescence of tight or occluding junctions where membrane glycoproteins bind cells together. • Freeze fracture is the only way to determine the prescence of such junctions. The method is also good for the study of intramembrane structures.
Freeze etching technique • Freeze etching is the sublimation of surface ice under vacuum to reveal details of the fractured face that were originally hidden. • A metal/carbon mix enables the sample to be imaged in a SEM (block-face) or TEM (replica). • It is used to investigate for instance cell organelles, membranes, layers and emulsions. • The technique is traditionally used for biological applications but started to develop significance in physics and material science.
For example: Ice or frozen specimens with a temperature of –120 °C have a saturation pressure of about 10–7 mbar. If this pressure is established in the chamber, condensation and evaporation are in equilibrium. The amount of evaporated molecules is equal to the amount of condensed molecules. At a higher pressure the condensation rate is higher than the sublimation rate – ice crystals grow on the specimen’s surface. This has to be avoided by all means. A colder (than the specimen) plate above the specimen reduces the local pressure and works as a condensation trap. Water molecules driven up from the specimen preferentially attach to the colder surface. At a lower pressure than the saturation pressure more molecules sublimate than condensate and freeze etching takes place. Performing freeze etching until the sample is completely ice free, is called freeze drying. This process only works for small samples to be performed in a reasonable time. It is done in several steps by heating up from around –120 °C to –60 °C maintaining the temperature of each step for a certain time. This can take up to days.
Autoradiography • An autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance. • The film or emulsion is apposed to the labelled tissue section to obtain the autoradiograph (also called an autoradiogram). • Theauto- prefix indicates that the radioactive substance is within the sample, as distinguished from the case of historadiographyor microradiography, in which the sample is X-rayed using an external source. • Some autoradiographs can be examined microscopically for localization of cells or organelles in which the process is termed micro-autoradiography. • For example, micro-autoradiography was used to examine whether atrazine was being metabolized by the hornwortplant or by epiphytic microorganisms in the biofilm layer surrounding the plant.
