Optical Microscope
The optical microscope is an optical instrument with a lens and an eyepiece which allows you to enlarge the image of a small object (which characterizes its magnification ) and to separate the details of this image (and its resolving power ) so it is observable by the human eye. It is used in biology to observe the cells, tissues, petrography to identify rocks, metals and metallography to examine the structure of a metal or alloy.
Do not confuse it with the magnifying binocular that does not require samples of thin plates, or reflective, and can observe natural parts unprepared by magnifying the image by a factor low, but keeping stereoscopic vision conducive to revealing macroscopic grain, nicks, cracks, etc..
History
It is difficult to say who invented the compound microscope. It is often said that the Dutch optician Hans Janssen and his son Zacharias Janssen fabricated the first microscope in 1590, but this is from a declaration by Zacharias Janssen himself in the middle of the seventeenth century. The announced date is rather unlikely since it has been shown that Zacharias Janssen was born about 1590.
Another favorite inventor under the microscope is Galilee. He developed a Occhiolino a compound microscope with a convex lens and concave another in 1609.
A drawing by Francesco Stelluti three bees appears on the seal of Pope Urban VIII (1623-1644) and is considered the first published image of microscopy First Approach The microscope is based on lenses to get an enlarged image of the sample to observe. The object to be observed is placed before the first lens called " objective ". If the object is beyond the focal length, it forms a real inverted image and of different sizes, the image is larger than the object if it is located at a distance less than twice the focal length Objective. The second lens is the eye : it is positioned so that the image is in focus plane. Thus, the eye observes an image "at infinity", so by relaxing the muscles responsible for the accommodation , which is a better visual comfort. This is a diopter focus, partly composed of doublets to correct certain aberrations optics. In contrast to other optical systems that are defined by their optical magnification ( telescope ) or magnification ( camera ), the appropriate term for the microscope, is his power over the angle subtended by the object seen through the instrument, the length of this object. The illumination technique most used in wide-field conventional microscopy is the Khler illumination , which ensures optimal image quality. From bottom to top: The eyepiece can be replaced by a camera, or - in the case of video microscopy - a video camera or a camera CCD to a digital acquisition. This allows for the observation on a video monitor (TV screen type) and ease of use and image processing (printing, data processing, telemedicine , etc.).. The resolution of a microscope means its ability to separate very similar details. Regardless of the sensor and the aberrations and imperfections of lenses, the resolution of optical microscope is fundamentally limited by the diffraction of light. In fact, because of diffraction, the image of a point is not a point but a spot (the Airy disk or more generally the point spread function - PSF). Thus, two separate but related images will be for two spots which may prevent recovery of distinguishing the two image points: the details are no longer resolved. According to the theory of Abbe , the resolution limit (transverse) of a microscope, that is to say the shortest distance below which two neighboring points will no longer be distinguished, can be expressed simply in the using the wavelength of illumination, the refractive index n at the output target, and the half cone angle of maximum light available. where NA is the product n sina or numerical aperture of the lens. We can therefore increase the resolution of two ways: The resolution limit of a conventional light microscope is about 0.2. The transmission electron microscope reach him, a limit 100 times smaller. Microscopy techniques can overcome the Abbe limit. They are sometimes called "super resolution" or nanoscale. These include: When using a conventional microscope, it is used in transmission, that is to say that the light passes through the sample observed. It is also possible to work "in reflection." In this case, the sample is illuminated on the same side as the observer, either through a microscope for the top right and bottom in the case of inverted microscopes used in metallography and crystallography. The light produced by the source passes through a first objective, arrives on the sample, and is reflected back through the objective observation which requires multiple sets of mirrors or prisms. Reflection microscopy allows examination of opaque objects, or too thick for transmission. In return, of course, she can only give information on the sample surface in the case of white light observation, in polarized light, it reveals the orientations of grains of minerals or metal components. A classic case is the metallography where one carries out observations of pieces of metal called micrographs in this way. As stated above the microscope is often reversed, observing the room placed on the plate holder (usually pierced with a circular hole). A contrast of transmitted light (dia - through), the incident illumination (epi - around) can observe objects in opaque colors and giving them a "more natural". The idea of such lighting is old, since 1740 , Descartes inspired Lieberkhm who created for his microscopic observations mirror silver around the lens, the focus of this mirror target preparation. Optical microscopy in bright field (or "bright") is the simplest and oldest techniques of microscopy. The wavelengths used (visible spectrum) limits the resolving power of this microscope to 0.2 microns for those of them who have the best optics. If the sample is illuminated from above, the microscope is "inverted microscope" The objective is then located beneath the preparation, and the focuser tube straightens the beams of light to the eye are "normally" positioned to the user. The dark field microscope that uses the principle of "dark-field microscopy" improves the contrast of transparent samples but not colored . The Rheinberg illumination is a variant of the dark-field illumination in which transparent color filters are inserted just before the condenser so that light rays are more or less oblique colored differently (the bottom of the image can be blue while the sample appears bright yellow). The resolution limit is the same as the dark field. Other color combinations are possible, but their effectiveness is quite variable . The dark-field microscopy is particularly suitable for fresh samples and allows microcinematography (eg bacteria moving.) She has no interest in colored objects (or sections stained smears). It is particularly useful for: The use of oblique illumination (per side) gives a three dimensional appearance and can highlight aspects invisible otherwise. This is the main advantage. The limitations are the same as the bright-field microscopy. In polarized light microscopy, the sample is placed between a polarizer and an analyzer to detect changes in light polarization after crossing the sample. This technique is very useful for observing the environment birefringent , including mineralogy. When certain compounds are illuminated by a light source of high energy, they then emit light at lower energy. This is the phenomenon of fluorescence. Fluorescence microscopy is to form an image by collecting the emitted light. This method is now of primary importance in the life sciences. It can be very sensitive, allowing detection of even single molecules. We use several fluorescent dyes to mark different structures or chemical compounds. This allows simultaneous detection of different compounds, while differentiating by color fluorescence. Phase contrast is a widely used technique that helps to highlight the differences in refractive index as difference in contrast. It was developed by the Dutch physicist Frederik Zernike in the 1930s (he received the Nobel Prize for it in 1953). The nucleus of a cell for example will appear dark in the surrounding cytoplasm. The contrast is excellent, however this technique can be used with thick objects. Often, a halo forms around small objects that can drown details. The system consists of a circular ring in the condenser which produces a cone of light. This cone is superimposed on a ring of similar size in the lens. Each goal has a ring of different size, so it is necessary to adjust the condenser lens at each change. The ring in the objective has special optical properties: it reduces the intensity of direct light and, more importantly, it creates an artificial phase difference of a quarter of a wavelength that causes interference with scattered light, which creates the image contrast. Interference contrast (IC, IC for English) is a technique used to visualize transparent objects by increasing their contrast. CI is currently under Nomarski invented in the 1950s is most prevalent. This technique provides greater contrast compared to the phase by eliminating the halo phenomenon peculiar to him. She won in microscopy in many areas today. The confocal microscope produces an image in a manner totally different from the normal bright field microscopy. The resolution is slightly better, but the most important point is that it can form an image of cross sections without being disturbed by the light outside the focal plane. It thus gives a clear picture of objects in three dimensions. The confocal microscope is often used in conjunction with fluorescence microscopy. The observed sample must meet certain conditions: In biology, it is necessary first to place the tissue section (or the liquid containing living organisms) between a slide and a glass slide. The goal is to approach the blade without the development, by mistake, destroying the preparation became very fragile. Because of the preparation, optical microscopy requires a significant amount of additional equipment for the sole destination for microscopic observation. Take the case of biopsy in Medicine and Biology ( Pathology ): diagnosis by microscopy, biological parts taken by biopsy during surgery, short deadlines. To prepare the plate, using a device called cryotome , a sort of "ham slicer, placed in a cryostat (freezer), which can cut very thin slices of the body that will be observed by cooling it rapidly, then the cutting using a razor blade special sharpened on another machine with a plate of glass using diamond pastes. If you want to work at room temperature, the delays are longer and require replacement of dehydration and water removed by paraffin (24 hours) for the sample retains its stiffness, then it is colored by several substances Shares of alternating very long time, too. Principle of optical microscope base
Constitution of the microscope
Limitations of optical microscope
Optical Microscopy super-resolved
Use and development of optical microscope
Reflection Microscopy
The incident light
Bright field microscopy
The illumination is done by transmission of white light, that is to say that the sample is illuminated from below and observed from above. The limitations of this technique are mainly low contrast of most biological samples and resolution due to the low blur created by matter outside the focal plane. In return, the technique is simple and the sample requires only minimal preparation. dark field microscopy
The dark field illumination light source uses a carefully aligned to minimize the amount of light transmitted directly and collect only the light scattered by the sample. It greatly increases the contrast, especially for transparent samples, while requiring little equipment and a simple sample preparation. However, this technique suffers from low light intensity collected and is still affected by the resolution limit. Oblique Illumination
polarized light microscopy
Fluorescence Microscopy
The phase-contrast microscope
The interference contrast microscope
The confocal microscope
The inverted microscope stand
Sample preparation
References
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