Optical microscopy – is the technical field of studying objects using optical microscopes. Optical microscopes are devices that use visible light rays as both eye works in the optical wavelength range. So, optical microscopes could not have diffraction limits resolution less than 0.2 micrometers (200 nanometers) and with maximum magnification up to 2000 times. The human eye is a biological optical system, characterized by a certain resolution - the smallest distance between the elements of the observed object, while they can still be distinguished from one another. For a normal eye, at a distance from the object to the so-called. best vision distance (D = 250 mm) the average normal resolution is 0.176 mm. Dimensions of microorganisms, most of the plant and animal cells, small crystals parts microstructure of metals and alloys, etc. are significantly less than this value. Various types of microscopes designed for the observation and study of such objects. Using microscopes we can determine shape, size, structure, and many other characteristics of microscopic objects.
It is believed that the Dutch ophthalmologist Hans Jansen and his son Zachary Jansen invented the first microscope in 1590, but it was a statement of the Zacharias Jansen in the middle of the XVII century. Another contender for the title of inventor of the microscope was Galileo Galilei. In 1609, he developed “occhiolino” - compound optical microscope with convex and concave lenses. Galileo introduced his microscope to the public in the Accademia dei Lincei, founded by Federico Cesi. Image of three bees Francesco Stelluti was the part of Pope Urban’s VIII seal, and it is considered the first published microscopic symbol. In late 1600s, Christiaan Huygens, another Dutchman invented a simple two-lens eyepiece system, which achromatically regulated and, therefore, was a huge step forward in the history of microscopes. Huygens eyepieces are made to this day, but the location of eyepieces is inconvenient compared to modern eyepieces.
Optical microscope system consists of the two basic elements - the objective and eyepiece. They fixed in a movable tube, which is located on the metal base on which located the object table. Magnification of an optical microscope without additional lenses between the lens and the eyepiece is the product of their magnifications. Modern microscopes almost always have lighting system (in particular, the condenser with iris diaphragm), macro-and micro-screws to adjust the sharpness, and position control system condenser. Depending on the purpose of the research in specialized microscopes, can be used more devices and systems.
Microscope objective is a complex optical system that forms an enlarged image of the object, and is the primary and most critical part of the microscope. Lens creates an image that is viewed through the eyepiece. Eyepieces can produce a substantial magnification so the optical distortions introduced by the lens and the eyepiece magnification will increase. It imposes on the quality of the lens greater demands than on the eyepiece. Lenses of biological microscopes and other microscopes (except stereoscopic) are largely standardized and interchangeable. In first instance mechanical (connecting) lens options affect interchangeability.
In 1858, Royal Microscopical Society standardized screw threads for objectives, lenses, and related nosepieces (ISO 8038). Nowadays, this thread used in virtually all microscopes, except stereo microscopes or special microscopes. Thread diameter for microscopes is 4/5 "(about 20 mm). One more thing that effects interchangeability of lenses is parfocal distance - the distance between the specimen plane and the shoulder of the flange of the in the microscope. Shoulder of the flange is part of a microscope that the objective lens is supported. Most modern microscopes are designed for lenses with a 45mm parfocal distance. Previously widely used lenses for 33 mm. With the increasing complexity of the optical scheme began to appear large lenses with large parfocal distance (for example, 60 mm and 95 mm). Free working distance from the lens to the object under study is called the working distance of the lens. Generally perfocal distance as smaller as bigger the lens magnification. Working distance of the lens and lens length are parfocal length of the lens.
Optical microscopes can be characterized into different categories and types. Let’s analyze how optical microscopes differ from each other. First type characterized by the number of eyepieces. There are monocular, binocular and trinocular microscopes. The image formed by the lens can be directed to the eyepiece, or divided into a plurality of identical images. Microscopes without division called monocular, they look with one eye. Ease of observation with two eyes predetermined widespread of binocular microscopes with two identical eyepieces. In addition, the microscope can be equipped with photographic equipment, which can be mounted either instead of the standard eyepiece or in a separate optical port. These microscopes are called Trinocular.
Some microscopes allow to illuminate the object through the lens of the microscope. In this case, are used a special lens that will also serve light condenser. In the optical path of the microscope set the semi-transparent mirror and the light source port. More often, this mechanism is used for lighting fluorescent microscopy in the ultraviolet.
Stereo microscopes are designed for craftsmanship under the microscope, such as watchmaking, microelectronics, micro modeling, neurosurgery, etc. Stereoscopes are the best for such work to properly evaluate the situation of the observed objects under a microscope in three dimensions, which requires stereo vision, a large depth of sharpness and significant space under the lens for the job. Stereo microscopes have low magnification (in few or ten times) and long working distance lens (the distance from the optical to the observation point, usually a few centimeters). Also, they don’t have built-in adjustable tables and lighting systems. For the convenience stereomicroscope doesn’t turn the image. Also, lens stereomicroscopes are often replaceable.
One more type of optical microscopes is metallographic microscope. Specificity of metallographic research is the necessity to observe the surface structure of opaque objects. Therefore, microscope built under the scheme of the reflected light where there is a special illuminator mounted on the lens side. The system of prisms and mirrors directs the light to the object, after that the light reflected from the opaque object and sent back into the lens. Modern metallographic microscopes are characterized by a large distance between the surface of the table and large lenses that allow you to work with larger samples. Maximum distance can reach tens of centimeters.
Another type of an optical microscope is polarizing microscope. The principle of the polarizing microscope is to get an image of the object when it is irradiated with polarized beams, which in turn must be obtained from conventional light using a special device - a polarizer. When polarized light passes through a material (either by reflection) it changes the polarization plane of light, so in the second polarizing filter change detected in excessive darkening.
There are many types of optical microscopy: bright-field microscopy, dark field microscopy, fluorescent microscopy, differential interference contrast microscopy and phase contrast microscopy. The operating principle of fluorescent microscopy based on the properties of fluorescent radiation. Microscopes are used to study transparent and opaque objects. Fluorescent light is reflected differently by different surfaces and materials that can successfully apply it for immunochemistry, immunological, and immunogenetic studies. Bright-field microscopy or light microscopy - a type of optical (light) microscopy, where the visualization of investigated object based on the selective absorption of elements of its structure with light of different wavelengths. Dark field microscopy - a method of image obtaining in optical and electron microscopy, in which the lens gets only scattered beam, and the reference beam is cut off intense. As a result, images of samples obtained light on a dark field. Cutting of the primary reference beam increases the image contrast.
Phase contrast microscopy – is a method of image acquisition in optical microscopes, where the phase shift of the electromagnetic wave transforms into contrast of intensity. To obtain phase contrast image light from the source splits into two coherent light beams, that have different optical paths, one of these beams is called a reference, another objective. Confocal microscopy uses a special method of optical mapping to increase optical resolution and contrast of the recording device. This method uses spotlights and diaphragm with miniature aperture, which set in front of the detector to remove scattered out of focus light. Confocal microscope allows to reconstruct the image on the basis of the three-dimensional structure of the sample. This method has gained wide recognition in the scientific and industrial community and is used for verification of semiconductors, biology and spintronics. Differential interference contrast microscopy is optical microscopy that used to create contrast in unstained transparent specimens. Differential interference contrast microscope determines the optical density of an object based on principle of interference and thus inaccessible to the eye to see the details. The image is similar to phase contrast, but there are no diffraction halos.
References
Abramowitz M, Davidson MW. (2007). Introduction to Microscopy. Retrieved from Molecular Expressions.: http://micro.magnet.fsu.edu/primer/anatomy/introduction.html
Pawley, J. (2006). Handbook of Biological Confocal Microscopy. New York: Springer.
Pluta, M. (1988). Advanced Light Microscopy, Vol. 1: Principles and Basic Properties. Amsterdam: Elsevier Science Ltd.
Ruzin, S. E. (1999). Plant Microtechnique and Microscopy. New York: Oxford University Press.
Zernike, F. (1955, 03 11). How I Discovered Phase Contrast. Science, pp. 345-349.
