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MICROSCOPY Microscopy is the technical field of using microscopes to view samples or objects. There are three well-known branches of microscopy, optical, electron and scanning probe microscopy. Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beam interacting with the subject of study, and the subsequent collection of this scattered radiation in order to build up an image. The optical microscope uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the microscopes. New designs of digital microscopes are now available which use a CCD camera to examine a sample and the image is shown directly on a computer screen without the need for expensive optics such as eye-pieces. Chromatic aberration Chromatic aberration is caused by a lens having a different refractive index for different wavelengths of light (the dispersion of the lens). Since the focal length “ f ” of a lens is dependent on the refractive index “ n ”, different wavelengths of light will be focused on different positions. Numerical aperture Numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. Where ‟ n „ is the index of refraction of the medium in which the lens is working and θ is the half-angle of the maximum cone of light that can enter or exit the lens. Refractive Index 1.0 for air, 1.33 for pure water, and up to 1.56 for oils NA is important because it indicates the resolving power of a lens. The size of the finest detail that can be resolved is proportional to λ / NA, where λ is the wavelength of the light. WAVELENGTH Wavelength is the distance between repeating units of a propagating wave of a given frequency. It is commonly designated by the Greek letter lambda (λ). Alens with a larger numerical aperture will be able to visualize finer details than a lens with a smaller numerical aperture. Lenses with larger numerical apertures also collect more light and will generally provide a brighter image. Optical resolution Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged. The ability of a lens to resolve detail is usually determined by the quality of the lens but is ultimately limited by diffraction The resolution of a microscope is defined as the minimum separation needed between two objects under examination in order for the microscope to discern them as separate objects. Thisminimum distance is labeled δ. If two objects are separated by a distance shorter than δ, they will appear as a single object in the microscope. DEPTH OF FIELD Depth of field (DOF) is the portion of a scene that appears sharp in the image. The DOF is determined by the subject distance (that is, the distance to the plane that is perfectly in focus), the lens focal length, and the lens f- number (relative aperture). Magnification Magnification is the process of enlarging something only in appearance, not in physical size. Magnification is also a number describing by which factor an object was magnified. When this number is less than one it refers to a reduction in size, sometimes called Minification. Real image Areal image is a representation of an actual object (source) formed by rays of light passing through the image. Ifa screen is placed in the plane of a real image, the image will generally become visible. Real images can be produced by concave mirrors and converging lenses. Virtual image A virtual image is an image in which the outgoing rays from a point on the object never actually intersect at a point. A simple example is a flat mirror where the image of oneself is perceived at twice the distance from yourself to the mirror. That is, if you are half a meter in front of the mirror, your image will appear at a distance of half a meter inside or behind the mirror. Oil Immersion Objective oilimmersion is a technique used to increase the resolution of a microscope. Thisis achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens. The refractive indices of the oil and of the glass in the first lens element are nearly the same, which means that the refraction of light will be small upon entering the lens In addition to improving resolution. The use of oil is also advantageous in that it reduces the reflective losses as light enters the lens. Cedar wood Oil is used in Oil immersion. Stereo microscope The stereo or dissecting microscope is designed differently , and serves a different purpose. Ituses two separate optical paths with two objectives and two eyepieces to provide slightly different viewing angles to the left and right eyes. this way it produces a three- In dimensional visualization of the sample being examined Stereo microscope The stereo microscope is often used to study the surfaces of solid specimens or to carry out close work such as sorting, dissection, microsurgery, watch-making, small circuit board manufacture or inspection, etc. Unlike compound microscopes, illumination in a stereo microscope most often uses reflected (episcopic) illumination rather than transmitted (diascopic) illumination, that is, light reflected from the surface of an object rather than light transmitted through an object. Digital Microscope Low power microscopy is also possible with digital microscopes, with a camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor. Oftencalled "USB" microscopes, they offer high magnifications (up to about 200×) without the need to use eyepieces, and at very low cost. Digital microscope A digital microscope uses optics and a charge-coupled device (CCD) camera to output a digital image to a monitor. A digital microscope differs from an optical microscope in that there is no provision to observe the sample directly through an eyepiece. Sincethe optical image is projected directly on the CCD camera, the entire system is designed for the monitor image Digital microscope Resolution of the image is dependent on the CCD used in the camera. Using a typical 2 Megapixel CCD, an image with 1600 x 1200 pixels is generated. The resolution of the image is dependent on the field of view of the lens used with the camera. The approximate pixel resolution can be determined by dividing the horizontal field of view (FOV) by 1600. Most common instruments have a relatively low resolution of 1.3 Megapixels, but higher resolution cameras are available. The images can be recorded and stored in the normal way on the computer. The camera is usually fitted with a light source, although extra sources (such as a fibre- optic light) can be used to highlight features of interest in the object. They also offer a large depth of field, a great advantage at high magnifications. Electron microscope The electron microscope uses a particle beam of electrons to illuminate a specimen and create a highly-magnified image. Electron microscopes have much greater resolving power than light microscopes and can obtain much higher magnifications of up to 2 million times, while the best light microscopes are limited to magnifications of 2000 times. First EM - Ruska 1933 EM New Version EM Image - Pollen EM Image- Ant Head EM Image- Cell Transmission Electron Microscope (TEM) The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to create an image. The electrons are emitted by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses. The Electron beam is then transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam Scanning Electron Microscope (SEM) Unlike the TEM, where electrons of the high voltage beam carry the image of the specimen, the electron beam of the Scanning Electron Microscope (SEM) does not at any time carry a complete image of the specimen. TheSEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (Raster scanning). Ateach point on the specimen the incident electron beam loses some energy, and that lost energy is converted into other forms, such as heat, emission of low- energy secondary electrons, light emission (cathodoluminescence) or x-ray emission. The display of the SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. SEM Image – Insect coated with Gold Reflection Electron Microscope (REM) Inthe Reflection Electron Microscope (REM) as in the TEM, an electron beam is incident on a surface, but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is detected. Scanning Transmission Electron Microscope (STEM) The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action occur before the electrons hit the specimen in the STEM, but afterward in the TEM Sample preparation in EM Chemical Fixation for biological specimens aims to stabilize the specimen's mobile macromolecular structure by chemical cross linking of proteins with aldehydes such as formaldehyde and glutaraldehyde, and lipids with osmium tetroxide. Cryofixation When freezing a specimen so rapidly, to liquid nitrogen or even liquid helium temperatures, the water forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its solution state. Dehydration Freeze drying, or replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. Embedding, biological specimens dehydration, tissue for After 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 epoxy propane and then infiltrated with a resin such as Araldite epoxy resin; tissues may also be embedded directly in water- miscible acrylic resin. Afterthe resin has been polymerized (hardened) the sample is thin sectioned (ultra thin sections) and stained - it is then ready for viewing. Sectioning Produces thin slices of specimen, semitransparent to electrons. These can be cut on an ultra microtome with a diamond knife to produce ultra thin slices about 60-90nm 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 (especially biological) materials are nearly "transparent" to electrons (weak phase objects). Typicallythin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl acetate followed by aqueous lead citrate. Negative Staining Negative staining is usually done with heavy metal salts commonly derived from molybdenum, uranium, or tungsten. Heavy ions are used since they will readily interact with the electron beam and produce phase contrast. Asmall drop of the sample is deposited on the carbon coated grid, allowed to settle for approximately one minute, blotted dry if necessary, and then covered with a small drop of the stain (for example 2% uranyl acetate). Aftera few seconds, this drop is also blotted dry, and the sample is ready for viewing. 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 (cryofixed), then fractured by simply 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 Conductive Coating Anultra thin 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. Such coatings include gold, gold/palladium, platinum, tungsten, graphite etc. and are especially important for the study of specimens with the scanning electron microscope. ESEM 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. Positivelycharged ions generated by beam interactions with the gas help to neutralize the negative charge on the specimen surface. Environmental Scanning Electron Microscope is used to study Wet and Oily specimens Fluorescence microscopy The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence or phosphorescence. FLUORESCENCE MICROSCOPE FLUORESCENCE MICROSCOPE- WORKING Endothelial cells under the fluorescent microscope Yeast cell membrane The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as Phosphorescence Fluorescence microscopy is a rapid expanding technique, both in the medical and biological sciences. The technique has made it possible to identify cells and cellular components with a high degree of specificity. Forexample, certain antibodies and disease conditions or impurities in inorganic material can be studied with the fluorescence microscopy. Autofluorescence A variety of specimens exhibit autofluorescence ( emission of visible light) when they are irradiated Incontrast, the study of animal tissues and pathogens is often complicated with either extremely faint or bright, nonspecific autofluorescence. Forthe latter studies, fluorochromes are added (also termed fluorophores), which are excited by specific wavelengths of irradiating light and emit light of defined and useful intensity Fluorophores It is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. 1. Energy is absorbed by the atom which becomes excited. 2. The electron jumps to a higher energy level. 3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing Confocal microscopy Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three- dimensional images by using a spatial pinhole to eliminate out-of- focus light Confocal Microscope Confocal Microscope Image Confocal Microscope Ray path 3D Confocal Microscope Ray path Confocal Microscope Image Basic concept The principle of confocal imaging was patented by Marvin Minsky in 1957. In a conventional fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a photodetector or a camera. 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 information. Confocal laser scanning microscopy (CLSM or LSCM) Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images. The key feature of confocal microscopy is its ability to produce in-focus images of thick specimens, a process known as optical sectioning. Images are acquired point-by- point and reconstructed with a computer, allowing three- dimensional reconstructions of topologically-complex objects. Image formation Ina Confocal laser scanning microscope, a laser beam passes through a light source aperture and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens. A beam splitter separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. Afterpassing a pinhole, the fluorescent light is detected by a photodetection device (a photomultiplier tube (PMT) or avalanche photodiode), transforming the light signal into an electrical one that is recorded by a computer. Atomic de Broglie microscope TheAtomic de Broglie microscope is an imaging system which is expected to provide resolution at the nanometer scale using neutral He atoms as probe particles. Such a device could provide the resolution at nanometer scale and be absolutely non- destructive, but it is not developed so well as optical microscope or an electron microscope. Atomic Force Microscope Atomic Force Microscope Atomic Force Microscopy - Image Dark field microscopy Darkfield microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample. Dark field Microscopy - Image Bright field Microscopy - Image Infrared microscopy The term infrared microscope covers two main types of diffraction-limited microscopy. The first provides optical visualization plus IR spectroscopic data collection. The second (more recent and more advanced) technique employs focal plane array detection for infrared chemical imaging, where the image contrast is determined by the response of individual sample regions to particular IR wavelengths selected by the user. Scanning probe microscopy This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope (AFM), the Scanning tunneling microscope and the photonic force microscope. Allsuch methods imply a solid probe tip in the vicinity (near field) of an object, which is supposed to be almost flat. Scanning tunneling microscopy (STM ) Scanning tunneling microscopy (STM) is a powerful technique for viewing surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer (at IBM Zürich), the Nobel Prize in Physics in 1986 STM probes the density of states of a material using tunneling current. For STM, good resolution is considered to be 0.1 nm lateral resolution and 0.01 nm depth resolution. The STM can be used not only in ultra high vacuum but also in air and various other liquid or gas ambients, and at temperatures ranging from near zero kelvin to a few hundred degrees Celsius The STM is based on the concept of quantum tunneling. When a conducting tip is brought very near to a metallic or semi conducting surface, a bias between the two can allow electrons to tunnel through the vacuum between them Scanning tunneling microscopy (STM ) Scanning Probe Microscopy (SPM) Itis a type of microscopy that forms images of surfaces using a physical probe that scans the specimen. An image of the surface is obtained by mechanically moving the probe in a raster scan of the specimen, line by line, and recording the probe- surface interaction as a function of position. PROBE FORCE MICROSCOPY Scanning voltage microscopy (SVM) It is also called Nanopotentiometry. It is a scientific experimental technique based on atomic force microscopy. Aconductive probe, usually only a few nanometers wide at the tip, is placed in full contact with an operational electronic or optoelectronic sample. Byconnecting the probe to a high-impedance voltmeter and rastering over the sample's surface, a map of the electric potential can be acquired. SVM is generally nondestructive to the sample although some damage may occur to the sample or the probe if the pressure required to maintain good electrical contact is too high. Ifthe input impedance of the voltmeter is sufficiently large, the SVM probe should not perturb the operation of the operational sample. SVM is particularly well suited to analyzing microelectronic devices (such as transistors or diodes) or quantum electronic devices (such as quantum well diode lasers) directly because nanometer spatial resolution is possible. SVM can also be used to verify theoretical simulation of complex electronic devices. Ultrasonic force Microscopy UltrasonicForce Microscopy (UFM) has been developed in order to improve the details and image contrast on "flat" areas of interest where the AFM images are limited in contrast. The combination of AFM-UFM allows a near field acoustic microscopic image to be generated. The AFM tip is used to detect the ultrasonic waves and overcomes the limitation of wavelength that occurs in acoustic microscopy. By using the elastic changes under the AFM tip, an image of much greater detail than the AFM topography can be generated. Stimulated Emission Depletion microscopy, or STED Stimulated Emission Depletion microscopy, or STED microscopy, is a technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction with standard confocal laser scanning microscopes and conventional far-field optical microscopes NMR MICROSCOPY Phase contrast Microscope - Image Phase contrast Microscope - Image Digital Pathology (virtual microscopy) Digital Pathology is an image- based information environment enabled by computer technology that allows for the management of information generated from a digital slide. Digitalpathology is enabled in part by virtual microscopy, which is the practice of converting glass slides into digital slides that can be viewed, managed, and analyzed.