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| | Hello! <br>I'm Hindi male :D. <br>I really love Computer programming!<br><br>my weblog; tumaternidad.com, [http://www.tumaternidad.com/foro/categories/embarazo.42 link home], |
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| {{Infobox laboratory equipment
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| |name = Microscope
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| |image = Optical microscope nikon alphaphot +.jpg|200px
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| |uses = Small sample observation
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| |notable_experiments = Discovery of [[Cell (biology)|cell]]s
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| |inventor = [[Hans Lippershey]]<br/>[[Zacharias Janssen]]
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| |related = [[Microscope]]<br>[[Electron microscope]]
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| }}
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| [[File:Microscope And Digital Camera.JPG|thumb|300px|right|A modern microscope with a [[mercury bulb]] for [[fluorescence microscopy]]. The microscope has a [[digital camera]], and is attached to a [[computer]].]]
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| The '''optical microscope''', often referred to as the "'''light microscope'''", is a type of [[microscope]] which uses [[Visible spectrum|visible light]] and a system of [[lens (optics)|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 [[Optical resolution|resolution]] and sample [[contrast (vision)|contrast]].
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| 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 image]]s. Purely [[digital microscope]]s 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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| Alternatives to optical microscopy which do not use visible light include [[scanning electron microscopy]] and [[transmission electron microscopy]].
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| ==Optical configurations==
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| [[File:Microscope simple diagram.png|thumb|right|150px|Diagram of a simple microscope]]
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| There are two basic configurations of the conventional optical microscope: the simple microscope and the compound microscope. The vast majority of modern [[research]] microscopes are compound microscopes while some cheaper commercial [[digital microscope]]s are simple single lens microscopes. A [[magnifying glass]] is, in essence, a single lens simple 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.
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| ===Simple microscope===
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| A '''simple microscope''' is a microscope that uses a lens or set of lenses to enlarge an object through angular magnification alone, giving the viewer an erect enlarged [[virtual image]].<ref>[http://www.msnucleus.org/membership/html/jh/biological/microscopes/lesson2/microscopes2c.html msnucleus.org MICROSCOPES Lesson 2 - Page 3, CLASSIFICATION OF MICROSCOPES]</ref><ref>[http://books.google.com/books?id=NKh9dQKnTdEC&pg=PA213&dq=eyepiece+simple++microscope&hl=en&sa=X&ei=575vUqDaDvLA4AOS-YH4CA&ved=0CGYQ6AEwBzgK#v=onepage&q=eyepiece%20simple%20%20microscope&f=false The IIT Foundation Series - Physics Class 8, 2/e By Trishna Knowledge Systems, page 213]</ref> Simple microscopes are not capable of high magnification. The use of a single convex lens or groups of lenses are still found in simple magnification devices such as the [[magnifying glass]], [[loupe]]s, and [[eyepiece]]s for telescopes and microscopes.
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| ===Compound microscope===
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| [[File:Microscope compound diagram.png|thumb|left|150px|Diagram of a compound microscope]]
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| A '''compound microscope''' is a microscope which uses a lens close to the object being viewed to collect light (called the [[Objective (optics)|objective]] lens) which focuses a [[real image]] of the object inside the microscope (image 1). That image is then magnified by a second lens or group of lenses (called the [[eyepiece]]) that gives the viewer an enlarged inverted virtual image of the object (image 2).<ref>[http://books.google.com/books?id=Y6-sE4gUX-QC&pg=PA6&dq=microscope+simple+compound+virtual&hl=en&sa=X&ei=YbtvUrO7F5fi4AO-soCYBw&ved=0CGYQ6AEwBjgU#v=onepage&q=microscope%20simple%20compound%20virtual&f=false Ian M. Watt, The Principles and Practice of Electron Microscopy, page 6]</ref> The use of a compound objective/eyepiece combination allows for much higher magnification, 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.
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| ==History==
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| {{See also|History of optics|Timeline of microscope technology}}
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| ===Invention===
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| It is difficult to say who invented the compound microscope. The [[Dutch people|Dutch]] spectacle-maker [[Zacharias Janssen]] is sometimes claimed to have invented it in 1590 (a claim made by his son and fellow countrymen, in different testimony in 1634 and 1655)<ref>Albert Van Helden, Sven Dupre, Rob Van Gent, The Origins of the Telescope - 2011, page 43</ref><ref>Brian Shmaefsky, Biotechnology 101 - 2006, page 171</ref><ref>Note: stories vary, including Zacharias Janssen had the help of his father Hans Janssen (or sometimes said to have been built entirely by his father). Zacharias' probable birth date of 1585 (The Origins of the Telescope, page 28) makes it unlikely he invented it in 1590 and the claim of invention is based on the testimony of Zacharias Janssen's son, Johannes Zachariassen, who may have fabricated the whole story (The Origins of the Telescope, page 43).</ref> Another claim is that Janssen's competitor, [[Hans Lippershey]], invented the compound microscope. 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 language|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.<ref>[http://brunelleschi.imss.fi.it/esplora/microscopio/dswmedia/risorse/testi_completi.pdf "Il microscopio di Galileo"], Instituto e Museo di Storia della Scienza (in Italian)</ref>
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| [[Christiaan Huygens]], another Dutchman, developed a simple 2-lens ocular system in the late 17th century that was [[Achromatic lens|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.
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| ===Popularization===
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| [[File:Stelluti bees1630.jpg|thumb|right|The oldest published image known to have been made with a microscope: bees by [[Francesco Stelluti]], 1630<ref>"''The Lying stones of Marrakech''", by Stephen Jay Gould, 2000</ref>]]
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| [[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 simple microscopes, with a single very small, 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.
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| ===Lighting techniques===
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| 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.
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| 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.
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| 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 (wave propagation)|interference]] rather than [[Absorption (electromagnetic radiation)|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 (wave propagation)|interference]]-based imaging technique.
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| ===Fluorescence microscopy===
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| Modern biological microscopy depends heavily on the development of [[fluorescent]] [[Hybridization probe|probe]]s 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.
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| 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 [[Green fluorescent protein|GFP]] which a live cell can [[protein expression|express]] making it fluorescent.
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| ==Components==
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| [[File:Optical microscope nikon alphaphot.jpg|thumb|right|300px|Basic optical transmission microscope elements (1990s)]]
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| 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<ref>http://www.microscope.com/education-center/how-to-guides/how-to-use-a-compound-microscope/</ref> (numbered below according to the image on the right):
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| * Eyepiece (ocular lens) (1)
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| * Objective turret, revolver, or revolving nose piece (to hold multiple objective lenses) (2)
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| * [[objective (optics)|Objective lenses]] (3)
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| * Focus knobs (to move the stage)
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| ** Coarse adjustment (4)
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| ** Fine adjustment (5)
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| * Stage (to hold the specimen) (6)
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| * Light source (a [[light]] or a [[mirror]]) (7)
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| * Diaphragm and [[condenser (microscope)|condenser]] (8)
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| * Mechanical stage (9)
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| ===Eyepiece (ocular lens)===
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| {{Main|Eyepiece}}
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| 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 [[apochromat]]ic objectives.
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| ===Objective turret (revolver or revolving nose piece)===
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| 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.
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| ===Objective===
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| {{Main|Objective (optics)}}
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| At the lower end of a typical compound optical microscope, there are one or more [[objective lens]]es 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 lens|parfocal]], which means that when one changes from one lens to another on a microscope, the sample stays in [[focus (optics)|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 length]]s 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.
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| ====Oil immersion objective====
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| [[File:Leica EpifluorescenceMicroscope ObjectiveLens.jpg|thumb|right|300px|Two Leica [[oil immersion]] microscope objective lenses: 100× (left) and 40× (right)]]
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| {{Main|Oil immersion}}
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| Some microscopes make use of [[oil-immersion objective]]s 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.<ref>{{cite web |url=http://www.olympusmicro.com/primer/anatomy/objectives.html |title=Microscope objectives |work=Olympus Microscopy Resource Center |first=Spring |last=Kenneth |coauthors=Keller, H. Ernst; Davidson, Michael W. |accessdate=29 Oct 2008}}</ref> 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×.
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| ===Focus knobs===
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| 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.
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| ===Frame===
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| 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.
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| 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.
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| ===Stage===
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| 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 [[Microscope slide|slides]] (rectangular glass plates with typical dimensions of 25×75 mm, on which the specimen is mounted).
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| 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.
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| 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.
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| 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.
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| 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.
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| ===Light source===
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| 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 [[LED]]s and [[laser]]s are becoming a more common provision.
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| ===Condenser===
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| The [[condenser (microscope)|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 (optics)|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.
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| ==Magnification==
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| 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×.
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| ===Magnification and micrographs===
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| 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]].
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| 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).
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| In the case of digital cameras the size of the pixels in the [[CMOS]] or [[Charge-coupled device|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.
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| ==Operation==
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| [[File:Microscope-optical path.svg|right|thumb|400px|Optical path in a typical microscope]]
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| 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.
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| 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.
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| 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.
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| 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.<ref>Stephen G. Lipson, Ariel Lipson, Henry Lipson, ''Optical Physics 4th Edition'', Cambridge University Press, ISBN 978-0-521-49345-1</ref>
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| 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 [[:media:Microscope diag.svg|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.
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| ===Illumination techniques===
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| {{main|Microscopy}}
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| Many techniques are available which modify the light path to generate an improved [[contrast (vision)|contrast]] image from a sample. Major techniques for generating increased contrast from the sample include [[Polarized light microscopy|cross-polarized light]], [[dark field]], [[phase contrast]] and [[differential interference contrast]] illumination. A recent technique ([[Sarfus]]) combines [[Polarized light microscopy|cross-polarized light]] and specific contrast-enhanced slides for the visualization of nanometric samples.
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| <gallery caption="Four examples of transilumination techniques used to generate contrast in a sample of [[tissue paper]]. 1.559 μm/pixel." align="center">
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| File:Paper Micrograph Bright.png|[[Bright field microscopy|Bright field]] illumination, sample contrast comes from [[absorbance]] of light in the sample.
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| File:Paper Micrograph Cross-Polarised.png|[[Polarized light microscopy|Cross-polarized light]] illumination, sample contrast comes from rotation of [[polarized]] light through the sample.
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| File:Paper Micrograph Dark.png|[[Dark field]] illumination, sample contrast comes from light [[scattered radiation|scattered]] by the sample.
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| File:Paper Micrograph Phase.png|[[Phase contrast]] illumination, sample contrast comes from [[Interference (wave propagation)|interference]] of different path lengths of light through the sample.
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| </gallery>
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| ===Other techniques===
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| 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.
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| *Reflected light, or incident, illumination (for analysis of surface structures)
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| *Fluorescence microscopy, both:
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| :*[[Epifluorescence microscopy]]
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| :*[[Confocal microscopy]]
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| *[[Ultraviolet–visible spectroscopy|Microspectroscopy]] (where a UV-visible spectrophotometer is integrated with an optical microscope)
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| *Ultraviolet microscopy
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| *Near-Infrared microscopy
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| *Multiple transmission microscopy<ref>N. C. Pégard and J. W. Fleischer, [http://www.opticsinfobase.org/abstract.cfm?uri=CLEO:%20S%20and%20I-2011-CThW6 "Contrast Enhancement by Multi-Pass Phase-Conjugation Microscopy,"] CLEO:2011, paper CThW6 (2011).</ref> for contrast enhancement and aberration reduction.
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| *Automation (for automatic scanning of a large sample or image capture)
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| ==Applications==
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| Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutic research, mineralogy and microbiology.<ref>[http://www.fy.chalmers.se/microscopy/students/imagecourse/O1.pdf O1 Optical Microscopy] By Katarina Logg. Chalmers Dept. Applied Physics. 2006-01-20</ref>
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| Optical microscopy is used for [[medical diagnosis]], the field being termed [[histopathology]] when dealing with tissues, or in [[smear test]]s on free cells or tissue fragments.
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| In industrial use, binocular microscopes are common. Aside from applications needing true [[Stereo microscope|depth perception]], the use of dual eyepieces reduces [[Asthenopia|eye strain]] associated with long workdays at a microscopy station. In certain applications, long-working-distance or long-focus microscopes<ref name="macrolensdb">{{cite web | url=http://www.macrolenses.de/ml_detail_sl.php?ObjektiveNr=315 | title=Long-focus microscope with camera adapter}}</ref> are beneficial. An item may need to be examined behind a [[Optical window|window]], or industrial subjects may be a hazard to the objective. Such optics resemble telescopes with close-focus capabilities.<ref name="questar">{{cite web | url=http://company7.com/questar/microscope.html/ | title=Questar Maksutov microscope}}</ref><ref name="fta">{{cite web | url=http://www.firsttenangstroms.com/accessories/longrangemicroscope/LongRangeMicroscope.html/ | title=FTA long-focus microscope}}</ref>
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| ==Optical microscope variants==
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| {{Main|Microscopy}}
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| {{see also|Stereo microscope|Comparison microscope|Confocal microscope|USB microscope|Digital microscope}}
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| 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:
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| * [[Stereo microscope]], a low powered microscope which provides a stereoscopic view of the sample, commonly used for dissection.
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| * [[Comparison microscope]], which has two separate light paths allowing direct comparison of two samples via one image in each eye.
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| * [[Inverted microscope]], for studying samples from below; useful for cell cultures in liquid, or for metallography.
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| * [[Student microscope]], designed for low cost, durability, and ease of use.
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| * Fiber optic connector inspection microscope, designed for connector end-face inspection
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| Other microscope variants are designed for different illumination techniques:
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| * [[Petrographic microscope]], whose design usually includes a polarizing filter, rotating stage and gypsum plate to facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation.
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| * [[Polarizing microscope]], similar to the petrographic microscope.
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| * [[Phase contrast microscope]], which applies the phase contrast illumination method.
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| * [[Epifluorescence microscope]], designed for analysis of samples which include fluorophores.
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| * [[Confocal microscope]], a widely used variant of epifluorescent illumination which uses a scanning laser to illuminate a sample for fluorescence.
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| ===Digital microscope===
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| [[File:2008Computex DnI Award AnMo Dino-Lite Digital Microscope.jpg|thumb|right|200px|A miniature [[USB microscope]].]]
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| {{Main|Digital microscope}}
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| 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 [[histology|histological]] stain.
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| {{Main|USB microscope}}
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| Low-powered digital microscopes, [[USB microscope]]s, are also commercially available. These are essentially [[webcam]]s 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.
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| ==Limitations==
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| At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by [[diffraction]] rings. These are called [[Airy disk]]s. 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-limited system|diffraction limit]]. Assuming that optical aberrations in the whole optical set-up are negligible, the resolution ''d'', can be stated as:
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| :<math>d = \frac { \lambda } { 2 NA }</math>
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| Usually a wavelength of 550 nm is assumed, which corresponds to [[green]] light. With [[Earth's atmosphere|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.<ref name="fb">{{cite journal|author=E.G. van Putten, D. Akbulut, J. Bertolotti, W.L. Vos, A. Lagendijk, and A.P. Mosk|arxiv=1103.3643|year=2011|doi=10.1103/PhysRevLett.106.193905|title=Scattering Lens Resolves Sub-100 nm Structures with Visible Light|journal=Physical Review Letters|volume=106|issue=19}}</ref>
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| ===Surpassing the resolution limit===
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| 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.<ref>{{cite journal|title=Real Time Holographic Microscopy Using a Peculiar Holographic Illuminating System and a Rotary Shearing Interferometer|author=D. Courjon and J. Bulabois|year=1979|volume=10|issue=3|journal=Journal of Optics}}</ref>
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| Using fluorescent samples more techniques are available. Examples include [[Vertico SMI]], [[near field scanning optical microscopy]] which uses [[evanescent waves]], and [[STED microscope|stimulated emission depletion]]. In 2005, a microscope capable of detecting a single molecule was described as a teaching tool.<ref>{{cite web|title = Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope|url = http://chemeducator.org/bibs/0010004/1040269mk.htm|accessdate = February 25, 2009}}</ref>
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| Despite significant progress in the last decade, techniques for surpassing the diffraction limit remain limited and specialized.
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| 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.
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| ====Structured illumination SMI====
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| 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.<ref>{{cite journal|doi=10.1117/12.336833|title=Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating|year=1999|last1=Heintzmann|first1=Rainer|volume=3568|pages=185–196}}</ref><ref>{{US patent|7342717}} Christoph Cremer, Michael Hausmann, Joachim Bradl, Bernhard Schneider ''Wave field microscope with detection point spread function'', priority date 10 July 1997</ref>
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| ====Localization microscopy SPDMphymod====
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| 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).<ref>{{cite journal|author=Lemmer P ''et al.''|doi=10.1007/s00340-008-3152-x|title=SPDM: light microscopy with single-molecule resolution at the nanoscale|year=2008|journal=Applied Physics B|volume=93|pages=1–12}}</ref><ref>{{cite journal|doi=10.1117/12.260797|title=Comparative study of three-dimensional localization accuracy in conventional, confocal laser scanning and axial tomographic fluorescence light microscopy|year=1996|last1=Bradl|first1=Joachim|volume=2926|pages=201–206}}</ref><ref>{{cite journal|author=R. Heintzmann, H. Münch, C. Cremer |year=1997|title=High-precision measurements in epifluorescent microscopy – simulation and experiment|journal=Cell Vision |volume=4|pages=252–253}}</ref><ref>{{US patent|6424421}} Christoph Cremer, Michael Hausmann, Joachim Bradl, Bernd Rinke ''Method and devices for measuring distances between object structures'', priority date 23 December 1996</ref>
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| [[File:3D Dual Color Super Resolution Microscopy Cremer 2010.png|thumb|600px|3D Dual Color Super Resolution Microscopy Cremer 2010.png|3D dual color super resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568 LIMON]]
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| Many standard fluorescent dyes like [[Green fluorescent protein|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.<ref>{{cite journal|author=Manuel Gunkel ''et al.''|pmid=19548231|year=2009|title=Dual color localization microscopy of cellular nanostructures|volume=4|issue=6|pages=927–38|doi=10.1002/biot.200900005|journal=Biotechnology journal}}</ref>
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| ====3D super resolution microscopy====
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| 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.<ref>{{cite journal|author=Rainer Kaufmann ''et al.''|doi=10.1111/j.1365-2818.2010.03436.x|title=Analysis of Her2/neu membrane protein clusters in different types of breast cancer cells using localization microscopy|year=2011|journal=Journal of Microscopy|volume=242|pages=46–54|pmid=21118230|issue=1}}</ref>
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| ====STED====
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| [[STED microscope|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.<ref>{{cite web|url = http://www.heise.de/english/newsticker/news/81528|title = German Future Prize for crossing Abbe's Limit|accessdate = February 24, 2009}}</ref>
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| ==Alternatives==
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| In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed which use other waves.
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| *[[Atomic force microscope]] (AFM)
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| *[[Scanning electron microscope]] (SEM)
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| *[[Scanning ion-conductance microscopy]] (SICM)
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| *[[Scanning tunneling microscope]] (STM)
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| *[[Transmission electron microscopy]] (TEM)
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| *Ultraviolet microscope
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| *[[X-ray microscope]]
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| 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
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| 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.
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| 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.
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| 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 alloy]]s, or the [[microstructure]] of [[polymers]].
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| ==See also==
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| {{colbegin|2}}
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| *[[Digital microscope]]
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| *[[Köhler illumination]]
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| *[[Microscope slide]]
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| {{colend}}
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| ==References==
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| {{reflist|30em}}
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| ==Further reading==
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| *"Metallographic and Materialographic Specimen Preparation, Light Microscopy, Image Analysis and Hardness Testing", Kay Geels in collaboration with Struers A/S, ASTM International 2006.
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| ==External links==
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| *[http://www.antique-microscopes.com Antique Microscopes.com] A collection of early microscopes
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| *[http://www.musoptin.com/mikro1.html Historical microscopes], an illustrated collection with more than 3000 photos of scientific microscopes by European makers {{de icon}}
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| *[http://golubcollection.berkeley.edu The Golub Collection], A collection of 17th through 19th Century microscopes, including extensive descriptions
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| *[http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html ''Molecular Expressions''], concepts in optical microscopy
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| *[http://www.doitpoms.ac.uk/tlplib/optical-microscopy/index.php Online tutorial of practical optical microscopy]
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| *[http://openwetware.org/wiki/Microscopy OpenWetWare]
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| *[http://ccdb.ucsd.edu/sand/main?stype=lite&keyword=light%20microscopy&Submit=Go&event=display&start=1 Cell Centered Database]
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| *[http://www.juliantrubin.com/bigten/leeuwenhoek_microscope.html Antonie van Leeuwenhoek: Father of Microscopy and Microbiology]
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| {{DEFAULTSORT:Optical Microscope}}
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| [[Category:Microscopy]]
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| [[Category:Dutch inventions]]
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| [[hu:Fénymikroszkóp]]
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