GK Answer Explanations
Negative staining is a technique used in electron microscopy
is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A transmission electron microscope can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.
Transmission electron microscopes use electrostatic and electromagnetic lenses to control the electron beam and focus it to form an image. These electron optical lenses are analogous to the glass lenses of an optical light microscope.
Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the image.
The first electromagnetic lens was developed in 1926 by
According to Dennis Gabor, the physicist Leó Szilárd tried in 1928 to convince Busch to build an electron microscope, for which he had filed a patent.
German physicist Ernst Ruska and the electrical engineer Max Knoll constructed the prototype electron microscope in 1931, capable of four-hundred-power magnification; the apparatus was the first demonstration of the principles of
Two years later, in 1933, Ruska built an electron microscope that exceeded the resolution attainable with an optical (light) microscope. Moreover, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained the patent for the electron microscope in May 1931.
In 1932, Ernst Lubcke of Siemens & Halske built and obtained images from a prototype electron microscope, applying concepts described in the Rudenberg patent applications. Five years later (1937), the firm financed the work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska (Ernst’s brother) to develop applications for the microscope, especially with biological specimens. Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope. The first practical electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus; and Siemens produced the first commercial transmission electron microscope (
) in 1939. Although contemporary transmission electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype
Materials to be viewed under an electron microscope may require processing to produce a suitable sample.
The technique required varies depending on the specimen and the analysis required:
Chemical fixation – for biological specimens aims to stabilize the specimen's mobile macromolecular structure by chemical crosslinking of proteins with aldehydes such as formaldehyde and glutaraldehyde, and lipids with osmium tetroxide.
Negative stain – suspensions containing nanoparticles or fine biological material (such as viruses and bacteria) are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or phosphotungstic acid. This mixture is applied to a suitably coated EM grid, blotted, then allowed to dry. Viewing of this preparation in the TEM should be carried out without delay for best results. The method is important in microbiology for fast but crude morphological identification, but can also be used as the basis for high resolution 3D reconstruction using EM tomography methodology when carbon films are used for support.
Negative staining is also used for observation of nanoparticles.
Cryofixation – freezing a specimen so rapidly, in liquid ethane, and maintained at liquid nitrogen or even liquid helium temperatures, so that the water forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its solution state. An entire field called cryo-electron microscopy has branched from this technique. With the development of cryo-electron microscopy of vitreous sections (CEMOVIS), it is now possible to observe samples from virtually any biological specimen close to its native state.
Dehydration – or replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. Also freeze drying.
Embedding, biological specimens – after dehydration, tissue for observation in the transmission electron microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 'transition solvent' such as propylene oxide (epoxypropane) or acetone and then infiltrated with an epoxy resin such as Araldite, Epon, or Durcupan; tissues may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerized (hardened) the sample is thin sectioned (ultrathin sections) and stained – it is then ready for viewing.
Embedding, materials – after embedding in resin, the specimen is usually ground and polished to a mirror-like finish using ultra-fine abrasives. The polishing process must be performed carefully to minimize scratches and other polishing artifacts that reduce image quality.
Metal shadowing – Metal (e.g. platinum) is evaporated from an overhead electrode and applied to the surface of a biological sample at an angle. The surface topography results in variations in the thickness of the metal that are seen as variations in brightness and contrast in the electron
– A surface shadowed with metal (e.g. platinum, or a mixture of carbon and platinum) at an angle is coated with pure carbon evaporated from carbon electrodes at right angles to the surface. This is followed by removal of the specimen material (e.g. in an acid bath, using enzymes or by mechanical separation) to produce a surface replica that records the surface ultrastructure and can be examined using transmission electron microscopy.
Sectioning – produces thin slices of specimen, semitransparent to electrons. These can be cut on an ultramicrotome with a diamond knife to produce ultra-thin sections about 60–90 nm thick. Disposable glass knives are also used because they can be made in the lab and are much cheaper.
Staining – uses heavy metals such as lead, uranium or tungsten to scatter imaging electrons and thus give contrast between different structures, since many (
) materials are nearly "transparent" to electrons (weak phase objects). In biology, specimens can be stained "en bloc" before embedding and also later after sectioning. Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl acetate followed by aqueous lead citrate.
Freeze-fracture or freeze-etch – a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixation), then fractured by breaking or by using a microtome while maintained at liquid nitrogen temperature. The cold fractured surface (sometimes "etched" by increasing the temperature to about −100 °C for several minutes to let some ice sublime) is then shadowed with evaporated platinum or gold at an average angle of 45° in a high vacuum evaporator. A second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve stability of the replica coating. The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The still-floating replica is thoroughly washed free from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM.
Ion beam milling – thins samples until they are transparent to electrons by firing ions (typically argon) at the surface from an angle and sputtering material from the surface. A subclass of this is focused ion beam milling, where gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing prior to SEM analysis of materials that are difficult to prepare using mechanical polishing.
Conductive coating – an ultrathin coating of electrically conducting material, deposited either by high vacuum evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static electric fields at the specimen due to the electron irradiation required during imaging. The coating materials include gold, gold/palladium, platinum, tungsten, graphite, etc.
Earthing – to avoid electrical charge accumulation on a conductive coated sample, it is usually electrically connected to the metal sample holder. Often an electrically conductive adhesive is used for this purpose.
Q.) Negative staining is a technique used in
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