Can you assess the importance of the invention of the microscope to science?


Ever since the first microscope was invented in 1590, they have improved our knowledge in basic biology and biomedical research.

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A comparison microscope is a device used to analyze side-by-side specimens. It consists of two microscopes connected by an optical bridge, which results in a split view window enabling two separate objects to be viewed simultaneously. This avoids the observer having to rely on memory when comparing two objects under a conventional microscope. In the 1920s forensic ballistics was waiting at its inception. In 1929, using a comparison microscope adapted for the purpose by Calvin Goddard and his partner Phillip Gravelle used similar techniques to absolve the Chicago Police Department of participation in the St. Valentine's Day Massacre. Philip O. Gravelle, a chemist, developed a comparison microscope for use in the identification of fired bullets and cartridge cases with the support and guidance of forensic ballistics pioneer Calvin Goddard. It was a significant advance in the science of firearms identification in forensic science. The firearm from which a bullet or cartridge case has been fired is identified by the comparison of the unique striae left on the bullet or cartridge case from the worn, machined metal of the barrel, breach block, extractor, or firing pin in the gun. It was Gravelle who mistrusted his memory. "As long as he could inspect only one bullet at a time with his microscope, and had to keep the picture of it in his memory until he placed the comparison bullet under the microscope, scientific precision could not be attained. He therefore developed the comparison microscope and Goddard made it work." Calvin Goddard perfected the comparison microscope and subsequently popularized its use.Sir Sydney Smith also appreciated the idea, emphasizing its importance in forensic science and firearms identification. He took the comparison microscope to Scotland and introduced it to the European scientists for firearms identification and other forensic science needs. The modern instrument has many optical, mechanical and electronic refinements, including fiber optic illumination, video capabilities, digital imaging, automatic exposure for conventional photography, etc. Despite this evolution, however, the basic tools and techniques have remained unchanged which are to determine whether or not ammunition components were fired by a single firearm based on unique and reproducible microscopic and class characteristics, or to reach a "no conclusion" result if insufficient marks are present. Since, ballistic identification has benefited from a long series of structural, scientific and technological advances, law enforcement agencies have established forensic laboratories and researchers have learned much more about how to match bullets and cartridge cases to the guns used to fire them, and comparison microscopes have become more sophisticated. By the end of the 1980s, ballistic identification was an established sub-specialty of forensic science. Visualization tools have also been developed to allows the firearms examiner to verify the degree of similarity between any two tool-marks in question. These are designed to simulate the operation of the comparison microscope but is capable of rendering a 2D view of the 3D surfaces in a manner similar to that of the conventional comparison microscope. The prevalence of hand-gun related crime in the United States compared to most other developed countries provided the impetus for the development of the comparison microscope.][ As with most firearms, the fired ammunition components may acquire sufficient unique and reproducible microscopic marks to be identifiable as having been fired by a single firearm. Making these comparisons is correctly referred to as firearms identification, or sometimes called as "ballistics". Historically, and currently, this forensic discipline ultimately requires a microscopic side-by-side comparison of fired bullets or cartridge cases, one pair at a time, by a forensic examiner to confirm or eliminate the two items as having been fired by a single firearm. For this purpose, the traditional tool of the firearms examiner has been what is often called the ballistics comparison microscope. The interior of a gun's barrel is machined to have grooves (called rifling) that force the bullet to rotate as it travels along it. These grooves and their counterpart, called "lands" imprint groove and land impressions on the surface of the bullet. Together with these land and groove impressions, imperfections on the barrel surface are incidentally transferred to the bullet's surface. Because these imperfections are randomly generated, during manufacture or due to use, they are unique to each barrel. These patterns or imperfections, therefore, amount to a "signature" that each barrel imprints on each of the bullets fired through it. It is this "signature" on the bullets imparted due to the unique imperfections on the barrel that enable the validation and identification of bullets as having originated from a particular gun. Comparison microscope is used to analyze the matching of the microscopic impressions found on the surface of bullets and casings. When a firearm or a bullet or cartridge case are recovered from a crime scene, forensic examiners compare the ballistic fingerprint of the recovered bullet or cartridge case with the ballistic fingerprint of a second bullet or cartridge case test-fired from the recovered firearm. If the ballistic fingerprint on the test-fired bullet or cartridge case matches the ballistic fingerprint on the recovered bullet or cartridge case, investigators know that the recovered bullet or cartridge case was also fired from the recovered gun. A confirmed link between a specific firearm and a bullet or cartridge case recovered from a crime scene constitutes a valuable lead, because investigators may be able to connect the firearm to a person, who may then become either a suspect or a source of information helpful to the investigation. Forensic innovator Calvin Goddard offered ballistic identification evidence in 1921 to help secure convictions of accused murderers and anarchists Nicola Sacco and Bartolomeo Vanzetti. On April 8, 1927, Sacco and Vanzetti were finally sentenced to death in the electric chair. A worldwide outcry arose and Governor Alvin T. Fuller finally agreed to postpone the executions and set up a committee to reconsider the case. By this time, firearms examination had improved considerably, and it was now known that an semi-automatic pistol could be traced by several different methods if both bullet and casing were recovered from the scene. Automatic pistols could now be traced by unique markings of the rifling on the bullet, by firing pin indentations on the fired primer, or by unique ejector and extractor marks on the casing. The committee appointed to review the case used the services of Calvin Goddard in 1927. Goddard used Philip Gravelle's newly invented comparison microscope and helixometer, a hollow, lighted magnifier probe used to inspect gun barrels, to make an examination of Sacco's .32 Colt, the bullet that killed Berardelli, and the spent casings recovered from the scene of the crime. In the presence of one of the defense experts, he fired a bullet from Sacco's gun into a wad of cotton and then put the ejected casing on the comparison microscope next to casings found at the scene. Then he looked at them carefully. The first two casings from the robbery did not match Sacco's gun, but the third one did. Even the defense expert agreed that the two cartridges had been fired from the same gun. The second original defense expert also concurred. The committee upheld the convictions. In October 1961, ballistics tests were run with improved technology using Sacco's Colt automatic. The results confirmed that the bullet that killed the victim, Berardelli in 1920 came from the same .32 Colt Auto taken from the pistol in Sacco's possession. Subsequent investigations in 1983 also supported Goddard's findings. Colonel Goddard was the key forensic expert in solving the 1929 St. Valentine's Day Massacre in which seven gangsters were killed by rival Al Capone mobsters dressed as Chicago police officers. It also led to the establishment of the United States' first independent criminological laboratory, which was located at Northwestern University and headed by Goddard. At this new lab, ballistics, fingerprinting, blood analysis and trace evidence were all brought under one roof. In 1929, using a comparison microscope adapted for the ballistics comparison by his partner, Phillip Gravelle, Goddard used similar techniques to absolve the Chicago Police Department of participation in the St. Valentine's Day Massacre. The case of Sacco and Vanzetti, which took place in Bridgewater, Massachusetts, is responsible for popularizing the use of the comparison microscope for bullet comparison. Forensic expert Calvin Goddard's conclusions were upheld when the evidence was re-examined in 1961.
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the 17th century. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast. Historically optical microscopes were easy to develop and are popular because they use visible light so that samples may be directly observed by eye. The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph. Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images. Purely digital microscopes are now available which use a CCD camera to examine a sample, showing the resulting image directly on a computer screen without the need for eyepieces. Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy. There are two basic configurations of the conventional optical microscope: the simple (single lens) and the compound (many lenses). The vast majority of modern research microscopes are compound microscopes while some cheaper commercial digital microscopes are simple single lens microscopes. A magnifying glass is, in essence, a basic single lens microscope. In general, microscope optics are static; to focus at different focal depths the lens to sample distance is adjusted, and to get a wider or narrower field of view a different magnification objective lens must be used. Most modern research microscopes also have a separate set of optics for illuminating the sample. A simple microscope is a microscope that uses only one lens for magnification, and is the original design of light microscope. Van Leeuwenhoek's microscopes consisted of a small, single converging lens mounted on a brass plate, with a screw mechanism to hold the sample or specimen to be examined. Microscopist Brian Ford has shown images from such basic instruments. Though now considered primitive, the use of a single, convex lens for viewing is still found in simple magnification devices, such as the magnifying glass and the loupe. A compound microscope is a microscope which uses multiple lenses to collect light from the sample and then a separate set of lenses to focus the light into the eye or camera. Compound microscopes are heavier, larger and more expensive than simple microscopes due to the increased number of lenses used in construction. The main advantages of multiple lenses are improved numerical aperture (see resolution limit below), reduced chromatic aberration and exchangeable objective lenses to adjust the magnification. A compound microscope also makes more advanced illumination setups, such as phase contrast possible. 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 17th century. 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 later by fellow Lincean Giovanni Faber. Faber coined the name from the Greek words μικρόν (micron) meaning "small", and σκοπεῖν (skopein) meaning "to look at", a name meant to be analogous with "telescope", another word coined by the Linceans. Christiaan Huygens, another Dutchman, developed a simple 2-lens ocular system in the late 17th century 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. Antonie van Leeuwenhoek (1632–1723) is credited with bringing the microscope to the attention of biologists, even though simple magnifying lenses were already being produced in the 16th century. 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 difficulties in configuring multiple lenses. Still, despite widespread claims, van Leeuwenhoek is not the inventor of the microscope. While basic microscope technology and optics have been available for over 400 years it is much more recently that techniques in sample illumination were developed to generate the high quality images seen today. In August 1893 August Köhler developed Köhler illumination. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination. Before development of Köhler illumination the image of the light source, for example a lightbulb filament, was always visible in the image of the sample. The Nobel Prize in physics was awarded to Dutch physicist Fritz Zernike in 1953 for his development of phase contrast illumination which allows imaging of transparent samples. By using interference rather than absorption of light, extremely transparent samples, such as live mammalian cells, can be imaged without having to use staining techniques. Just two years later, in 1955, Georges Nomarski published the theory for differential interference contrast microscopy, another interference-based imaging technique. Modern biological microscopy depends heavily on the development of fluorescent probes for specific structures within a cell. In contrast to normal transilluminated light microscopy, in fluorescence microscopy the sample is illuminated through the objective lens with a narrow set of wavelengths of light. This light interacts with fluorophores in the sample which then emit light of a longer wavelength. It is this emitted light which makes up the image. Since the mid 20th century chemical fluorescent stains, such as DAPI which binds to DNA, have been used to label specific structures within the cell. More recent developments include immunofluorescence, which uses fluorescently labelled antibodies to recognise specific proteins within a sample, and fluorescent proteins like GFP which a live cell can express making it fluorescent. All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path. In addition, the vast majority of microscopes have the same 'structural' components (numbered below according to the image on the right): The eyepiece, or ocular lens, is a cylinder containing two or more lenses; its function is to bring the image into 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 2×, 50× and 10×. 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. Objective turret, revolver, or revolving nose piece is the part that holds the set of objective lenses. It allows the user to switch between objective lenses. At the lower end of a typical compound optical microscope, there are one or more objective lenses that collect light from the sample. The objective is usually in a cylinder housing containing a glass single or multi-element compound lens. Typically there will be around three objective lenses screwed into a circular nose piece which may be rotated to select the required objective lens. These arrangements are designed to be parfocal, which means that when one changes from one lens to another on a microscope, the sample stays in focus. Microscope objectives are characterized by two parameters, namely, magnification and numerical aperture. The former typically ranges from 5× to 100× while the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm, respectively. Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field in the resulting image. Some high performance objective lenses may require matched eyepieces to deliver the best optical performance. Some microscopes make use of oil-immersion objectives or water-immersion objectives for greater resolution at high magnification. These are used with index-matching material such as immersion oil or water and a matched cover slip between the objective lens and the sample. The refractive index of the index-matching material is higher than air allowing the objective lens to have a larger numerical aperture (greater than 1) so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. Numerical apertures as high as 1.6 can be achieved. The larger numerical aperture allows collection of more light making detailed observation of smaller details possible. An oil immersion lens usually has a magnification of 40 to 100×. Adjustment knobs move the stage up and down with separate adjustment for coarse and fine focusing. The same controls enable the microscope to adjust to specimens of different thickness. In older designs of microscopes, the focus adjustment wheels move the microscope tube up or down relative to the stand and had a fixed stage. The whole of the optical assembly is traditionally attached to a rigid arm, which in turn is attached to a robust U-shaped foot to provide the necessary rigidity. The arm angle may be adjustable to allow the viewing angle to be adjusted. The frame provides a mounting point for various microscope controls. Normally this will include controls for focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled wheel to control fine focus. Other features may be lamp controls and/or controls for adjusting the condenser. The stage is a platform below the objective which supports the specimen being viewed. In the center of the stage is a hole through which light passes to illuminate the specimen. The stage usually has arms to hold slides (rectangular glass plates with typical dimensions of 25×75 mm, on which the specimen is mounted). At magnifications higher than 100× moving a slide by hand is not practical. A mechanical stage, typical of medium and higher priced microscopes, allows tiny movements of the slide via control knobs that reposition the sample/slide as desired. If a microscope did not originally have a mechanical stage it may be possible to add one. All stages move up and down for focus. With a mechanical stage slides move on two horizontal axes for positioning the specimen to examine specimen details. Focusing starts at lower magnification in order to center the specimen by the user on the stage. Moving to a higher magnification requires the stage to be moved higher vertically for re-focus at the higher magnification and may also require slight horizontal specimen position adjustment. Horizontal specimen position adjustments are the reason for having a mechanical stage. Due to the difficulty in preparing specimens and mounting them on slides, for children it's best to begin with prepared slides that are centered and focus easily regardless of the focus level used. Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes, however, have their own adjustable and controllable light source – often a halogen lamp, although illumination using LEDs and lasers are becoming a more common provision. The condenser is a lens designed to focus light from the illumination source onto the sample. The condenser may also include other features, such as a diaphragm and/or filters, to manage the quality and intensity of the illumination. For illumination techniques like dark field, phase contrast and differential interference contrast microscopy additional optical components must be precisely aligned in the light path. The actual power or magnification of a compound optical microscope is the product of the powers of the ocular (eyepiece) and the objective lens. The maximum normal magnifications of the ocular and objective are 10× and 100× respectively, giving a final magnification of 1,000×. When using a camera to capture a micrograph the effective magnification of the image must take into account the size of the image. This is independent of whether it is on a print from a film negative or displayed digitally on a computer screen. In the case of photographic film cameras the calculation is simple; the final magnification is the product of: the objective lens magnification, the camera optics magnification and the enlargement factor of the film print relative to the negative. A typical value of the enlargement factor is around 5× (for the case of 35mm film and a 15x10 cm (6×4 inch) print). In the case of digital cameras the size of the pixels in the CMOS or CCD detector and the size of the pixels on the screen have to be known. The enlargement factor from the detector to the pixels on screen can then be calculated. As with a film camera the final magnification is the product of: the objective lens magnification, the camera optics magnification and the enlargement factor. The optical components of a modern microscope are very complex and for a microscope to work well, the whole optical path has to be very accurately set up and controlled. Despite this, the basic operating principles of a microscope are quite simple. The objective lens is, at its simplest, a very high powered magnifying glass i.e. a lens with a very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen. It is this real image that is viewed by the eyepiece lens that provides further enlargement. In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling the eye to focus on the virtual image. In all microscopes the image is intended to be viewed with the eyes focused at infinity (mind that the position of the eye in the above figure is determined by the eye's focus). Headaches and tired eyes after using a microscope are usually signs that the eye is being forced to focus at a close distance rather than at infinity. Many techniques are available which modify the light path to generate an improved contrast image from a sample. Major techniques for generating increased contrast from the sample include cross-polarized light, dark field, phase contrast and differential interference contrast illumination. A recent technique (Sarfus) combines cross-polarized light and specific contrast-enhanced slides for the visualization of nanometric samples. Bright field illumination, sample contrast comes from absorbance of light in the sample. Cross-polarized light illumination, sample contrast comes from rotation of polarized light through the sample. Dark field illumination, sample contrast comes from light scattered by the sample. Phase contrast illumination, sample contrast comes from interference of different path lengths of light through the sample. Modern microscopes allow more than just observation of transmitted light image of a sample; there are many techniques which can be used to extract other kinds of data. Most of these require additional equipment in addition to a basic compound microscope. Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutic research, mineralogy and microbiology. Optical microscopy is used for medical diagnosis, the field being termed histopathology when dealing with tissues, or in smear tests on free cells or tissue fragments. In industrial use, binocular microscopes are common. Aside from applications needing true depth perception, the use of dual eyepieces reduces eye strain associated with long workdays at a microscopy station. In certain applications, long-working-distance or long-focus microscopes are beneficial. An item may need to be examined behind a window, or industrial subjects may be a hazard to the objective. Such optics resemble telescopes with close-focus capabilities. There are many variants of the basic compound optical microscope design for specialized purposes. Some of these are physical design differences allowing specialization for certain purposes: Other microscope variants are designed for different illumination techniques: A digital microscope is a microscope equipped with a digital camera allowing observation of a sample via a computer. Microscopes can also be partly or wholly computer-controlled with various levels of automation. Digital microscopy allows greater analysis of a microscope image, for example measurements of distances and areas and quantitaton of a fluorescent or histological stain. Low-powered digital microscopes, USB microscopes, are also commercially available. These are essentially webcams with a high-powered macro lens and generally do not use transillumination. The camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor. They offer modest magnifications (up to about 200×) without the need to use eyepieces, and at very low cost. High power illumination is usually provided by an LED source or sources adjacent to the camera lens. At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between two closely spaced Airy disks (or, in other words the ability of the microscope to reveal adjacent structural detail as distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine details. The extent and magnitude of the diffraction patterns are affected by both the wavelength of light (λ), the refractive materials used to manufacture the objective lens and the numerical aperture (NA) of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field, known as the diffraction limit. Assuming that optical aberrations in the whole optical set-up are negligible, the resolution d, can be stated as: Usually a wavelength of 550 nm is assumed, which corresponds to green light. With air as the external medium, the highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable with conventional lenses is about 200 nm. A new type of lens using multiple scattering of light allowed to improve the resolution to below 100 nm. Multiple techniques are available for reaching resolutions higher than the transmitted light limit described above. Techniques for surpassing the resolution limit for bright field microscopy include ultraviolet microscopes, which use shorter wavelengths of light so the diffraction limit is lower. Holographic techniques, as described by Courjon and Bulabois in 1979, are also capable of breaking this resolution limit, although resolution was restricted in their experimental analysis. Using fluorescent samples more techniques are available. Examples include Vertico SMI, near field scanning optical microscopy which uses evanescent waves, and stimulated emission depletion. In 2005, a microscope capable of detecting a single molecule was described as a teaching tool. While most techniques focus on increases in lateral resolution there are also some techniques which aim to allow analysis of extremely thin samples. For example sarfus methods place the thin sample on a contrast-enhancing surface and thereby allows to directly visualize films as thin as 0.3 nanometers. SMI (spatially modulated illumination microscopy) is a light optical process of the so-called point spread function (PSF) engineering. These are processes which modify the PSF of a microscope in a suitable manner to either increase the optical resolution, to maximize the precision of distance measurements of fluorescent objects that are small relative to the wavelength of the illuminating light, or to extract other structural parameters in the nanometer range. SPDM (spectral precision distance microscopy), the basic localization microscopy technology is a light optical process of fluorescence microscopy which allows position, distance and angle measurements on "optically isolated" particles (e.g. molecules) well below the theoretical limit of resolution for light microscopy. "Optically isolated" means that at a given point in time, only a single particle/molecule within a region of a size determined by conventional optical resolution (typically approx. 200–250 nm diameter) is being registered. This is possible when molecules within such a region all carry different spectral markers (e.g. different colors or other usable differences in the light emission of different particles). Many standard fluorescent dyes like GFP, Alexa dyes, Atto dyes, Cy2/Cy3 and fluorescein molecules can be used for localization microscopy, provided certain photo-physical conditions are present. Using this so-called SPDMphymod (physically modifiable fluorophores) technology a single laser wavelength of suitable intensity is sufficient for nanoimaging. 3D super resolution microscopy with standard fluorescent dyes can be achieved by combination of localization microscopy for standard fluorescent dyes SPDMphymod and structured illumination SMI. Stimulated emission depletion is a simple example of how higher resolution surpassing the diffraction limit is possible, but it has major limitations. STED is a fluorescence microscopy technique which uses a combination of light pulses to induce fluorescence in a small sub-population of fluorescent molecules in a sample. Each molecule produces a diffraction-limited spot of light in the image, and the centre of each of these spots corresponds to the location of the molecule. As the number of fluorescing molecules is low the spots of light are unlikely to overlap and therefore can be placed accurately. This process is then repeated many times to generate the image. Stefan Hell of the Max Planck Institute for Biophysical Chemistry was awarded the 10th German Future Prize in 2006 for his development of the STED microscope. In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed which use other waves. The use of electrons and X-rays in place of light allows much higher resolution – the wavelength of the radiation is shorter so the diffraction limit is lower. To make the short-wavelength probe non-destructive, the atomic beam imaging system (atomic nanoscope) has been proposed and widely discussed in the literature, but it is not yet competitive with conventional imaging systems. STM and AFM are scanning probe techniques using a small probe which is scanned over the sample surface. Resolution in these cases is limited by the size of the probe; micromachining techniques can produce probes with tip radii of 5–10 nm. Additionally, methods such as electron or X-ray microscopy use a vacuum or partial vacuum, which limits their use for live and biological samples (with the exception of an environmental scanning electron microscope). The specimen chambers needed for all such instruments also limits sample size, and sample manipulation is more difficult. Color cannot be seen in images made by these methods, so some information is lost. They are however, essential when investigating molecular or atomic effects, such as age hardening in aluminium alloys, or the microstructure of polymers.
An electron microscope (EM) is a type of microscope that uses an electron beam to illuminate a specimen and produce a magnified image. An EM has greater resolving power than a light microscope and can reveal the structure of smaller objects because electrons have wavelengths about 100,000 times shorter than visible light photons. They can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x. The electron microscope uses 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 a light optical 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, the electron microscope is often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs, using specialized digital cameras or frame grabbers to capture the image. The first electromagnetic lens was developed in 1926 by Hans Busch (). 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. The 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 electron microscopy. 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 (TEM) in 1939. Although contemporary electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype. The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to create an image. The electron beam is produced 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 +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed on a monitor or computer. Resolution of the TEM is limited primarily by spherical aberration, but a new generation of aberration correctors have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical aberration for the high-resolution transmission electron microscopy (HRTEM) has allowed the production of images with resolution below 0.5 angstrom (50 picometres) and magnifications above 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development. An important mode of TEM utilization is electron diffraction. The advantages of electron diffraction over X-ray crystallography are that the specimen need not be a single crystal or even a polycrystalline powder, and also that the Fourier transform reconstruction of the object's magnified structure occurs physically and thus avoids the need for solving the phase problem faced by the X-ray crystallographers after obtaining their X-ray diffraction patterns of a single crystal or polycrystalline powder. The major disadvantage of the transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Biological specimens typically require to be chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers and similar materials may require special `staining' with heavy atom labels in order to achieve the required image contrast. 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. The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). When the electron beam interacts with the specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission, which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. The image displayed by an 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. In the SEM image of an ant shown at right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs. Generally, the image resolution of an SEM is about an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimetres in size and (depending on instrument design and settings) has a great depth of field, and so can produce images that are good representations of the three-dimensional shape of the sample. Another advantage of SEM is its variety called environmental scanning electron microscope (ESEM) can produce images of sufficient quality and resolution with the samples being wet or contained in low vacuum or gas. This greatly facilitates imaging biological samples that are unstable in the high vacuum of conventional electron microscopes. The most common configuration for an SEM produces a single value per pixel, with the results usually rendered as black-and-white images. However, often these images are then colorized through the use of feature-detection software, or simply by hand-editing using a graphics editor. This is usually for aesthetic effect or for clarifying structure, and generally does not add information about the specimen. In some configurations more information is gathered per pixel, often by the use of multiple detectors. The attributes of topography and material contrast can be obtained by a pair of backscattered electron detectors and such attributes can be superimposed on a single color image by assigning a different primary color to each attribute. Similarly, a combination of backscattered and secondary electron signals can be assigned to different colors and superimposed on a single color micrograph displaying simultaneously the properties of the specimen. In a similar method, secondary electron and backscattered electron detectors are superimposed and a colour is assigned to each of the images captured by each detector, with an end result of a combined colour image where colours are related to the density of the components. This method is known as Density-dependent colour SEM (DDC-SEM). Micrographs produced by DDC-SEM retain topographical information, which is better captured by the secondary electrons detector and combine it to the information about density, obtained by the backscattered electron detector. Some types of detectors used in SEM have analytical capabilities, and can provide several items of data at each pixel. Examples are the Energy-dispersive X-ray spectroscopy (EDS) detectors used in elemental analysis and Cathodoluminescence microscope (CL) systems that analyse the intensity and spectrum of electron-induced luminescence in (for example) geological specimens. In SEM systems using these detectors it is common to color code the signals and superimpose them in a single color image, so that differences in the distribution of the various components of the specimen can be seen clearly and compared. Optionally, the standard secondary electron image can be merged with the one or more compositional channels, so that the specimen's structure and composition can be compared. Such images can be made while maintaining the full integrity of the original signal, which is not modified in any way. In the 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. This technique is typically coupled with reflection high energy electron diffraction (RHEED) and reflection high-energy loss spectroscopy (RHELS). Another variation is spin-polarized low-energy electron microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains. 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 (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion. Often TEM can be equipped with the scanning option and then it can function both as TEM and STEM. 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: Electron microscopes are expensive to build and maintain, but the capital and running costs of confocal light microscope systems now overlaps with those of basic electron microscopes. Microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field cancelling systems. The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. One exception is the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr or 2.7 kPa) and/or wet environment. Scanning electron microscopes operating in conventional high-vacuum mode usually image conductive specimens; therefore non-conductive materials require conductive coating (gold/palladium alloy, carbon, osmium, etc.) Low-voltage mode of modern microscopes makes possible observation of non-conductive specimens without coating. Non-conductive materials can be imaged also by a variable pressure (or environmental) scanning electron microscope. Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for example) require no special treatment before being examined in the electron microscope. Samples of hydrated materials, including almost all biological specimens have to be prepared in various ways to stabilize them, reduce their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may result in artifacts, but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods. It is generally believed by scientists working in the field that as results from various preparation techniques have been compared and that there is no reason that they should all produce similar artifacts, it is reasonable to believe that electron microscopy features correspond with those of living cells. Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique. Biology and life sciences John H L Watson's recollections at the University of Toronto when he worked with Hillier and Prebus: [1]
A microscope (from the Ancient Greek: , mikrós, "small" and , skopeîn, "to look" or "see") is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope. There are many types of microscopes, the most common and first to be invented is the optical microscope which uses light to image the sample. Other major types of microscopes are the electron microscope (both the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscope. The first microscope to be developed was the optical microscope, although the original inventor is not easy to identify. One legend states the microscope was invented by Roger Bacon, along with the telescope sometime in the 1200's. An early microscope was made in 1590 in Middelburg, Netherlands. Two eyeglass makers are variously given credit: Hans Lippershey (who developed an early telescope) and Zacharias Janssen. Giovanni Faber coined the name microscope for Galileo Galilei's compound microscope in 1625 (Galileo had called it the "occhiolino" or "little eye"). The first detailed account of the interior construction of living tissue based on the use of a microscope did not appear until 1644, in Giambattista Odierna's L'occhio della mosca, or The Fly's Eye. It was not until the 1660s and 1670s that the microscope was used extensively for research in Italy, The Netherlands and England. Marcelo Malpighi in Italy began the analysis of biological structures beginning with the lungs. Robert Hooke's Micrographia had a huge impact, largely because of its impressive illustrations. The greatest contribution came from Antonie van Leeuwenhoek who discovered red blood cells and spermatozoa and helped popularise microscopy as a technique. On 9 October 1676, Van Leeuwenhoek reported the discovery of micro-organisms. In 1893 August Köhler developed a key technique for sample illumination, Köhler illumination, which is central to modern light microscopy. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination. Further developments in sample illumination came from Fritz Zernike in 1953 and George Nomarski 1955 for their development of phase contrast and differential interference contrast illumination which allow imaging of transparent samples. In the early 1900s a significant alternative to light microscopy was developed, using electrons rather than light to generate the image. Ernst Ruska started development of the first electron microscope in 1931 which was the transmission electron microscope (TEM). The transmission electron microscope works on the same principle as 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 a 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. Electron microscopes quickly became popular following the Second World War. Ernst Ruska, working at Siemens developed the first commercial transmission electron microscope and major scientific conferences on electron microscopy started being held in the 1950s. In 1965 the first commercial scanning electron microscope was developed by Professor Sir Charles Oatley and his postgraduate student Gary Stewart and marketed by the Cambridge Instrument Company as the "Stereoscan". The 1980s saw the development of the first scanning probe microscopes. The first was the scanning tunneling microscope in 1981, developed by Gerd Binnig and Heinrich Rohrer. This was closely followed in 1986 with Gerd Binnig, Quate, and Gerber's invention of the atomic force microscope. The most recent developments in light microscope largely centre on the rise of fluorescence microscopy in biology. During the last decades of the 20th century, particularly in the post-genomic era, many techniques for fluorescent labeling of cellular structures were developed. The main groups of techniques are small chemical staining of cellular structures, for example DAPI to label DNA, use of antibodies conjugated to fluorescent reporters, see immunofluorescence, and fluorescent proteins, such as green fluorescent protein. These techniques use these different fluorophores for analysis of cell structure at a molecular level in both live and fixed samples. 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. Much current research (in the early 21st century) on optical microscope techniques is focused on development of superresolution analysis of fluorescently labeled samples. Structured illumination can improve resolution by around two to four times and techniques like stimulated Emission Depletion microscopy are approaching the resolution of electron microscopes. Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the image, i.e., light or photons(optical microscopes), electrons (electron microscopes) or a probe (scanning probe microscopes). Alternatively, microscopes can be classed on whether they analyse the sample via a scanning point (confocal optical microscopes, scanning electron microscopes and scanning probe microscopes) or analyse the sample all at once (wide field optical microscope and transmission electron microscopes). Wide field optical microscopes and transmission electron microscopes use the theory of lenses (optics for light microscopes and electromagnet lenses for electron microscopes) in order to magnify the image generated by the passage of a wave transmitted through the sample, or reflected by the sample. The waves used are electromagnetic (in optical microscopes) or electron beams (in electron microscopes). Resolution in these microscopes is limited by the wavelength of the radiation used to image the sample, where shorter wavelengths allow for a higher resolution. Scanning optical and electron microscopes, like the confocal microscope and scanning electron microscope, use lenses to focus a spot of light or electrons onto the sample then analyze the reflected or transmitted waves. The point is then scanned over the sample to analyze a rectangular region. Magnification of the image is achieved by displaying the data from scanning a physically small sample area on a relatively large screen. These microscopes have the same resolution limit as wide field optical, probe, and electron microscopes. Scanning probe microscopes also analyze a single point in the sample and then scan the probe over a rectangular sample region to build up an image. As these microscopes do not use electromagnetic or electron radiation for imaging they are not subject to the same resolution limit as the optical and electron microscopes described above. The most common type of microscope (and the first invented) is the optical microscope. This is an optical instrument containing one or more lenses producing an enlarged image of a sample placed in the focal plane. Optical microscopes have refractive glass and occasionally of plastic or quartz, to focus light into the eye or another light detector. Mirror-based optical microscopes operate in the same manner. Typical magnification of a light microscope, assuming visible range light, is up to 1500x with a theoretical resolution limit of around 0.2 micrometres or 200 nanometres. Specialized techniques (e.g., scanning confocal microscopy, Vertico SMI) may exceed this magnification but the resolution is diffraction limited. The use of shorter wavelengths of light, such as the ultraviolet, is one way to improve the spatial resolution of the optical microscope, as are devices such as the near-field scanning optical microscope.
Sarfus, a recent optical technique increases the sensitivity of standard optical microscope to a point it becomes possible to directly visualize nanometric films (down to 0.3 nanometre) and isolated nano-objects (down to 2 nm-diameter). The technique is based on the use of non-reflecting substrates for cross-polarized reflected light microscopy. Ultraviolet light enables the resolution of microscopic features, as well as to image samples that are transparent to the eye. Near infrared light can be used to visualize circuitry embedded in bonded silicon devices, since silicon is transparent in this region of wavelengths. In fluorescence microscopy, many wavelengths of light, ranging from the ultraviolet to the visible can be used to cause samples to fluoresce to allow viewing by eye or with the use of specifically sensitive cameras. Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. The use of phase contrast does not require staining to view the slide. This microscope technique made it possible to study the cell cycle in live cells. The traditional optical microscope has more recently evolved into the digital microscope. In addition to, or instead of, directly viewing the object through the eyepieces, a type of sensor similar to those used in a digital camera is used to obtain an image, which is then displayed on a computer monitor. These sensors may use CMOS or charge-coupled device (CCD) technology, depending on the application. Three major variants of electron microscopes exist: Of these techniques AFM and STM are the most commonly used. Scanning acoustic microscopes use sound waves to measure variations in acoustic impedance. Similar to Sonar in principle, they are used for such jobs as detecting defects in the subsurfaces of materials including those found in integrated circuits.
Timeline of microscope technology .

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected and that contain information about the sample's surface topography and composition. The electron beam is generally scanned in a raster scan pattern, and the beam's position is combined with the detected signal to produce an image. SEM can achieve resolution better than 1 nanometer. Specimens can be observed in high vacuum, in low vacuum, and (in environmental SEM) in wet conditions. The most common mode of detection is by secondary electrons emitted by atoms excited by the electron beam. The number of secondary electrons is a function of the angle between the surface and the beam. On a flat surface, the plume of secondary electrons is mostly contained by the sample, but on a tilted surface, the plume is partially exposed and more electrons are emitted. By scanning the sample and detecting the secondary electrons, an image displaying the tilt of the surface is created. An account of the early history of SEM has been presented by McMullan. Although Max Knoll produced a photo with a 50 mm object-field-width showing channeling contrast by the use of an electron beam scanner, it was Manfred von Ardenne who in 1937 invented a true microscope with high magnification by scanning a very small raster with a demagnified and finely focused electron beam. Ardenne applied the scanning principle not only to achieve magnification but also to purposefully eliminate the chromatic aberration otherwise inherent in the electron microscope. He further discussed the various detection modes, possibilities and theory of SEM, together with the construction of the first high magnification instrument of a SEM. Further work was reported by Zworykin's group, followed by the Cambridge groups in the 1950s and early 1960s headed by Charles Oatley, all of which finally led to the marketing of the first commercial instrument by Cambridge Scientific Instrument Company as the "Stereoscan" in 1965 (delivered to DuPont). The types of signals produced by a SEM include secondary electrons (SE), back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence) (CL), specimen current and transmitted electrons. Secondary electron detectors are standard equipment in all SEMs, but it is rare that a single machine would have detectors for all possible signals. The signals result from interactions of the electron beam with atoms at or near the surface of the sample. In the most common or standard detection mode, secondary electron imaging or SEI, the SEM can produce very high-resolution images of a sample surface, revealing details less than 1 nm in size. Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample. This is exemplified by the micrograph of pollen shown above. A wide range of magnifications is possible, from about 10 times (about equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the magnification limit of the best light microscopes. Back-scattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. BSE are often used in analytical SEM along with the spectra made from the characteristic X-rays, because the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen. BSE images can provide information about the distribution of different elements in the sample. For the same reason, BSE imaging can image colloidal gold immuno-labels of 5 or 10 nm diameter, which would otherwise be difficult or impossible to detect in secondary electron images in biological specimens. Characteristic X-rays are emitted when the electron beam removes an inner shell electron from the sample, causing a higher-energy electron to fill the shell and release energy. These characteristic X-rays are used to identify the composition and measure the abundance of elements in the sample. All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub. Several models of SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an object of that size to 45°. For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. They are therefore usually coated with an ultrathin coating of electrically conducting material, deposited on the sample either by low-vacuum sputter coating or by high-vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium, and graphite. Additionally, coating may increase signal/noise ratio for samples of low atomic number (Z). The improvement arises because secondary electron emission for high-Z materials is enhanced. An alternative to coating for some biological samples is to increase the bulk conductivity of the material by impregnation with osmium using variants of the OTO staining method (O-osmium, T-thiocarbohydrazide, O-osmium). Nonconducting specimens may be imaged uncoated using environmental SEM (ESEM) or low-voltage mode of SEM operation. Environmental SEM instruments place the specimen in a relatively high-pressure chamber where the working distance is short and the electron optical column is differentially pumped to keep vacuum adequately low at the electron gun. The high-pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal. Low-voltage SEM is typically conducted in an FEG-SEM because the field emission guns (FEG) is capable of producing high primary electron brightness and small spot size even at low accelerating potentials. Operating conditions to prevent charging of non-conductive specimens must be adjusted such that the incoming beam current was equal to sum of outcoming secondary and backscattered electrons currents. It usually occurs at accelerating voltages of 0.3–4 kV. Embedding in a resin with further polishing to a mirror-like finish can be used for both biological and materials specimens when imaging in backscattered electrons or when doing quantitative X-ray microanalysis. The main preparation techniques are not required in the environmental SEM outlined below, but some biological specimens can benefit from fixation. For SEM, a specimen is normally required to be completely dry, since the specimen chamber is at high vacuum. Hard, dry materials such as wood, bone, feathers, dried insects, or shells can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure. Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives, and optionally followed by postfixation with osmium tetroxide. The fixed tissue is then dehydrated. Because air-drying causes collapse and shrinkage, this is commonly achieved by replacement of water in the cells with organic solvents such as ethanol or acetone, and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide by critical point drying. The carbon dioxide is finally removed while in a supercritical state, so that no gas-liquid interface is present within the sample during drying. The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape, and sputter-coated with gold or gold/palladium alloy before examination in the microscope. If the SEM is equipped with a cold stage for cryo microscopy, cryofixation may be used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens. Cryo-fixed specimens may be cryo-fractured under vacuum in a special apparatus to reveal internal structure, sputter-coated, and transferred onto the SEM cryo-stage while still frozen. Low-temperature scanning electron microscopy is also applicable to the imaging of temperature-sensitive materials such as ice (see e.g. illustration at left) and fats. Freeze-fracturing, freeze-etch or freeze-and-break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The preparation method reveals the proteins embedded in the lipid bilayer. Back scattered electron imaging, quantitative X-ray analysis, and X-ray mapping of specimens often requires that the surfaces be ground and polished to an ultra smooth surface. Specimens that undergo WDS or EDS analysis are often carbon coated. In general, metals are not coated prior to imaging in the SEM because they are conductive and provide their own pathway to ground. Fractography is the study of fractured surfaces that can be done on a light microscope or commonly, on an SEM. The fractured surface is cut to a suitable size, cleaned of any organic residues, and mounted on a specimen holder for viewing in the SEM. Integrated circuits may be cut with a focused ion beam (FIB) or other ion beam milling instrument for viewing in the SEM. The SEM in the first case may be incorporated into the FIB. Metals, geological specimens, and integrated circuits all may also be chemically polished for viewing in the SEM. Special high-resolution coating techniques are required for high-magnification imaging of inorganic thin films. In a typical SEM, an electron beam is thermionically emitted from an electron gun fitted with a tungsten filament cathode. Tungsten is normally used in thermionic electron guns because it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be heated for electron emission, and because of its low cost. Other types of electron emitters include lanthanum hexaboride () cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is upgraded and FEG, which may be of the cold-cathode type using tungsten single crystal emitters or the thermally assisted Schottky type, using emitters of zirconium oxide. The electron beam, which typically has an energy ranging from 0.2 keV to 40 keV, is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column, typically in the final lens, which deflect the beam in the x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface. When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume, which extends from less than 100 nm to around 5 µm into the surface. The size of the interaction volume depends on the electron's landing energy, the atomic number of the specimen and the specimen's density. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the emission of electromagnetic radiation, each of which can be detected by specialized detectors. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifiers of various types are used to amplify the signals, which are displayed as variations in brightness on a computer monitor (or, for vintage models, on a cathode ray tube). Each pixel of computer videomemory is synchronized with the position of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. In older microscopes image may be captured by photography from a high-resolution cathode ray tube, but in modern machines image is saved to a computer data storage. Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude from about 10 to 500,000 times. Unlike optical and transmission electron microscopes, image magnification in the SEM is not a function of the power of the objective lens. SEMs may have condenser and objective lenses, but their function is to focus the beam to a spot, and not to image the specimen. Provided the electron gun can generate a beam with sufficiently small diameter, a SEM could in principle work entirely without condenser or objective lenses, although it might not be very versatile or achieve very high resolution. In a SEM, as in scanning probe microscopy, magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. Assuming that the display screen has a fixed size, higher magnification results from reducing the size of the raster on the specimen, and vice versa. Magnification is therefore controlled by the current supplied to the x, y scanning coils, or the voltage supplied to the x, y deflector plates, and not by objective lens power. The most common configuration for an SEM produces a single value per pixel, with the results usually rendered as black-and-white images. However, often these images are then colorized through the use of feature-detection software, or simply by hand-editing using a graphics editor. This is usually for aesthetic effect or for clarifying structure, and generally does not add information about the specimen. In some configurations more information is gathered per pixel, often by the use of multiple detectors. The attributes of topography and material contrast can be obtained by a pair of backscattered electron detectors and such attributes can be superimposed on a single color image by assigning a different primary color to each attribute. Similarly, a combination of backscattered and secondary electron signals can be assigned to different colors and superimposed on a single color micrograph displaying simultaneously the properties of the specimen. In a similar method, secondary electron and backscattered electron detectors are superimposed and a colour is assigned to each of the images captured by each detector, with an end result of a combined colour image where colours are related to the density of the components. This method is known as Density-dependent colour SEM (DDC-SEM). Micrographs produced by DDC-SEM retain topographical information, which is better captured by the secondary electrons detector and combine it to the information about density, obtained by the backscattered electron detector. Some types of detectors used in SEM have analytical capabilities, and can provide several items of data at each pixel. Examples are the Energy-dispersive X-ray spectroscopy (EDS) detectors used in elemental analysis and Cathodoluminescence microscope (CL) systems that analyse the intensity and spectrum of electron-induced luminescence in (for example) geological specimens. In SEM systems using these detectors it is common to color code the signals and superimpose them in a single color image, so that differences in the distribution of the various components of the specimen can be seen clearly and compared. Optionally, the standard secondary electron image can be merged with the one or more compositional channels, so that the specimen's structure and composition can be compared. Such images can be made while maintaining the full integrity of the original signal, which is not modified in any way. The most common imaging mode collects low-energy (<50 eV) secondary electrons that are ejected from the k-shell of the specimen atoms by inelastic scattering interactions with beam electrons. Due to their low energy, these electrons originate within a few nanometers from the sample surface. The electrons are detected by an Everhart-Thornley detector, which is a type of scintillator-photomultiplier system. The secondary electrons are first collected by attracting them towards an electrically biased grid at about +400 V, and then further accelerated towards a phosphor or scintillator positively biased to about +2,000 V. The accelerated secondary electrons are now sufficiently energetic to cause the scintillator to emit flashes of light (cathodoluminescence), which are conducted to a photomultiplier outside the SEM column via a light pipe and a window in the wall of the specimen chamber. The amplified electrical signal output by the photomultiplier is displayed as a two-dimensional intensity distribution that can be viewed and photographed on an analogue video display, or subjected to analog-to-digital conversion and displayed and saved as a digital image. This process relies on a raster-scanned primary beam. The brightness of the signal depends on the number of secondary electrons reaching the detector. If the beam enters the sample perpendicular to the surface, then the activated region is uniform about the axis of the beam and a certain number of electrons "escape" from within the sample. As the angle of incidence increases, the "escape" distance of one side of the beam will decrease, and more secondary electrons will be emitted. Thus steep surfaces and edges tend to be brighter than flat surfaces, which results in images with a well-defined, three-dimensional appearance. Using the signal of secondary electrons image resolution less than 0.5 nm is possible. Backscattered electrons (BSE) consist of high-energy electrons originating in the electron beam, that are reflected or back-scattered out of the specimen interaction volume by elastic scattering interactions with specimen atoms. Since heavy elements (high atomic number) backscatter electrons more strongly than light elements (low atomic number), and thus appear brighter in the image, BSE are used to detect contrast between areas with different chemical compositions. The Everhart-Thornley detector, which is normally positioned to one side of the specimen, is inefficient for the detection of backscattered electrons because few such electrons are emitted in the solid angle subtended by the detector, and because the positively biased detection grid has little ability to attract the higher energy BSE electrons. Dedicated backscattered electron detectors are positioned above the sample in a "doughnut" type arrangement, concentric with the electron beam, maximizing the solid angle of collection. BSE detectors are usually either of scintillator or of semiconductor types. When all parts of the detector are used to collect electrons symmetrically about the beam, atomic number contrast is produced. However, strong topographic contrast is produced by collecting back-scattered electrons from one side above the specimen using an asymmetrical, directional BSE detector; the resulting contrast appears as illumination of the topography from that side. Semiconductor detectors can be made in radial segments that can be switched in or out to control the type of contrast produced and its directionality. Backscattered electrons can also be used to form an electron backscatter diffraction (EBSD) image that can be used to determine the crystallographic structure of the specimen. The nature of the SEM's probe, energetic electrons, makes it uniquely suited to examining the optical and electronic properties of semiconductor materials. The high-energy electrons from the SEM beam will inject charge carriers into the semiconductor. Thus, beam electrons lose energy by promoting electrons from the valence band into the conduction band, leaving behind holes. In a direct bandgap material, recombination of these electron-hole pairs will result in cathodoluminescence; if the sample contains an internal electric field, such as is present at a p-n junction, the SEM beam injection of carriers will cause electron beam induced current (EBIC) to flow. Cathodoluminescence and EBIC are referred to as "beam-injection" techniques, and are very powerful probes of the optoelectronic behavior of semiconductors, in particular for studying nanoscale features and defects. Cathodoluminescence, the emission of light when atoms excited by high-energy electrons return to their ground state, is analogous to UV-induced fluorescence, and some materials such as zinc sulfide and some fluorescent dyes, exhibit both phenomena. Cathodoluminescence is most commonly experienced in everyday life as the light emission from the inner surface of the cathode ray tube in television sets and computer CRT monitors. In the SEM, CL detectors either collect all light emitted by the specimen or can analyse the wavelengths emitted by the specimen and display an emission spectrum or an image of the distribution of cathodoluminescence emitted by the specimen in real color. X-rays, which are produced by the interaction of electrons with the sample, may also be detected in an SEM equipped for energy-dispersive X-ray spectroscopy or wavelength dispersive X-ray spectroscopy. The spatial resolution of the SEM depends on the size of the electron spot, which in turn depends on both the wavelength of the electrons and the electron-optical system that produces the scanning beam. The resolution is also limited by the size of the interaction volume, or the extent to which the material interacts with the electron beam. The spot size and the interaction volume are both large compared to the distances between atoms, so the resolution of the SEM is not high enough to image individual atoms, as is possible in the shorter wavelength (i.e. higher energy) transmission electron microscope (TEM). The SEM has compensating advantages, though, including the ability to image a comparatively large area of the specimen; the ability to image bulk materials (not just thin films or foils); and the variety of analytical modes available for measuring the composition and properties of the specimen. Depending on the instrument, the resolution can fall somewhere between less than 1 nm and 20 nm. By 2009, The world's highest SEM resolution at high-beam energies (0.4 nm at 30 kV) is obtained with the Hitachi S-5500][. At low-beam energies, the best resolution (by 2009) is achieved by the Magellan XHR system from FEI Company (0.9 nm at 1 kV)][. Conventional SEM requires samples to be imaged under vacuum, because a gas atmosphere rapidly spreads and attenuates electron beams. As a consequence, samples that produce a significant amount of vapour, e.g. wet biological samples or oil-bearing rock, must be either dried or cryogenically frozen. Processes involving phase transitions, such as the drying of adhesives or melting of alloys, liquid transport, chemical reactions, and solid-air-gas systems, in general cannot be observed. Some observations of living insects have been possible, however. The first commercial development of the ESEM in the late 1980s allowed samples to be observed in low-pressure gaseous environments (e.g. 1–50 Torr or 0.1–6.7 kPa) and high relative humidity (up to 100%). 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. The first commercial ESEMs were produced by the ElectroScan Corporation in USA in 1988. ElectroScan was taken over by Philips (who later sold their electron-optics division to FEI Company) in 1996. ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. Uncoated Plastics and Elastomers can be routinely examined, as can uncoated biological samples. Coating can be difficult to reverse, may conceal small features on the surface of the sample and may reduce the value of the results obtained. X-ray analysis is difficult with a coating of a heavy metal, so carbon coatings are routinely used in conventional SEMs, but ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive specimens. ESEM may be the preferred for electron microscopy of unique samples from criminal or civil actions, where forensic analysis may need to be repeated by several different experts. SEMs do not naturally provide 3D images contrary to SPMs. However 3D data can be obtained using a SEM with different methods such as: Possible applications are roughness measurement, measurement of fractal dimension, corrosion measurement and step height evaluation. The following are examples of images taken using an SEM. Colored SEM image of soybean cyst nematode and egg. The artificial coloring makes the image easier for non-specialists to view and understand the structures and surfaces revealed in micrographs. SEM image of a house fly compound eye surface at 450× magnification. Compound eye of Antarctic krill Euphausia superba. Arthropod eyes are a common subject in SEM micrographs due to the depth of focus that an SEM image can capture. Colored picture. Ommatidia of Antarctic krill eye, a higher magnification of the krill's eye. SEMs cover a range from light microscopy up to the magnifications available with a TEM. Colored picture. SEM image of normal circulating human blood. This is an older and noisy micrograph of a common subject for SEM micrographs: red blood cells. SEM image of a hederelloid from the Devonian of Michigan (largest tube diameter is 0.75 mm). The SEM is used extensively for capturing detailed images of micro and macro fossils. Backscattered electron (BSE) image of an antimony-rich region in a fragment of ancient glass. Museums use SEMs for studying valuable artifacts in a nondestructive manner. SEM image of the corrosion layer on the surface of an ancient glass fragment; note the laminar structure of the corrosion layer. SEM image of a photoresist layer used in semiconductor manufacturing taken on a field emission SEM. These SEMs are important in the semiconductor industry for their high-resolution capabilities. SEM image of the surface of a kidney stone showing tetragonal crystals of Weddellite (calcium oxalate dihydrate) emerging from the amorphous central part of the stone. Horizontal length of the picture represents 0.5 mm of the figured original. Two images of the same depth hoar snow crystal, viewed through a light microscope (left) and as a SEM image (right). Note how the SEM image allows for clear perception of the fine structure details which are hard to fully make out in the light microscope image.
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nm can be observed. In cell and molecular biology, a large number of molecular events in cellular surfaces such as cell adhesion, binding of cells by hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often overwhelmed by the background fluorescence due to the much larger population of non-bound molecules. The idea of using total internal reflection to illuminate cells contacting the surface of glass was first described by E.J. Ambrose in 1956. This idea was then extended by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s as TIRFM. A TIRFM uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. The evanescent wave is generated only when the incident light is totally internally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells as shown in the figure above. Note, however, that the region visualised is at least a few hundred nanometers wide, so the cytoplasmic zone immediately beneath the plasma membrane is necessarily visualised in addition to the plasma membrane during TIRF microscopy. The selective visualisation of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution. TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology.

The scientific method is a body of techniques for investigating phenomena, acquiring new knowledge, or correcting and integrating previous knowledge. To be termed scientific, a method of inquiry must be based on empirical and measurable evidence subject to specific principles of reasoning. The Oxford English Dictionary defines the scientific method as: "a method or procedure that has characterized natural science since the 17th century, consisting in systematic observation, measurement, and experiment, and the formulation, testing, and modification of hypotheses."

The chief characteristic which distinguishes the scientific method from other methods of acquiring knowledge is that scientists seek to let reality speak for itself,]discuss[ supporting a theory when a theory's predictions are confirmed and challenging a theory when its predictions prove false. Although procedures vary from one field of inquiry to another, identifiable features distinguish scientific inquiry from other methods of obtaining knowledge. Scientific researchers propose hypotheses as explanations of phenomena, and design experimental studies to test these hypotheses via predictions which can be derived from them. These steps must be repeatable, to guard against mistake or confusion in any particular experimenter. Theories that encompass wider domains of inquiry may bind many independently derived hypotheses together in a coherent, supportive structure. Theories, in turn, may help form new hypotheses or place groups of hypotheses into context.

Biomedical research (or experimental medicine), in general simply known as medical research, is the basic research, applied research, or translational research conducted to aid and support the body of knowledge in the field of medicine. Medical research can be divided into two general categories: the evaluation of new treatments for both safety and efficacy in what are termed clinical trials, and all other research that contributes to the development of new treatments. The latter is termed preclinical research if its goal is specifically to elaborate knowledge for the development of new therapeutic strategies. A new paradigm to biomedical research is being termed translational research, which focuses on iterative feedback loops between the basic and clinical research domains to accelerate knowledge translation from the bedside to the bench, and back again. Medical research may involve doing research on public health, biochemistry, clinical research, microbiology, physiology, oncology, surgery and research on many other non-communicable diseases such as diabetes and cardiovascular diseases.

The increased longevity of humans over the past century can be significantly attributed to advances resulting from medical research. Among the major benefits have been vaccines for measles and polio, insulin treatment for diabetes, classes of antibiotics for treating a host of maladies, medication for high blood pressure, improved treatments for AIDS, statins and other treatments for atherosclerosis, new surgical techniques such as microsurgery, and increasingly successful treatments for cancer. New, beneficial tests and treatments are expected as a result of the Human Genome Project. Many challenges remain, however, including the appearance of antibiotic resistance and the obesity epidemic.

The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast.

The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph. Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images. Purely digital microscopes are now available which use a CCD camera to examine a sample, showing the resulting image directly on a computer screen without the need for eyepieces.

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