Microscope

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Microscope
Compound Microscope (cropped).JPG
Microscope
UsesSmall sample observation
Notable experimentsDiscovery of cells
Related items Optical microscope Electron microscope

A microscope (from the Ancient Greek : μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see") is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope.

Microscopy technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

Science systematic enterprise that builds and organizes knowledge

Science is a systematic enterprise that builds and organizes knowledge in the form of testable explanations and predictions about the universe.

Contents

There are many types of microscopes, and they may be grouped in different ways. One way is to describe the way the instruments interact with a sample to create images, either by sending a beam of light or electrons to a sample in its optical path, 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, which uses light to pass through a sample to produce an image. Other major types of microscopes are the fluorescence microscope, the electron microscope (both the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscopes. [1]

Optical microscope Microscope that uses visible light

The optical microscope, often referred to as the light microscope, is a type of microscope that commonly uses visible light and a system of lenses to magnify images of small objects. 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 many complex designs aim to improve resolution and sample contrast. Often used in the classroom and at home unlike the electron microscope which is used for closer viewing.

Light electromagnetic radiation in or near visible spectrum

Light is electromagnetic radiation within a certain portion of the electromagnetic spectrum. The word usually refers to visible light, which is the visible spectrum that is visible to the human eye and is responsible for the sense of sight. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), or 4.00 × 10−7 to 7.00 × 10−7 m, between the infrared and the ultraviolet. This wavelength means a frequency range of roughly 430–750 terahertz (THz).

Fluorescence microscope

A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

History

18th-century microscopes from the Musee des Arts et Metiers, Paris Old-microscopes.jpg
18th-century microscopes from the Musée des Arts et Métiers, Paris

Although objects resembling lenses date back 4000 years and there are Greek 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 in the 13th century. [2] [3] [4] 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. [5] The inventor is unknown although many claims have been made over the years. Several revolve around the spectacle-making centers in the Netherlands including claims it was invented in 1590 by Zacharias Janssen (claim made by his son) and/or Zacharias' father, Hans Martens, [6] [7] claims it was invented by their neighbor and rival spectacle maker, Hans Lippershey (who applied for the first telescope patent in 1608), [8] and claims it was invented by expatriate Cornelis Drebbel who was noted to have a version in London in 1619. [9] [10] Galileo Galilei (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. [11] [12] [13] Giovanni Faber coined the name microscope for the compound microscope Galileo submitted to the Accademia dei Lincei in 1625 [14] (Galileo had called it the "occhiolino" or "little eye").

The Greeks or Hellenes are an ethnic group native to Greece, Cyprus, southern Albania, Italy, Turkey, Egypt and, to a lesser extent, other countries surrounding the Mediterranean Sea. They also form a significant diaspora, with Greek communities established around the world.

Magnifying glass lens which makes things appear larger

A magnifying glass is a convex lens that is used to produce a magnified image of an object. The lens is usually mounted in a frame with a handle. A magnifying glass can be used to focus light, such as to concentrate the sun's radiation to create a hot spot at the focus for fire starting.

Objective (optics) optical element that gathers light from the object being observed and focuses the light rays to produce a real image

In optical engineering, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be a single lens or mirror, or combinations of several optical elements. They are used in microscopes, telescopes, cameras, slide projectors, CD players and many other optical instruments. Objectives are also called object lenses, object glasses, or objective glasses.

Rise of modern light microscopes

Carl Zeiss binocular compound microscope, 1914 Binocular compound microscope, Carl Zeiss Jena, 1914 (6779276516).jpg
Carl Zeiss binocular compound microscope, 1914

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. [15]

Histology study of the microscopic anatomy of cells and tissues of plants and animals

Histology, also microanatomy, is the branch of biology which studies the tissues of animals and plants using microscopy. It is commonly studied using a light microscope or electron microscope, the specimen having been sectioned, stained, and mounted on a microscope slide. Histological studies may be conducted using tissue culture, where live animal cells are isolated and maintained in an artificial environment for various research projects. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of staining. Histology is one of the major preclinical subjects in medical school. Medical students are expected to be familiar with the morphological features and function of all cells and tissues of the human body from an early stage of their studies, so histology often stretches over several semesters.

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. 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. [16] Then, Van Leeuwenhoek re-discovered red blood cells (after Jan Swammerdam) and spermatozoa, and helped popularise the use of microscopes to view biological ultrastructure. On 9 October 1676, van Leeuwenhoek reported the discovery of micro-organisms. [15]

Marcello Malpighi Italian physician

Marcello Malpighi was an Italian biologist and physician, who is referred to as the "Father of microscopical anatomy, histology, physiology and embryology". Malpighi's name is born by several physiological features related to the biological excretory system, such as the Malpighian corpuscles and Malpighian pyramids of the kidneys and the Malpighian tubule system of insects. The splenic lymphoid nodules are often called the "Malpighian bodies of the spleen" or Malpighian corpuscles. The botanical family Malpighiaceae is also named after him. He was the first person to see capillaries in animals, and he discovered the link between arteries and veins that had eluded William Harvey. Malpighi was one of the earliest people to observe red blood cells under a microscope, after Jan Swammerdam. His treatise De polypo cordis (1666) was important for understanding blood composition, as well as how blood clots. In it, Malpighi described how the form of a blood clot differed in the right against the left sides of the heart.

Robert Hooke English natural philosopher, architect and polymath

Robert Hooke FRS was an English natural philosopher, architect and polymath.

<i>Micrographia</i> book by Robert Hooke

Micrographia: or Some Physiological Descriptions of Minute Bodies Made by Magnifying Glasses. With Observations and Inquiries Thereupon. is a historically significant book by Robert Hooke about his observations through various lenses. It is particularly notable for being the first book to illustrate insects, plants etc. as seen through microscopes. Published in January 1665, the first major publication of the Royal Society, it became the first scientific best-seller, inspiring a wide public interest in the new science of microscopy. It is also notable for coining the biological term cell.

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. [5] 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, 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.

A condenser is an optical lens which renders a divergent beam from a point source into a parallel or converging beam to illuminate an object.

August Köhler German scientist

August Karl Johann Valentin Köhler was a German professor and early staff member of Carl Zeiss AG in Jena, Germany. He is best known for his development of the microscopy technique of Köhler illumination, an important principle in optimizing microscopic resolution power by evenly illuminating the field of view. This invention revolutionized light microscope design and is widely used in traditional as well as modern digital imaging techniques today.

Köhler illumination is a method of specimen illumination used for transmitted and reflected light optical microscopy. Köhler illumination acts to generate an even illumination of the sample and ensures that an image of the illumination source is not visible in the resulting image. Köhler illumination is the predominant technique for sample illumination in modern scientific light microscopy. It requires additional optical elements which are more expensive and may not be present in more basic light microscopes.

Electron microscopes

Electron microscope constructed by Ernst Ruska in 1933 Ernst Ruska Electron Microscope - Deutsches Museum - Munich-edit.jpg
Electron microscope constructed by Ernst Ruska in 1933

In the early 20th century a significant alternative to the light microscope was developed, an instrument that uses a beam of electrons rather than light 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. [17] 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. 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. [18] 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. [19]

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

Fluorescence microscope with the filter cube turret above the objective lenses, coupled with a camera. Olympus-BX61-fluorescence microscope.jpg
Fluorescence microscope with the filter cube turret above the objective lenses, coupled with a camera.

The most recent developments in light microscope largely centre on the rise of fluorescence microscopy in biology. [20] During the last decades of the 20th century, particularly in the post-genomic era, many techniques for fluorescent staining of cellular structures were developed. [20] 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. [21] These techniques use these different fluorophores for analysis of cell structure at a molecular level in both live and fixed samples.

The rise of fluorescence microscopy drove the development of a major modern microscope design, the confocal microscope. The principle was patented in 1957 by Marvin Minsky, although laser technology limited practical application of the technique. It was not until 1978 when Thomas and Christoph Cremer developed the first practical confocal laser scanning microscope and the technique rapidly gained popularity through the 1980s.

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 can improve resolution by around two to four times and techniques like stimulated emission depletion (STED) microscopy are approaching the resolution of electron microscopes. [22] 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. [23]

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. [24] 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. [24]

Types

Types of microscopes illustrated by the principles of their beam paths MicroscopesOverview.svg
Types of microscopes illustrated by the principles of their beam paths
Evolution of spatial resolution achieved with optical, transmission (TEM) and aberration-corrected electron microscopes (ACTEM). MicroscopyResolution.png
Evolution of spatial resolution achieved with optical, transmission (TEM) and aberration-corrected electron microscopes (ACTEM).

Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the image, i.e., light or photons (optical microscopes), electrons (electron microscopes) or a probe (scanning probe microscopes). Alternatively, microscopes can be 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 for light microscopes and electromagnet lenses for electron microscopes) in order to magnify the image generated by the passage of a wave transmitted through the sample, or reflected by the sample. The waves used are electromagnetic (in optical microscopes) or electron beams (in electron microscopes). Resolution in these microscopes is limited by the wavelength of the radiation used to image the sample, where shorter wavelengths allow for a higher resolution. [20]

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

The most common type of microscope (and the first invented) is the optical microscope. This is an optical instrument containing one or more lenses producing an enlarged image of a sample placed in the focal plane. Optical microscopes have refractive glass (occasionally plastic or quartz), 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 1250x with a theoretical resolution limit of around 0.250  micrometres or 250  nanometres. [20] This limits practical magnification to ~1500x. 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 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.

Unstained cells viewed by typical brightfield (left) compared to phase contrast microscopy (right). Brightfield phase contrast cell image.jpg
Unstained cells viewed by typical brightfield (left) compared to phase contrast microscopy (right).

Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. [20] The use of phase contrast does not require staining to view the slide. This microscope technique made it possible to study the cell cycle in live cells.

The traditional optical microscope has more recently evolved into the digital microscope. In addition to, or instead of, directly viewing the object through the eyepieces, a type of sensor similar to those used in a digital camera is used to obtain an image, which is then displayed on a computer monitor. These sensors may use CMOS or charge-coupled device (CCD) technology, depending on the application.

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. [26]

Modern transmission electron microscope Electron Microscope.jpg
Modern transmission electron microscope

Electron

Transmission electron micrograph of a dividing cell undergoing cytokinesis Cytokinesis-electron-micrograph.jpg
Transmission electron micrograph of a dividing cell undergoing cytokinesis

The two major types of electron microscopes are transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs). [20] [21] 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. [20] This requires careful sample preparation, since electrons are scattered strongly by most materials. [21] The samples must also be very thin (50–100 nm) in order for the electrons to pass through it. [20] [21] Cross-sections of cells stained with osmium and heavy metals reveal clear organelle membranes and proteins such as ribosomes. [21] 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. [21] 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 require coating with a substance such as a heavy metal. [20] This allows three-dimensional views of the surface of samples. [20] [21]

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). [27] 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. [28] 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. [27]

Leaf surface viewed by a scanning electron microscope. Leaf epidermis.jpg
Leaf surface viewed by a scanning electron microscope.

Other types

Scanning acoustic microscopes use sound waves to measure variations in acoustic impedance. Similar to Sonar in principle, they are used for such jobs as detecting defects in the subsurfaces of materials including those found in integrated circuits. On February 4, 2013, Australian engineers built a "quantum microscope" which provides unparalleled precision. [29]

See also

Related Research Articles

Electron microscope type of 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 higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode and magnifications of up to about 10,000,000× whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000×.

Scanning electron microscope

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the intensity of the detected signal to produce an image. In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using an Everhart-Thornley detector. The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography. SEM can achieve resolution better than 1 nanometer.

Diffraction-limited system

The resolution of an optical imaging system – a microscope, telescope, or camera – can be limited by factors such as imperfections in the lenses or misalignment. However, there is a principal limit to the resolution of any optical system, due to the physics of diffraction. An optical system with resolution performance at the instrument's theoretical limit is said to be diffraction-limited.

Photoemission electron microscopy is a type of electron microscopy that utilizes local variations in electron emission to generate image contrast. The excitation is usually produced by ultraviolet light, synchrotron radiation or X-ray sources. PEEM measures the coefficient indirectly by collecting the emitted secondary electrons generated in the electron cascade that follows the creation of the primary core hole in the absorption process. PEEM is a surface sensitive technique because the emitted electrons originate from a shallow layer. In physics, this technique is referred to as PEEM, which goes together naturally with low-energy electron diffraction (LEED), and low-energy electron microscopy (LEEM). In biology, it is called photoelectron microscopy (PEM), which fits with photoelectron spectroscopy (PES), transmission electron microscopy (TEM), and scanning electron microscopy (SEM).

X-ray microscope

An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations.

Confocal microscopy optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

Two-photon excitation microscopy

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in depth. It differs from traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, as the wavelengths of the two exciting photons are longer than the wavelength of the resulting emitted light. Two-photon excitation microscopy typically uses near-infrared excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.

Dark-field microscopy

Dark-field microscopy describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen is generally dark.

Stereo microscope

The stereo, stereoscopic or dissecting microscope is an optical microscope variant designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. The instrument uses two separate optical paths with two objectives and eyepieces to provide slightly different viewing angles to the left and right eyes. This arrangement produces a three-dimensional visualization of the sample being examined. Stereomicroscopy overlaps macrophotography for recording and examining solid samples with complex surface topography, where a three-dimensional view is needed for analyzing the detail.

Scanning confocal electron microscopy (SCEM) is an electron microscopy technique analogous to scanning confocal optical microscopy (SCOM). In this technique, the studied sample is illuminated by a focussed electron beam, as in other scanning microscopy techniques, such as scanning transmission electron microscopy or scanning electron microscopy. However, in SCEM, the collection optics is arranged symmetrically to the illumination optics to gather only the electrons that pass the beam focus. This results in superior depth resolution of the imaging. The technique is relatively new and is being actively developed.

Optical sectioning

Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome. Many different techniques for optical sectioning are used and several microscopy techniques are specifically designed to improve the quality of optical sectioning.

The technique of vibrational analysis with scanning probe microscopy allows probing vibrational properties of materials at the submicrometer scale, and even of individual molecules. This is accomplished by integrating scanning probe microscopy (SPM) and vibrational spectroscopy. This combination allows for much higher spatial resolution than can be achieved with conventional Raman/FTIR instrumentation. The technique is also nondestructive, requires non-extensive sample preparation, and provides more contrast such as intensity contrast, polarization contrast and wavelength contrast, as well as providing specific chemical information and topography images simultaneously.

Endomicroscopy is a technique for obtaining histology-like images from inside the human body in real-time, a process known as ‘optical biopsy’. It generally refers to fluorescence confocal microscopy, although multi-photon microscopy and optical coherence tomography have also been adapted for endoscopic use. Commercially available clinical and pre-clinical endomicroscopes can achieve a resolution on the order of a micrometre, have a field-of-view of several hundred µm, and are compatible with fluorophores which are excitable using 488 nm laser light. The main clinical applications are currently in imaging of the tumour margins of the brain and gastro-intestinal tract, particularly for the diagnosis and characterisation of Barrett’s Esophagus, pancreatic cysts and colorectal lesions. A number of pre-clinical and transnational applications have been developed for endomicroscopy as it enables researchers to perform live animal imaging. Major pre-clinical applications are in gastro-intestinal tract, toumous margin detection, uterine complications, ischaemia, live imaging of cartilage and tendon, organoid imaging etc.

Light sheet fluorescence microscopy

Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction. A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because LSFM scans samples by using a plane of light instead of a point, it can acquire images at speeds 100 to 1000 times faster than those offered by point-scanning methods.

Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can provide information about dynamic biological processes.

Wide-field multiphoton microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by stage- or beam-scanning the sample. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 µm). The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy.

The operation of a photon scanning tunneling microscope (PSTM) is analogous to the operation of an electron scanning tunneling microscope (ESTM), with the primary distinction being that PSTM involves tunneling of photons instead of electrons from the sample surface to the probe tip. A beam of light is focused on a prism at an angle greater than the critical angle of the refractive medium in order to induce total internal reflection (TIR) within the prism. Although the beam of light is not propagated through the surface of the refractive prism under TIR, an evanescent field of light is still present at the surface.

References

First atomic force microscope Atomic Force Microscope Science Museum London.jpg
First atomic force microscope
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