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87-462: With conventional microscopy techniques, it is not possible to distinguish features that are located at distances less than about half the wavelength used (i.e. about 200 nm for visible light ). This diffraction limit is based on the wave nature of light . In conventional microscopes the limit is determined by the used wavelength and the numerical aperture of the optical system. The RESOLFT concept surmounts this limit by temporarily switching

174-539: A t {\displaystyle \Delta d={\frac {\lambda }{2n\cdot \sin \alpha \cdot {\sqrt {1+{\frac {I}{Isat}}}}}}} which can be regarded as an extension of Abbe ’s equation. The diffraction-unlimited nature of the RESOLFT family of concepts is reflected by the fact that the minimal resolvable distance Δ d {\displaystyle \Delta d} can be continuously decreased by increasing ς = I I s

261-420: A t {\displaystyle \Delta d\simeq {\frac {\lambda }{\pi n{\sqrt {\frac {I}{Isat}}}}}} , whereby I s a t {\displaystyle I_{sat}} is the characteristic intensity required for saturating the transition (half of the molecules remain in state A and half in state B), and I {\displaystyle I} denotes the intensity applied. If

348-479: A t {\displaystyle \varsigma ={\frac {I}{Isat}}} . Hence the quest for nanoscale resolution comes down to maximizing this quantity. This is possible by increasing I {\displaystyle I} or by lowering I s a t {\displaystyle I_{sat}} . Different processes are used when switching the molecular states. However, all have in common that at least two distinguishable states are used. Typically

435-618: A pulsed infrared laser is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by two-photon excitation , which means that no out-of-focus fluorescence is generated, and no pinhole is necessary to clean up the image. This allows imaging deep in scattering tissue, where a confocal microscope would not be able to collect photons efficiently. Two-photon microscopes with wide-field detection are frequently used for functional imaging, e.g. calcium imaging , in brain tissue. They are marketed as Multiphoton microscopes by several companies, although

522-522: A 1000-fold compared to multiphoton scanning microscopy . In scattering tissue, however, image quality rapidly degrades with increasing depth. Fluorescence microscopy is a powerful technique to show specifically labeled structures within a complex environment and to provide three-dimensional information of biological structures. However, this information is blurred by the fact that, upon illumination, all fluorescently labeled structures emit light, irrespective of whether they are in focus or not. So an image of

609-912: A RESOLFT-type microscope. During illumination with light, these proteins change their conformation. In the process they gain or lose their ability to emit fluorescence. The fluorescing state corresponds to state A, the non-fluorescing to state B and the RESOLFT concept applies again. The reversible transition (e.g. from B back to A) takes place either spontaneously or again driven by light. Inducing conformational changes in proteins can be achieved already at much lower switching light intensities as compared to stimulated emission or ground state depletion (some W/cm). In combination with 4Pi microscopy images with isotropic resolution below 40 nm have been taken of living cells at low light levels. Just as with proteins, also some organic dyes can change their structure upon illumination. The ability to fluoresce of such organic dyes can be turned on and off through visible light. Again

696-502: A Z-stack) plus the knowledge of the PSF, which can be derived either experimentally or theoretically from knowing all contributing parameters of the microscope. A multitude of super-resolution microscopy techniques have been developed in recent times which circumvent the diffraction limit . This is mostly achieved by imaging a sufficiently static sample multiple times and either modifying the excitation light or observing stochastic changes in

783-514: A booklet published in 1874. He developed the laws of image of non-luminous objects by 1872. Zeiss Optical Works began selling his improved microscopes in 1872, by 1877 they were selling microscopes with homogenous immersion objective, and in 1886 his apochromatic objective microscopes were being sold. He created the Abbe number , a measure of any transparent material's variation of refractive index with wavelength and Abbe's criterion, which tests

870-432: A cell will actually show up as a globule in the most often used differential interference contrast system according to Georges Nomarski . However, it has to be kept in mind that this is an optical effect , and the relief does not necessarily resemble the true shape. Contrast is very good and the condenser aperture can be used fully open, thereby reducing the depth of field and maximizing resolution. The system consists of

957-414: A certain structure is always blurred by the contribution of light from structures that are out of focus. This phenomenon results in a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture. However, blurring is not caused by random processes, such as light scattering, but can be well defined by the optical properties of

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1044-444: A circular annulus in the condenser, which produces a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different size ring, so for every objective another condenser setting has to be chosen. The ring in the objective has special optical properties: it, first of all, reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about

1131-419: A fine beam over the sample (for example confocal laser scanning microscopy and scanning electron microscopy ). Scanning probe microscopy involves the interaction of a scanning probe with the surface of the object of interest. The development of microscopy revolutionized biology , gave rise to the field of histology and so remains an essential technique in the life and physical sciences . X-ray microscopy

1218-739: A flat panel display. A 3D X-ray microscope employs a range of objectives, e.g., from 4X to 40X, and can also include a flat panel. The field of microscopy ( optical microscopy ) dates back to at least the 17th-century. Earlier microscopes, single lens magnifying glasses with limited magnification, date at least as far back as the wide spread use of lenses in eyeglasses in the 13th century but more advanced compound microscopes first appeared in Europe around 1620 The earliest practitioners of microscopy include Galileo Galilei , who found in 1610 that he could close focus his telescope to view small objects close up and Cornelis Drebbel , who may have invented

1305-644: A focused laser beam (e.g. 488 nm) that is scanned across the sample to excite fluorescence in a point-by-point fashion. The emitted light is directed through a pinhole to prevent out-of-focus light from reaching the detector, typically a photomultiplier tube . The image is constructed in a computer, plotting the measured fluorescence intensities according to the position of the excitation laser. Compared to full sample illumination, confocal microscopy gives slightly higher lateral resolution and significantly improves optical sectioning (axial resolution). Confocal microscopy is, therefore, commonly used where 3D structure

1392-448: A glass window: one sees not the glass but merely the dirt on the glass. There is a difference, as glass is a denser material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase, but clever optical solutions have been devised to change this difference in phase into a difference in amplitude (light intensity). To improve specimen contrast or highlight structures in

1479-558: A humble home – his father was a foreman in a spinnery. Supported by his father's employer, Abbe was able to attend secondary school and to obtain the general qualification for university entrance with fairly good grades, at the Eisenach Gymnasium, which he graduated from in 1857. By the time he left school, his scientific talent and his strong will had already become obvious. Thus, in spite of the family's strained financial situation, his father decided to support Abbe's studies at

1566-404: 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 beam scanning. In wide-field multiphoton microscopy

1653-404: A lower frequency. This effect is known as fluorescence . Often specimens show their characteristic autofluorescence image, based on their chemical makeup. This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent dyes can be used to stain structures or chemical compounds. One powerful method

1740-424: A monocular eyepiece. It is essential that both eyes are open and that the eye that is not observing down the microscope is instead concentrated on a sheet of paper on the bench besides the microscope. With practice, and without moving the head or eyes, it is possible to accurately trace the observed shapes by simultaneously "seeing" the pencil point in the microscopical image. It is always less tiring to observe with

1827-413: A quarter wavelength. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image. One disadvantage of phase-contrast microscopy is halo formation (halo-light ring). Superior and much more expensive is the use of interference contrast . Differences in optical density will show up as differences in relief. A nucleus within

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1914-420: A sample, special techniques must be used. A huge selection of microscopy techniques are available to increase contrast or label a sample. Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Limitations include low contrast of most biological samples and low apparent resolution due to

2001-603: A single-pixel photodetector to eliminate the need for a detector array and readout time limitations The method is at least 1000 times faster than the state-of-the-art CCD and CMOS cameras. Consequently, it is potentially useful for scientific, industrial, and biomedical applications that require high image acquisition rates, including real-time diagnosis and evaluation of shockwaves, microfluidics , MEMS , and laser surgery . Most modern instruments provide simple solutions for micro-photography and image recording electronically. However such capabilities are not always present and

2088-466: A smaller area where the intensity is below the amount for efficient switching to the dark state. Consequently, also the area where molecules can reside in state A is diminished. The (fluorescence) signal during a following readout originates from a very small spot and one can obtain very sharp images. In the RESOLFT concept, the resolution can be approximated by Δ d ≃ λ π n I I s

2175-426: A special prism ( Nomarski prism , Wollaston prism ) in the condenser that splits light in an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism in the objective. In a homogeneous specimen, there is no difference between the two beams, and no contrast

2262-519: A very powerful tool for investigation of nanomaterials . This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope (AFM), the scanning tunneling microscope , the photonic force microscope and the recurrence tracking microscope . All such methods use the physical contact of a solid probe tip to scan the surface of an object, which is supposed to be almost flat. Ernst Karl Abbe Ernst Karl Abbe HonFRMS (23 January 1840 – 14 January 1905)

2349-405: Is a variant of dark field illumination in which transparent, colored filters are inserted just before the condenser so that light rays at high aperture are differently colored than those at low aperture (i.e., the background to the specimen may be blue while the object appears self-luminous red). Other color combinations are possible, but their effectiveness is quite variable. Dispersion staining

2436-591: Is a widely used technique that shows differences in refractive index as difference in contrast. It was developed by the Dutch physicist Frits Zernike in the 1930s (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is formed even around small objects, which obscures detail. The system consists of

2523-495: Is an optical technique that results in a colored image of a colorless object. This is an optical staining technique and requires no stains or dyes to produce a color effect. There are five different microscope configurations used in the broader technique of dispersion staining. They include brightfield Becke line, oblique, darkfield, phase contrast, and objective stop dispersion staining. More sophisticated techniques will show proportional differences in optical density. Phase contrast

2610-451: Is being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast requires a polarized light source to function; two polarizing filters have to be fitted in the light path, one below the condenser (the polarizer), and the other above the objective (the analyzer). Note: In cases where

2697-442: Is bright, that is, generates a fluorescence signal, and the other state (B) is dark, and gives no signal. One transition between them can be induced by light (e.g. A→B, bright to dark). The sample is illuminated inhomogeneously with the illumination intensity at one position being very small (zero under ideal conditions). Only at this place are the molecules never in the dark state B (if A is the pre-existing state) and remain fully in

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2784-765: Is fair to say that Abbe was the first to reach this conclusion experimentally. In 1878, he built the first homogenous immersion system for the microscope. The objectives that the Abbe Zeiss collaboration were producing were of ideal ray geometry, allowing Abbe to find that the aperture sets the upper limit of microscopic resolution, not the curvature and placement of the lenses. Abbe's first publication of Eq. 1 occurred in 1882. In this publication, Abbe states that both his theoretical and experimental investigations confirmed Eq. 1 . Abbe's contemporary Henry Edward Fripp, English translator of Abbe's and Helmholtz's papers, puts their contributions on equal footing. He also perfected

2871-428: Is important. A subclass of confocal microscopes are spinning disc microscopes which are able to scan multiple points simultaneously across the sample. A corresponding disc with pinholes rejects out-of-focus light. The light detector in a spinning disc microscope is a digital camera, typically EM-CCD or sCMOS . A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light,

2958-456: Is the additive noise. Knowing this point spread function means that it is possible to reverse this process to a certain extent by computer-based methods commonly known as deconvolution microscopy. There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in nonrestorative and restorative methods. While the nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only

3045-417: Is the combination of antibodies coupled to a fluorophore as in immunostaining . Examples of commonly used fluorophores are fluorescein or rhodamine . The antibodies can be tailor-made for a chemical compound. For example, one strategy often in use is the artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, forming antibodies which bind to

3132-436: Is the standard operation mode in fluorescence microscopy and depicts state A. In state B the dye is permanently kept in its electronic ground state through stimulated emission . If the dye can fluoresce in state A and not in state B, the RESOLFT concept applies. (Main article GSD microscopy ) GSD microscopy (Ground State Depletion microscopy) also uses fluorescent markers. In state A, the molecule can freely be driven between

3219-414: Is three-dimensional and non-destructive, allowing for repeated imaging of the same sample for in situ or 4D studies, and providing the ability to "see inside" the sample being studied before sacrificing it to higher resolution techniques. A 3D X-ray microscope uses the technique of computed tomography ( microCT ), rotating the sample 360 degrees and reconstructing the images. CT is typically carried out with

3306-508: The Abbe sine condition . So monumental and advanced were Abbe's calculations and achievements that Frits Zernike based his phase contrast work on them, for which he was awarded the Nobel Prize in 1953, and Hans Busch used them to work on the development of the electron microscope . During his association with Carl Zeiss ' microscope works, not only was he at the forefront of the field of optics but also labor reform. He founded

3393-562: The interference method by Fizeau , in 1884. Abbe, Zeiss, Zeiss' son, Roderich Zeiss , and Otto Schott formed, in 1884, the Jenaer Glaswerk Schott & Genossen . This company, which in time would in essence merge with Zeiss Optical Works, was responsible for research and production of 44 initial types of optical glass. Working with telescopes , he built an image reversal system in 1895. In order to produce high quality objectives, Abbe made significant contributions to

3480-529: The social democratic Jenaische Zeitung (newspaper) in 1890 and in 1900, introduced the eight-hour workday , in remembrance of the 14-hour workday of his own father. In addition, he created a pension fund and a discharge compensation fund. In 1889, Ernst Abbe set up and endowed the Carl Zeiss Foundation for research in science. The aim of the foundation was "to secure the economic, scientific, and technological future and in this way to improve

3567-543: The 1947 book Schriften der Heidelberger Aktionsgruppe zur Demokratie und Zum Freien Sozialismus ( Writings of the Heidelberg Action Group on Democracy and Free Socialism ). The crater Abbe on the Moon was named in his honour. Abbe was a pioneer in optics, lens design, and microscopy, and an authority of his time. He left us with numerous publications of his findings, inventions, and discoveries. Below

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3654-695: The Göttingen observatory and at Physikalischer Verein in Frankfurt (an association of citizens interested in physics and chemistry that was founded by Johann Wolfgang von Goethe in 1824 and still exists today). On 8 August 1863 he qualified as a university lecturer at the University of Jena. In 1870, he accepted a contract as an associate professor of experimental physics , mechanics and mathematics in Jena. In 1871, he married Else Snell, daughter of

3741-447: The RESOLFT concept using saturated excitation to produce "negative" images, i.e. fluorescence occurs from everywhere except at a very small region around the geometrical focus of the microscope. Also non point-like patterns are used for illumination. Mathematical image reconstruction is necessary to obtain positive images again. Some fluorescent proteins can be switched on and off by light of an appropriate wavelength. They can be used in

3828-527: The Theory of the Microscope and the nature of Microscopic Vision", Abbe states that the resolution of a microscope is inversely dependent on its aperture, but without proposing a formula for the resolution limit of a microscope. In 1876, Abbe was offered a partnership by Zeiss and began to share in the considerable profits. Although the first theoretical derivations of Eq. 1 were published by others, it

3915-562: The Universities of Jena (1857–1859) and Göttingen (1859–1861). During his time as a student, Abbe gave private lessons to improve his income. His father's employer continued to fund him. Abbe was awarded his PhD in Göttingen on 23 March 1861. While at school, he was influenced by Bernhard Riemann and Wilhelm Eduard Weber , who also happened to be one of the Göttingen Seven . This was followed by two short assignments at

4002-512: The University of Jena in 1891. Abbe died 14 January 1905 in Jena. He was an atheist. In 1866, he became a research director at the Zeiss Optical Works , and in 1868 he invented the apochromatic lens , a microscope lens which eliminates both the primary and secondary color distortion. By 1870, Abbe invented the Abbe condenser , used for microscope illumination. In 1871, he designed the first refractometer , which he described in

4089-412: The applied light intensities can be quite low (some 100 W/cm). 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 microscopy: optical , electron , and scanning probe microscopy , along with

4176-418: The area where molecules reside in state A (bright state) can be made arbitrarily small despite the diffraction-limit. Upon weak illumination we see that the area where molecules remain in state A is still quite large because the illumination is so low that most molecules reside in state A. The shape of the illumination profile does not need to be altered. Increasing the illumination brightness already results in

4263-534: The blur of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are significant advantages. The use of oblique (from the side) illumination gives the image a three-dimensional appearance and can highlight otherwise invisible features. A more recent technique based on this method is Hoffmann's modulation contrast , a system found on inverted microscopes for use in cell culture. Oblique illumination enhances contrast even in clear specimens; however, because light enters off-axis,

4350-411: The bright state A. The area where molecules are mostly in the bright state can be made very small (smaller than the conventional diffraction limit) by increasing the transition light intensity (see below). Any signal detected is thus known to come only from molecules in the small area around the illumination intensity minimum. A high resolution image can be constructed by scanning the sample, i.e., shifting

4437-407: The compound microscope around 1620. Antonie van Leeuwenhoek developed a very high magnification simple microscope in the 1670s and is often considered to be the first acknowledged microscopist and microbiologist . Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of

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4524-403: The diagnosis and correction of optical aberrations , both spherical aberration and coma aberration , which is required for an objective to reach the resolution limit of Eq. 1 . In addition to spherical aberration, Abbe discovered that the rays in optical systems must have constant angular magnification over their angular distribution to produce a diffraction limited spot, a principle known as

4611-445: The diffraction limit. To realize such assumption, Knowledge of and chemical control over fluorophore photophysics is at the core of these techniques, by which resolutions of ~20 nanometers are obtained. Serial time encoded amplified microscopy (STEAM) is an imaging method that provides ultrafast shutter speed and frame rate, by using optical image amplification to circumvent the fundamental trade-off between sensitivity and speed, and

4698-482: The emerging field of X-ray microscopy . Optical microscopy and electron microscopy involve the diffraction , reflection , or refraction of electromagnetic radiation /electron beams interacting with the specimen , and the collection of the scattered radiation or another signal in order to create an image. This process may be carried out by wide-field irradiation of the sample (for example standard light microscopy and transmission electron microscopy ) or by scanning

4785-407: The exhibit of interest. The image is shown on a computer screen, so eye-pieces are unnecessary. Limitations of standard optical microscopy ( bright field microscopy ) lie in three areas; Live cells in particular generally lack sufficient contrast to be studied successfully, since the internal structures of the cell are colorless and transparent. The most common way to increase contrast is to stain

4872-460: The fluorescence property used marks the distinction of the states, however this is not essential, as absorption or scattering properties could also be exploited. (Main article STED microscopy ) Within the STED microscopy (STimulated Emission Depletion microscopy) a fluorescent dye molecule is driven between its electronic ground state and its excited state while sending out fluorescence photons. This

4959-666: The gains of using 3-photon instead of 2-photon excitation are marginal. Using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, high resolution optical sections can be taken. Single plane illumination, or light sheet illumination, is also accomplished using beam shaping techniques incorporating multiple-prism beam expanders . The images are captured by CCDs. These variants allow very fast and high signal to noise ratio image capture. Wide-field multiphoton microscopy refers to an optical non-linear imaging technique in which

5046-501: The ground and the first excited state and fluorescence can be sent out. In the dark state B the ground state of the molecule is depopulated, a transition to a long lived excited state takes place from which fluorescence is not emitted. As long as the molecule is in the dark state, it's not available for cycling between ground and excited state, fluorescence is hence turned off. SPEM (Saturated Pattern Excitation Microscopy) and SSIM (Saturated Structured Illumination Microscopy) are exploiting

5133-436: 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 camera 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

5220-487: The hypothesis, that a systematic trend exists in a set of observations (in terms of resolving power this criterion stipulates that an angular separation cannot be less than the ratio of the wavelength to the aperture diameter, see angular resolution ). Already a professor in Jena , he was hired by Carl Zeiss to improve the manufacturing process of optical instruments, which back then was largely based on trial and error. Abbe

5307-399: The illumination profile across the surface. The transition back from B to A can be either spontaneous or driven by light of another wavelength. The molecules have to be switchable several times in order to be present in state A or B at different times during scanning the sample. The method also works if the bright and the dark state are reversed, one then obtains a negative image. In RESOLFT

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5394-412: The image formation in the microscope imaging system. If one considers a small fluorescent light source (essentially a bright spot), light coming from this spot spreads out further from our perspective as the spot becomes more out of focus. Under ideal conditions, this produces an "hourglass" shape of this point source in the third (axial) dimension. This shape is called the point spread function (PSF) of

5481-403: The image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in the final image of many biological samples and continues to be affected by low apparent resolution. Rheinberg illumination

5568-476: The image. The deconvolution methods described in the previous section, which removes the PSF induced blur and assigns a mathematically 'correct' origin of light, are used, albeit with slightly different understanding of what the value of a pixel mean. Assuming most of the time , one single fluorophore contributes to one single blob on one single taken image, the blobs in the images can be replaced with their calculated position, vastly improving resolution to well below

5655-425: The intrinsic fluorescence of the protein or by using transmission microscopy. Both methods require an ultraviolet microscope as proteins absorbs light at 280 nm. Protein will also fluorescence at approximately 353 nm when excited with 280 nm light. Since fluorescence emission differs in wavelength (color) from the excitation light, an ideal fluorescent image shows only the structure of interest that

5742-507: The job security of their employees." He made it a point that the success of an employee was based solely on their ability and performance, not on their origin, religion, or political views. In 1896, he reorganized the Zeiss optical works into a cooperative with profit-sharing. His social views were so respected as to be used by the Prussian state as a model and idealized by Alfred Weber in

5829-603: The late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and electron microscopy. Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution of traditional microscopy to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes. Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative elemental analysis. This type of electron microscope, also known as analytical electron microscope, can be

5916-541: The mathematician and physicist Karl Snell, one of Abbe's teachers, with whom he had two daughters. He attained full professor status by 1879. He became director of the Jena astronomical and meteorological observatory in 1878. In 1889, he became a member of the Bavarian Academy of Sciences and Humanities . He also was a member of the Saxon Academy of Sciences. He was relieved of his teaching duties at

6003-466: The microscope focused so that the image is seen at infinity and with both eyes open at all times. Microspectroscopy:spectroscopy with a microscope As resolution depends on the wavelength of the light. Electron microscopy has been developed since the 1930s that use electron beams instead of light. Because of the much smaller wavelength of the electron beam, resolution is far higher. Though less common, X-ray microscopy has also been developed since

6090-570: The microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources, the image is said to be "convolved by the point spread function". The mathematically modeled PSF of a terahertz laser pulsed imaging system is shown on the right. The output of an imaging system can be described using the equation: s ( x , y ) = P S F ( x , y ) ∗ o ( x , y ) + n {\displaystyle s(x,y)=PSF(x,y)*o(x,y)+n} Where n

6177-417: The minima are produced by focusing optics with a numerical aperture NA = n sin ⁡ α {\displaystyle {\mbox{NA}}=n\sin \alpha } , the minimal distance at which two identical objects can be discerned is Δ d = λ 2 n ⋅ sin ⁡ α ⋅ 1 + I I s

6264-399: The molecules to a state in which they cannot send a (fluorescence-) signal upon illumination. This concept is different from for example electron microscopy where instead the used wavelength is much smaller. RESOLFT microscopy is an optical microscopy with very high resolution that can image details in samples that cannot be imaged with conventional or confocal microscopy . Within RESOLFT

6351-409: The more experienced microscopist may prefer a hand drawn image to a photograph. This is because a microscopist with knowledge of the subject can accurately convert a three-dimensional image into a precise two-dimensional drawing. In a photograph or other image capture system however, only one thin plane is ever in good focus. The creation of accurate micrographs requires a microscopical technique using

6438-471: The optical design of a microscope produces an appreciable lateral separation of the two beams we have the case of classical interference microscopy , which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects. An additional technique using interference is interference reflection microscopy (also known as reflected interference contrast, or RIC). It relies on cell adhesion to

6525-428: The organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the function of the original protein in vivo . Growth of protein crystals results in both protein and salt crystals. Both are colorless and microscopic. Recovery of the protein crystals requires imaging which can be done by

6612-422: The position of an object will appear to shift as the focus is changed. This limitation makes techniques like optical sectioning or accurate measurement on the z-axis impossible. Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly transmitted (unscattered) light entering

6699-499: The principles of STED microscopy and GSD microscopy are generalized. Structures that are normally too close to each other to be distinguished are read out sequentially. Within this framework all methods can be explained that operate on molecules that have at least two distinguishable states, where reversible switching between the two states is possible, and where at least one such transition can be optically induced. In most cases fluorescent markers are used, where one state (A) which

6786-455: The protein. The antibodies are then coupled chemically to a fluorophore and used to trace the proteins in the cells under study. Highly efficient fluorescent proteins such as the green fluorescent protein (GFP) have been developed using the molecular biology technique of gene fusion , a process that links the expression of the fluorescent compound to that of the target protein. This combined fluorescent protein is, in general, non-toxic to

6873-508: The range of excitation wavelengths , a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector. See also: total internal reflection fluorescence microscope Neuroscience Confocal laser scanning microscopy uses

6960-425: The restorative methods can actually reassign light to its proper place of origin. Processing fluorescent images in this manner can be an advantage over directly acquiring images without out-of-focus light, such as images from confocal microscopy , because light signals otherwise eliminated become useful information. For 3D deconvolution, one typically provides a series of images taken from different focal planes (called

7047-419: The sample. The resulting image can be detected directly by the eye, imaged on a photographic plate , or captured digitally . The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage, and support, makes up the basic light microscope. The most recent development is the digital microscope , which uses a CCD camera to focus on

7134-422: The slide to produce an interference signal. If there is no cell attached to the glass, there will be no interference. Interference reflection microscopy can be obtained by using the same elements used by DIC, but without the prisms. Also, the light that is being detected is reflected and not transmitted as it is when DIC is employed. When certain compounds are illuminated with high energy light, they emit light of

7221-419: The structures with selective dyes, but this often involves killing and fixing the sample. Staining may also introduce artifacts , which are apparent structural details that are caused by the processing of the specimen and are thus not features of the specimen. In general, these techniques make use of differences in the refractive index of cell structures. Bright-field microscopy is comparable to looking through

7308-527: Was a German businessman, optical engineer, physicist, and social reformer. Together with Otto Schott and Carl Zeiss , he developed numerous optical instruments. He was also a co-owner of Carl Zeiss AG , a German manufacturer of scientific microscopes, astronomical telescopes, planetariums, and other advanced optical systems. Abbe was born 23 January 1840 in Eisenach , Saxe-Weimar-Eisenach , to Georg Adam Abbe and Elisabeth Christina Barchfeldt. He came from

7395-511: Was labeled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye. To block the excitation light from reaching the observer or the detector, filter sets of high quality are needed. These typically consist of an excitation filter selecting

7482-504: Was so impressed as to offer a professorship at the University of Berlin , which he refused due to his ties to Zeiss. Abbe was in the camp of the wide aperturists, arguing that microscopic resolution is ultimately limited by the aperture of the optics, but also argued that depending on application there are other parameters that should be weighted over the aperture in the design of objectives. In Abbe's 1874 paper, titled "A Contribution to

7569-466: Was the first to define the term numerical aperture , as the sine of the half angle multiplied by the refractive index of the medium filling the space between the cover glass and front lens. Abbe is credited by many for discovering the resolution limit of the microscope, and the formula (published in 1873) although in a publication in 1874, Helmholtz states this formula was first derived by Joseph Louis Lagrange , who had died 61 years prior. Helmholtz

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