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A microscope () is a
laboratory instrument A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
used to examine objects that are too small to be seen by the naked eye.
Microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of mi ...
is the
science Science is a systematic endeavor that Scientific method, builds and organizes knowledge in the form of Testability, testable explanations and predictions about the universe. Science may be as old as the human species, and some of the earli ...
of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope. There are many types of microscopes, and they may be grouped in different ways. One way is to describe the method an instrument uses to interact with a sample and produce images, either by sending a beam of
light Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 te ...
or
electron The electron (, or in nuclear reactions) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary partic ...
s through a sample in its optical path, by detecting photon emissions from a sample, or by scanning across and a short distance from the surface of a sample using a probe. The most common microscope (and the first to be invented) is the
optical microscope The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of micros ...
, which uses
lens A lens is a transmissive optical device which focuses or disperses a light beam by means of refraction. A simple lens consists of a single piece of transparent material, while a compound lens consists of several simple lenses (''elements'' ...
es to refract visible light that passed through a thinly sectioned sample to produce an observable image. Other major types of microscopes are the fluorescence microscope,
electron microscope An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a ...
(both the transmission electron microscope and the scanning electron microscope) and various types of scanning probe microscopes.


History

Although objects resembling lenses date back 4,000 years and there are
Greek Greek may refer to: Greece Anything of, from, or related to Greece, a country in Southern Europe: *Greeks, an ethnic group. *Greek language, a branch of the Indo-European language family. **Proto-Greek language, the assumed last common ancestor ...
accounts of the optical properties of water-filled spheres (5th century BC) followed by many centuries of writings on optics, the earliest known use of simple microscopes ( magnifying glasses) dates back to the widespread use of lenses in
eyeglasses Glasses, also known as eyeglasses or spectacles, are vision eyewear, with lenses (clear or tinted) mounted in a frame that holds them in front of a person's eyes, typically utilizing a bridge over the nose and hinged arms (known as temples or ...
in the 13th century. The earliest known examples of compound microscopes, which combine an objective lens near the specimen with an eyepiece to view a real image, appeared in Europe around 1620. The inventor is unknown, even though many claims have been made over the years. Several revolve around the spectacle-making centers in the
Netherlands ) , anthem = ( en, "William of Nassau") , image_map = , map_caption = , subdivision_type = Sovereign state , subdivision_name = Kingdom of the Netherlands , established_title = Before independence , established_date = Spanish Netherl ...
, including claims it was invented in 1590 by Zacharias Janssen (claim made by his son) or Zacharias' father, Hans Martens, or both, claims it was invented by their neighbor and rival spectacle maker, Hans Lippershey (who applied for the first
telescope A telescope is a device used to observe distant objects by their emission, absorption, or reflection of electromagnetic radiation. Originally meaning only an optical instrument using lenses, curved mirrors, or a combination of both to obse ...
patent in 1608), and claims it was invented by
expatriate An expatriate (often shortened to expat) is a person who resides outside their native country. In common usage, the term often refers to educated professionals, skilled workers, or artists taking positions outside their home country, either ...
Cornelis Drebbel, who was noted to have a version in London in 1619.
Galileo Galilei Galileo di Vincenzo Bonaiuti de' Galilei (15 February 1564 – 8 January 1642) was an Italian astronomer, physicist and engineer, sometimes described as a polymath. Commonly referred to as Galileo, his name was pronounced (, ). He w ...
(also sometimes cited as compound microscope inventor) seems to have found after 1610 that he could close focus his telescope to view small objects and, after seeing a compound microscope built by Drebbel exhibited in Rome in 1624, built his own improved version. Giovanni Faber coined the name ''microscope'' for the compound microscope Galileo submitted to the Accademia dei Lincei in 1625 (Galileo had called it the ''occhiolino'' 'little eye').


Rise of modern light microscopes

The first detailed account of the microscopic anatomy of organic 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''. The microscope was still largely a novelty until the 1660s and 1670s when naturalists in Italy, the Netherlands and England began using them to study biology. Italian scientist Marcello Malpighi, called the father of histology by some historians of biology, began his analysis of biological structures with the lungs. The publication in 1665 of Robert Hooke's '' Micrographia'' had a huge impact, largely because of its impressive illustrations. A significant contribution came from Antonie van Leeuwenhoek who achieved up to 300 times magnification using a simple single lens microscope. He sandwiched a very small glass ball lens between the holes in two metal plates riveted together, and with an adjustable-by-screws needle attached to mount the specimen. Then, Van Leeuwenhoek re-discovered red blood cells (after Jan Swammerdam) and
spermatozoa A spermatozoon (; also spelled spermatozoön; ; ) is a motile sperm cell (biology), cell, or moving form of the ploidy, haploid cell (biology), cell that is the male gamete. A spermatozoon Fertilization, joins an ovum to form a zygote. (A zygote ...
, and helped popularise the use of microscopes to view biological ultrastructure. On 9 October 1676, van Leeuwenhoek reported the discovery of micro-organisms. The performance of a light microscope depends on the quality and correct use of the condensor lens system to focus light on the specimen and the objective lens to capture the light from the specimen and form an image. Early instruments were limited until this principle was fully appreciated and developed from the late 19th to very early 20th century, and until electric lamps were available as light sources. In 1893 August Köhler developed a key principle of sample illumination,
Köhler illumination Köhler illumination is a method of specimen illumination used for transmitted and reflected light (trans- and epi-illuminated) optical microscopy. Köhler illumination acts to generate an even illumination of the sample and ensures that an image o ...
, which is central to achieving the theoretical limits of resolution for the light microscope. This method of sample illumination produces even lighting and overcomes the limited contrast and resolution imposed by early techniques of sample illumination. Further developments in sample illumination came from the discovery of phase contrast by Frits Zernike in 1953, and differential interference contrast illumination by Georges Nomarski in 1955; both of which allow imaging of unstained, transparent samples.


Electron microscopes

In the early 20th century a significant alternative to the light microscope was developed, an instrument that uses a beam of
electron The electron (, or in nuclear reactions) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary partic ...
s rather than
light Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 te ...
to generate an image. The German physicist, Ernst Ruska, working with electrical engineer Max Knoll, developed the first prototype electron microscope in 1931, a transmission electron microscope (TEM). The transmission electron microscope works on similar principles to 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 for 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. Although TEMs were being used for research before WWII, and became popular afterwards, the SEM was not commercially available until 1965. Transmission electron microscopes became popular following the
Second World War World War II or the Second World War, often abbreviated as WWII or WW2, was a world war that lasted from 1939 to 1945. It involved the World War II by country, vast majority of the world's countries—including all of the great power ...
. Ernst Ruska, working at Siemens, developed the first commercial transmission electron microscope and, in the 1950s, major scientific conferences on electron microscopy started being held. 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". One of the latest discoveries made about using an electron microscope is the ability to identify a virus. Since this microscope produces a visible, clear image of small organelles, in an electron microscope there is no need for reagents to see the virus or harmful cells, resulting in a more efficient way to detect pathogens.


Scanning probe microscopes

From 1981 to 1983 Gerd Binnig and Heinrich Rohrer worked at IBM in Zurich, Switzerland to study the quantum tunnelling phenomenon. They created a practical instrument, a scanning probe microscope from quantum tunnelling theory, that read very small forces exchanged between a probe and the surface of a sample. The probe approaches the surface so closely that electrons can flow continuously between probe and sample, making a current from surface to probe. The microscope was not initially well received due to the complex nature of the underlying theoretical explanations. In 1984 Jerry Tersoff and D.R. Hamann, while at AT&T's Bell Laboratories in Murray Hill, New Jersey began publishing articles that tied theory to the experimental results obtained by the instrument. This was closely followed in 1985 with functioning commercial instruments, and in 1986 with Gerd Binnig, Quate, and Gerber's invention of the atomic force microscope, then Binnig's and Rohrer's Nobel Prize in Physics for the SPM. New types of scanning probe microscope have continued to be developed as the ability to machine ultra-fine probes and tips has advanced.


Fluorescence microscopes

The most recent developments in light microscope largely centre on the rise of fluorescence microscopy in
biology Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditar ...
. During the last decades of the 20th century, particularly in the post- genomic era, many techniques for fluorescent staining of cellular structures were developed. The main groups of techniques involve targeted chemical staining of particular cell structures, for example, the chemical compound 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 A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The firs ...
technology limited practical application of the technique. It was not until 1978 when Thomas and
Christoph Cremer Christoph Cremer (born in Freiburg im Breisgau, Germany) is a German physicist and emeritus at the Ruprecht-Karls-University Heidelberg, former honorary professor at the University of Mainz and was a former group leader at Institute of Molecul ...
developed the first practical confocal laser scanning microscope and the technique rapidly gained popularity through the 1980s.


Super resolution microscopes

Much current research (in the early 21st century) on optical microscope techniques is focused on development of superresolution analysis of fluorescently labelled samples.
Structured illumination A structured light pattern designed for surface inspection An Automatix Seamtracker arc welding robot equipped with a camera and structured laser light source, enabling the robot to follow a welding seam automatically Structured light is the p ...
can improve resolution by around two to four times and techniques like stimulated emission depletion (STED) microscopy are approaching the resolution of electron microscopes. This occurs because the diffraction limit is occurred from light or excitation, which makes the resolution must be doubled to become super saturated. Stefan Hell was awarded the 2014 Nobel Prize in Chemistry for the development of the STED technique, along with Eric Betzig and William Moerner who adapted fluorescence microscopy for single-molecule visualization.


X-ray microscopes

X-ray microscopes are instruments that use electromagnetic radiation usually in the soft X-ray band to image objects. Technological advances in X-ray lens optics in the early 1970s made the instrument a viable imaging choice. They are often used in tomography (see micro-computed tomography) to produce three dimensional images of objects, including biological materials that have not been chemically fixed. Currently research is being done to improve optics for hard X-rays which have greater penetrating power.


Types

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 Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 te ...
or photons (optical microscopes),
electron The electron (, or in nuclear reactions) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary partic ...
s (electron microscopes) or a probe (scanning probe microscopes). Alternatively, microscopes can be classified based on whether they analyze the sample via a scanning point (confocal optical microscopes, scanning electron microscopes and scanning probe microscopes) or analyze the sample all at once (wide field optical microscopes and transmission electron microscopes). Wide field optical microscopes and transmission electron microscopes both use the theory of lenses (
optics Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultra ...
for light microscopes and electromagnet lenses for electron microscopes) in order to magnify the image generated by the passage of a
wave In physics, mathematics, and related fields, a wave is a propagating dynamic disturbance (change from equilibrium) of one or more quantities. Waves can be periodic, in which case those quantities oscillate repeatedly about an equilibrium (r ...
transmitted through the sample, or reflected by the sample. The waves used are electromagnetic (in
optical microscope The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of micros ...
s) or
electron The electron (, or in nuclear reactions) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family, and are generally thought to be elementary partic ...
beams (in
electron microscopes An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
). Resolution in these microscopes is limited by the
wavelength In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats. It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tr ...
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 signals generated by the beam interacting with the sample. 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.


Optical microscope

The most common type of microscope (and the first invented) is the
optical microscope The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of micros ...
. This is an
optical Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultrav ...
instrument containing one or more
lenses A lens is a transmissive optical device which focuses or disperses a light beam by means of refraction. A simple lens consists of a single piece of transparent material, while a compound lens consists of several simple lenses (''elements ...
producing an enlarged image of a sample placed in the focal plane. Optical microscopes have refractive glass (occasionally plastic or
quartz Quartz is a hard, crystalline mineral composed of silica ( silicon dioxide). The atoms are linked in a continuous framework of SiO4 silicon-oxygen tetrahedra, with each oxygen being shared between two tetrahedra, giving an overall chemical ...
), to focus light on the eye or on to 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 1,250× with a theoretical resolution limit of around 0.250 
micrometre The micrometre ( international spelling as used by the International Bureau of Weights and Measures; SI symbol: μm) or micrometer ( American spelling), also commonly known as a micron, is a unit of length in the International System of ...
s or 250  nanometres. This limits practical magnification to ~1,500×. 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 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 is a recent optical technique that increases the sensitivity of a standard optical microscope to a point where it is 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 Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiati ...
light enables the resolution of microscopic features as well as the imaging of 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, which allows viewing by eye or with specifically sensitive cameras. Phase-contrast microscopy is an optical microscopic illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into
amplitude The amplitude of a periodic variable is a measure of its change in a single period (such as time or spatial period). The amplitude of a non-periodic signal is its magnitude compared with a reference value. There are various definitions of a ...
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 The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA ( DNA replication) and some of its organelles, and sub ...
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 A digital camera is a camera that captures photographs in digital memory. Most cameras produced today are digital, largely replacing those that capture images on photographic film. Digital cameras are now widely incorporated into mobile devic ...
is used to obtain an image, which is then displayed on a computer monitor. These sensors may use CMOS or
charge-coupled device A charge-coupled device (CCD) is an integrated circuit containing an array of linked, or coupled, capacitors. Under the control of an external circuit, each capacitor can transfer its electric charge to a neighboring capacitor. CCD sensors are ...
(CCD) technology, depending on the application. Digital microscopy with very low light levels to avoid damage to vulnerable biological samples is available using sensitive photon-counting digital cameras. It has been demonstrated that a light source providing pairs of entangled photons may minimize the risk of damage to the most light-sensitive samples. In this application of ghost imaging to photon-sparse microscopy, the sample is illuminated with infrared photons, each of which is spatially correlated with an entangled partner in the visible band for efficient imaging by a photon-counting camera.


Electron microscope

The two major types of electron microscopes are transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs). They both have series of electromagnetic and electrostatic lenses to focus a high energy beam of electrons on a sample. In a TEM the electrons pass through the sample, analogous to basic optical microscopy. This requires careful sample preparation, since electrons are scattered strongly by most materials. The samples must also be very thin (below 100 nm) in order for the electrons to pass through it. Cross-sections of cells stained with osmium and heavy metals reveal clear organelle membranes and proteins such as ribosomes. With a 0.1 nm level of resolution, detailed views of viruses (20 – 300 nm) and a strand of DNA (2 nm in width) can be obtained. In contrast, the SEM has raster coils to scan the surface of bulk objects with a fine electron beam. Therefore, the specimen do not necessarily need to be sectioned, but coating with a nanometric metal or carbon layer may be needed for nonconductive samples. SEM allows fast surface imaging of samples, possibly in thin water vapor to prevent drying.


Scanning probe

The different types of scanning probe microscopes arise from the many different types of interactions that occur when a small probe is scanned over and interacts with a specimen. These interactions or modes can be recorded or mapped as function of location on the surface to form a characterization map. The three most common types of scanning probe microscopes are atomic force microscopes (AFM), near-field scanning optical microscopes (MSOM or SNOM, scanning near-field optical microscopy), and scanning tunneling microscopes (STM). An atomic force microscope has a fine probe, usually of silicon or silicon nitride, attached to a cantilever; the probe is scanned over the surface of the sample, and the forces that cause an interaction between the probe and the surface of the sample are measured and mapped. A near-field scanning optical microscope is similar to an AFM but its probe consists of a light source in an optical fiber covered with a tip that has usually an aperture for the light to pass through. The microscope can capture either transmitted or reflected light to measure very localized optical properties of the surface, commonly of a biological specimen. Scanning tunneling microscopes have a metal tip with a single apical atom; the tip is attached to a tube through which a current flows. The tip is scanned over the surface of a conductive sample until a tunneling current flows; the current is kept constant by computer movement of the tip and an image is formed by the recorded movements of the tip.


Other types

Scanning acoustic microscope A scanning acoustic microscope (SAM) is a device which uses focused sound to investigate, measure, or image an object (a process called scanning acoustic tomography). It is commonly used in failure analysis and non-destructive evaluation. It als ...
s use sound waves to measure variations in acoustic impedance. Similar to
Sonar Sonar (sound navigation and ranging or sonic navigation and ranging) is a technique that uses sound propagation (usually underwater, as in submarine navigation) to navigate, measure distances ( ranging), communicate with or detect objects on ...
in principle, they are used for such jobs as detecting defects in the subsurfaces of materials including those found in integrated circuits. On February 4, 2013, Australian engineers built a "quantum microscope" which provides unparalleled precision.


Mobile apps

Mobile app A mobile application or app is a computer program or software application designed to run on a mobile device such as a phone, tablet, or watch. Mobile applications often stand in contrast to desktop applications which are designed to run on ...
microscopes can optionally be used as
optical microscope The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of micros ...
when the flashlight is activated. However, mobile app microscopes are harder to use due to visual noise, are often limited to 40x, and the resolution limits of the camera lens itself.


See also

* Fluorescence interference contrast microscopy * Laser capture microdissection * Microscope image processing * Microscope slide *
Multifocal plane microscopy Multifocal plane microscopy (MUM) or multiplane microscopy or multifocus microscopy is a form of light microscopy that allows the tracking of the 3D dynamics in live cells at high temporal and spatial resolution by simultaneously imaging differen ...
* Royal Microscopical Society


References


External links


Milestones in Light Microscopy
''Nature Publishing''


Nikon MicroscopyU, tutorials from Nikon


{{Authority control Microbiology equipment Microscopy Scientific instruments