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Fluorescence microscope

From Wikipedia, in a visual modern way
An upright fluorescence microscope (Olympus BX61) with the fluorescence filter cube turret above the objective lenses, coupled with a digital camera.
An upright fluorescence microscope (Olympus BX61) with the fluorescence filter cube turret above the objective lenses, coupled with a digital camera.
Fluorescence and confocal microscopes operating principle

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances.[1][2] "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a 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.[3]

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Optical microscope

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 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.

Fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

Scattering

Scattering

Scattering is a term used in physics to describe a wide range of physical processes where moving particles or radiation of some form, such as light or sound, are forced to deviate from a straight trajectory by localized non-uniformities in the medium through which they pass. In conventional use, this also includes deviation of reflected radiation from the angle predicted by the law of reflection. Reflections of radiation that undergo scattering are often called diffuse reflections and unscattered reflections are called specular (mirror-like) reflections. Originally, the term was confined to light scattering. As more "ray"-like phenomena were discovered, the idea of scattering was extended to them, so that William Herschel could refer to the scattering of "heat rays" in 1800. John Tyndall, a pioneer in light scattering research, noted the connection between light scattering and acoustic scattering in the 1870s. Near the end of the 19th century, the scattering of cathode rays and X-rays was observed and discussed. With the discovery of subatomic particles and the development of quantum theory in the 20th century, the sense of the term became broader as it was recognized that the same mathematical frameworks used in light scattering could be applied to many other phenomena.

Reflection (physics)

Reflection (physics)

Reflection is the change in direction of a wavefront at an interface between two different media so that the wavefront returns into the medium from which it originated. Common examples include the reflection of light, sound and water waves. The law of reflection says that for specular reflection the angle at which the wave is incident on the surface equals the angle at which it is reflected.

Attenuation

Attenuation

In physics, attenuation is the gradual loss of flux intensity through a medium. For instance, dark glasses attenuate sunlight, lead attenuates X-rays, and water and air attenuate both light and sound at variable attenuation rates.

Absorption (electromagnetic radiation)

Absorption (electromagnetic radiation)

In physics, absorption of electromagnetic radiation is how matter takes up a photon's energy — and so transforms electromagnetic energy into internal energy of the absorber. A notable effect is attenuation, or the gradual reduction of the intensity of light waves as they propagate through a medium. Although the absorption of waves does not usually depend on their intensity, in certain conditions (optics) the medium's transparency changes by a factor that varies as a function of wave intensity, and saturable absorption occurs.

Optical sectioning

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.

Principle

The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope are a light source (xenon arc lamp or mercury-vapor lamp are common; more advanced forms are high-power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter), and the emission filter (see figure below). The filters and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.[1] In this manner, the distribution of a single fluorophore (color) is imaged at a time. Multi-color images of several types of fluorophores must be composed by combining several single-color images.[1]

Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective). These microscopes are widely used in biology and are the basis for more advanced microscope designs, such as the confocal microscope and the total internal reflection fluorescence microscope (TIRF).

Epifluorescence microscopy

Schematic of a fluorescence microscope.
Schematic of a fluorescence microscope.

The majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the diagram. Light of the excitation wavelength illuminates the specimen through the objective lens. The fluorescence emitted by the specimen is focused to the detector by the same objective that is used for the excitation which for greater resolution will need objective lens with higher numerical aperture. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence method therefore gives a high signal-to-noise ratio. The dichroic beamsplitter acts as a wavelength specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any remaining excitation light back towards the source.

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Wavelength

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, troughs, or zero crossings, and is a characteristic of both traveling waves and standing waves, as well as other spatial wave patterns. The inverse of the wavelength is called the spatial frequency. Wavelength is commonly designated by the Greek letter lambda (λ). The term wavelength is also sometimes applied to modulated waves, and to the sinusoidal envelopes of modulated waves or waves formed by interference of several sinusoids.

Fluorophore

Fluorophore

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Xenon arc lamp

Xenon arc lamp

A xenon arc lamp is a highly specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure. It produces a bright white light to simulate sunlight, with applications in movie projectors in theaters, in searchlights, and for specialized uses in industry and research. For instance, Xenon arc lamps with mercury lamps are the two most common lamps used in wide-field fluorescence microscopes.

Mercury-vapor lamp

Mercury-vapor lamp

A mercury-vapor lamp is a gas-discharge lamp that uses an electric arc through vaporized mercury to produce light. The arc discharge is generally confined to a small fused quartz arc tube mounted within a larger soda lime or borosilicate glass bulb. The outer bulb may be clear or coated with a phosphor; in either case, the outer bulb provides thermal insulation, protection from the ultraviolet radiation the light produces, and a convenient mounting for the fused quartz arc tube.

Laser

Laser

A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The term is an acronym for light amplification by stimulated emission of radiation. The first laser was built in 1960 by Theodore Maiman at Hughes Research Laboratories, based on theoretical work by Charles H. Townes and Arthur Leonard Schawlow.

Excitation filter

Excitation filter

An excitation filter is a high quality optical-glass filter commonly used in fluorescence microscopy and spectroscopic applications for selection of the excitation wavelength of light from a light source. Most excitation filters select light of relatively short wavelengths from an excitation light source, as only those wavelengths would carry enough energy to cause the object the microscope is examining to fluoresce sufficiently. The excitation filters used may come in two main types — short pass filters and band pass filters. Variations of these filters exist in the form of notch filters or deep blocking filters. Other forms of excitation filters include the use of monochromators, wedge prisms coupled with a narrow slit and the use of holographic diffraction gratings, etc. [for beam diffraction of white laser light into the required excitation wavelength ].

Dichroic filter

Dichroic filter

A dichroic filter, thin-film filter, or interference filter is a color filter used to selectively pass light of a small range of colors while reflecting other colors. By comparison, dichroic mirrors and dichroic reflectors tend to be characterized by the colors of light that they reflect, rather than the colors they pass.

Confocal microscopy

Confocal microscopy

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.

Total internal reflection fluorescence microscope

Total internal reflection fluorescence microscope

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

Objective (optics)

Objective (optics)

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, binoculars, telescopes, cameras, slide projectors, CD players and many other optical instruments. Objectives are also called object lenses, object glasses, or objective glasses.

Fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

Numerical aperture

Numerical aperture

In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one material to another, provided there is no refractive power at the interface. The exact definition of the term varies slightly between different areas of optics. Numerical aperture is commonly used in microscopy to describe the acceptance cone of an objective, and in fiber optics, in which it describes the range of angles within which light that is incident on the fiber will be transmitted along it.

Light sources

Fluorescence microscopy requires intense, near-monochromatic, illumination which some widespread light sources, like halogen lamps cannot provide.[4] Four main types of light source are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers, supercontinuum sources, and high-power LEDs. Lasers are most widely used for more complex fluorescence microscopy techniques like confocal microscopy and total internal reflection fluorescence microscopy while xenon lamps, and mercury lamps, and LEDs with a dichroic excitation filter are commonly used for widefield epifluorescence microscopes. By placing two microlens arrays into the illumination path of a widefield epifluorescence microscope,[5] highly uniform illumination with a coefficient of variation of 1-2% can be achieved.

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Halogen lamp

Halogen lamp

A halogen lamp is an incandescent lamp consisting of a tungsten filament sealed in a compact transparent envelope that is filled with a mixture of an inert gas and a small amount of a halogen, such as iodine or bromine. The combination of the halogen gas and the tungsten filament produces a halogen-cycle chemical reaction, which redeposits evaporated tungsten on the filament, increasing its life and maintaining the clarity of the envelope. This allows the filament to operate at a higher temperature than a standard incandescent lamp of similar power and operating life; this also produces light with higher luminous efficacy and color temperature. The small size of halogen lamps permits their use in compact optical systems for projectors and illumination. The small glass envelope may be enclosed in a much larger outer glass bulb, which has a lower temperature, protects the inner bulb from contamination, and makes the bulb mechanically more similar to a conventional lamp.

Xenon arc lamp

Xenon arc lamp

A xenon arc lamp is a highly specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure. It produces a bright white light to simulate sunlight, with applications in movie projectors in theaters, in searchlights, and for specialized uses in industry and research. For instance, Xenon arc lamps with mercury lamps are the two most common lamps used in wide-field fluorescence microscopes.

Mercury-vapor lamp

Mercury-vapor lamp

A mercury-vapor lamp is a gas-discharge lamp that uses an electric arc through vaporized mercury to produce light. The arc discharge is generally confined to a small fused quartz arc tube mounted within a larger soda lime or borosilicate glass bulb. The outer bulb may be clear or coated with a phosphor; in either case, the outer bulb provides thermal insulation, protection from the ultraviolet radiation the light produces, and a convenient mounting for the fused quartz arc tube.

Excitation filter

Excitation filter

An excitation filter is a high quality optical-glass filter commonly used in fluorescence microscopy and spectroscopic applications for selection of the excitation wavelength of light from a light source. Most excitation filters select light of relatively short wavelengths from an excitation light source, as only those wavelengths would carry enough energy to cause the object the microscope is examining to fluoresce sufficiently. The excitation filters used may come in two main types — short pass filters and band pass filters. Variations of these filters exist in the form of notch filters or deep blocking filters. Other forms of excitation filters include the use of monochromators, wedge prisms coupled with a narrow slit and the use of holographic diffraction gratings, etc. [for beam diffraction of white laser light into the required excitation wavelength ].

Laser

Laser

A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The term is an acronym for light amplification by stimulated emission of radiation. The first laser was built in 1960 by Theodore Maiman at Hughes Research Laboratories, based on theoretical work by Charles H. Townes and Arthur Leonard Schawlow.

Supercontinuum

Supercontinuum

In optics, a supercontinuum is formed when a collection of nonlinear processes act together upon a pump beam in order to cause severe spectral broadening of the original pump beam, for example using a microstructured optical fiber. The result is a smooth spectral continuum. There is no consensus on how much broadening constitutes a supercontinuum; however researchers have published work claiming as little as 60 nm of broadening as a supercontinuum. There is also no agreement on the spectral flatness required to define the bandwidth of the source, with authors using anything from 5 dB to 40 dB or more. In addition the term supercontinuum itself did not gain widespread acceptance until this century, with many authors using alternative phrases to describe their continua during the 1970s, 1980s and 1990s.

Confocal microscopy

Confocal microscopy

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.

Total internal reflection fluorescence microscope

Total internal reflection fluorescence microscope

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

Microlens

Microlens

A microlens is a small lens, generally with a diameter less than a millimetre (mm) and often as small as 10 micrometres (µm). The small sizes of the lenses means that a simple design can give good optical quality but sometimes unwanted effects arise due to optical diffraction at the small features. A typical microlens may be a single element with one plane surface and one spherical convex surface to refract the light. Because micro-lenses are so small, the substrate that supports them is usually thicker than the lens and this has to be taken into account in the design. More sophisticated lenses may use aspherical surfaces and others may use several layers of optical material to achieve their design performance.

Coefficient of variation

Coefficient of variation

In probability theory and statistics, the coefficient of variation (CV), also known as relative standard deviation (RSD), is a standardized measure of dispersion of a probability distribution or frequency distribution. It is often expressed as a percentage, and is defined as the ratio of the standard deviation to the mean . The CV or RSD is widely used in analytical chemistry to express the precision and repeatability of an assay. It is also commonly used in fields such as engineering or physics when doing quality assurance studies and ANOVA gauge R&R, by economists and investors in economic models, and in neuroscience.

Sample preparation

3D-animation of the diatom Corethron sp.
Displays overlays from four fluorescent channels
(a) Green: [DiOC6(3) fluorescence] - stains cellular membranes indicating the core cell bodies
(b) Cyan: [PLL-A546 fluorescence] - generic counterstain for visualising eukaryotic cell surfaces
(c) Blue: [Hoechst fluorescence] - stains DNA, identifies nuclei
(d) Red: [chlorophyll autofluorescence] - resolves chloroplasts [6]
The animation starts by overlaying all available fluorescent channels, and then clarifies the visualisation by switching channels on and off
A sample of herring sperm stained with SYBR green in a cuvette illuminated by blue light in an epifluorescence microscope. The SYBR green in the sample binds to the herring sperm DNA and, once bound, fluoresces giving off green light when illuminated by blue light.
A sample of herring sperm stained with SYBR green in a cuvette illuminated by blue light in an epifluorescence microscope. The SYBR green in the sample binds to the herring sperm DNA and, once bound, fluoresces giving off green light when illuminated by blue light.

In order for a sample to be suitable for fluorescence microscopy it must be fluorescent. There are several methods of creating a fluorescent sample; the main techniques are labelling with fluorescent stains or, in the case of biological samples, expression of a fluorescent protein. Alternatively the intrinsic fluorescence of a sample (i.e., autofluorescence) can be used.[1] In the life sciences fluorescence microscopy is a powerful tool which allows the specific and sensitive staining of a specimen in order to detect the distribution of proteins or other molecules of interest. As a result, there is a diverse range of techniques for fluorescent staining of biological samples.

Biological fluorescent stains

Many fluorescent stains have been designed for a range of biological molecules. Some of these are small molecules which are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains such as DAPI and Hoechst (excited by UV wavelength light) and DRAQ5 and DRAQ7 (optimally excited by red light) which all bind the minor groove of DNA, thus labeling the nuclei of cells. Others are drugs, toxins, or peptides which bind specific cellular structures and have been derivatised with a fluorescent reporter. A major example of this class of fluorescent stain is phalloidin, which is used to stain actin fibers in mammalian cells. A new peptide, known as the Collagen Hybridizing Peptide, can also be conjugated with fluorophores and used to stain denatured collagen fibers. Staining of the plant cell walls is performed using stains or dyes that bind cellulose or pectin. The quest for fluorescent probes with a high specificity that also allow live imaging of plant cells is ongoing.[7]

There are many fluorescent molecules called fluorophores or fluorochromes such as fluorescein, Alexa Fluors, or DyLight 488, which can be chemically linked to a different molecule which binds the target of interest within the sample.

Immunofluorescence

Main article: Immunofluorescence

Immunofluorescence is a technique which uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the molecule of interest. A fluorophore can be directly conjugated to the primary antibody. Alternatively a secondary antibody, conjugated to a fluorophore, which binds specifically to the first antibody can be used. For example, a primary antibody raised in a mouse which recognises tubulin combined with a secondary anti-mouse antibody derivatised with a fluorophore could be used to label microtubules in a cell.

Fluorescent proteins

See also: Fluorescent protein

The modern understanding of genetics and the techniques available for modifying DNA allow scientists to genetically modify proteins to also carry a fluorescent protein reporter. In biological samples this allows a scientist to directly make a protein of interest fluorescent. The protein location can then be directly tracked, including in live cells.

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Herring

Herring

Herring are forage fish, mostly belonging to the family of Clupeidae.

Cuvette

Cuvette

In laboratories, a cuvette is a small tube-like container with straight sides and a circular or square cross-section. It is sealed at one end, and made of a clear, transparent material such as plastic, glass, or fused quartz. Cuvettes are designed to hold samples for spectroscopic measurement, where a beam of light is passed through the sample within the cuvette to measure the absorbance, transmittance, fluorescence intensity, fluorescence polarization, or fluorescence lifetime of the sample. This measurement is done with a spectrophotometer.

DNA

DNA

Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Fluorescent protein

Fluorescent protein

Fluorescent proteins include:Green fluorescent protein (GFP) Yellow fluorescent protein (YFP) Red fluorescent protein (RFP)

Autofluorescence

Autofluorescence

Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores).

Nucleic acid

Nucleic acid

Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomer components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is the ribose derivative deoxyribose, the polymer is DNA.

DAPI

DAPI

DAPI, or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore provides a marker for membrane viability.

Hoechst stain

Hoechst stain

Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These bis-benzimides were originally developed by Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33,342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation–emission spectra.

Actin

Actin

Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of over 100 μM; its mass is roughly 42 kDa, with a diameter of 4 to 7 nm.

Mammal

Mammal

A mammal is a vertebrate animal of the class Mammalia. Mammals are characterized by the presence of milk-producing mammary glands for feeding their young, a neocortex region of the brain, fur or hair, and three middle ear bones. These characteristics distinguish them from reptiles and birds, which they diverged from in the Carboniferous Period over 300 million years ago. Around 6,400 extant species of mammals have been described and divided into 29 orders.

Fluorophore

Fluorophore

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Cell wall

Cell wall

A cell wall is a structural layer surrounding some types of cells, just outside the cell membrane. It can be tough, flexible, and sometimes rigid. It provides the cell with both structural support and protection, and also acts as a filtering mechanism. Cell walls are absent in many eukaryotes, including animals, but are present in some other ones like fungi, algae and plants, and in most prokaryotes. A major function is to act as pressure vessels, preventing over-expansion of the cell when water enters.

Limitations

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence. Photobleaching can severely limit the time over which a sample can be observed by fluorescence microscopy. Several techniques exist to reduce photobleaching such as the use of more robust fluorophores, by minimizing illumination, or by using photoprotective scavenger chemicals.

Fluorescence microscopy with fluorescent reporter proteins has enabled analysis of live cells by fluorescence microscopy, however cells are susceptible to phototoxicity, particularly with short wavelength light. Furthermore, fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.

Unlike transmitted and reflected light microscopy techniques, fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence. For example, observing a tissue sample prepared with a fluorescent DNA stain by fluorescence microscopy only reveals the organization of the DNA within the cells and reveals nothing else about the cell morphologies.

Computational techniques that propose to estimate the fluorescent signal from non-fluorescent images (such as brightfield) may reduce these concerns.[8] In general, these approaches involve training a deep convolutional neural network on stained cells and then estimating the fluorescence on unstained samples. Thus by decoupling the cells under investigation from the cells used to train the network, imaging can performed quicker and with reduced phototoxicity.

Sub-diffraction techniques

See also: Super resolution microscopy and Correlative Light-Electron Microscopy

The wave nature of light limits the size of the spot to which light can be focused due to the diffraction limit. This limitation was described in the 19th century by Ernst Abbe and "limits an optical microscope's resolution to approximately half of the wavelength of the light used." Fluorescence microscopy is central to many techniques which aim to reach past this limit by specialized optical configurations.

Several improvements in microscopy techniques have been invented in the 20th century and have resulted in increased resolution and contrast to some extent. However they did not overcome the diffraction limit. In 1978 first theoretical ideas have been developed to break this barrier by using a 4Pi microscope as a confocal laser scanning fluorescence microscope where the light is focused ideally from all sides to a common focus which is used to scan the object by 'point-by-point' excitation combined with 'point-by-point' detection.[9] However, the first experimental demonstration of the 4pi microscope took place in 1994.[10] 4Pi microscopy maximizes the amount of available focusing directions by using two opposing objective lenses or two-photon excitation microscopy using redshifted light and multi-photon excitation.

Integrated correlative microscopy combines a fluorescence microscope with an electron microscope. This allows one to visualize ultrastructure and contextual information with the electron microscope while using the data from the fluorescence microscope as a labelling tool.[11]

The first technique to really achieve a sub-diffraction resolution was STED microscopy, proposed in 1994. This method and all techniques following the RESOLFT concept rely on a strong non-linear interaction between light and fluorescing molecules. The molecules are driven strongly between distinguishable molecular states at each specific location, so that finally light can be emitted at only a small fraction of space, hence an increased resolution.

As well in the 1990s another super resolution microscopy method based on wide field microscopy has been developed. Substantially improved size resolution of cellular nanostructures stained with a fluorescent marker was achieved by development of SPDM localization microscopy and the structured laser illumination (spatially modulated illumination, SMI).[12] Combining the principle of SPDM with SMI resulted in the development of the Vertico SMI microscope.[13][14] Single molecule detection of normal blinking fluorescent dyes like green fluorescent protein (GFP) can be achieved by using a further development of SPDM the so-called SPDMphymod technology which makes it possible to detect and count two different fluorescent molecule types at the molecular level (this technology is referred to as two-color localization microscopy or 2CLM).[15]

Alternatively, the advent of photoactivated localization microscopy could achieve similar results by relying on blinking or switching of single molecules, where the fraction of fluorescing molecules is very small at each time. This stochastic response of molecules on the applied light corresponds also to a highly nonlinear interaction, leading to subdiffraction resolution.

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Ernst Abbe

Ernst Abbe

Ernst Karl Abbe was a German physicist, optical scientist, entrepreneur, 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.

Two-photon excitation microscopy

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 thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of NIR 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 this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.

Correlative light-electron microscopy

Correlative light-electron microscopy

Correlative light-electron microscopy (CLEM) is the combination of an optical microscope - usually a fluorescence microscope - with an electron microscope. In an integrated CLEM system, the sample is imaged using an electron beam and an optical light path simultaneously. Traditionally, samples would be imaged using two separate microscopy modalities, potentially at different facilities and using different sample preparation methods. Integrated CLEM is thus considered to be beneficial because the methodology is quicker and easier, and it reduces the chance of changes in the sample during the process of data collection. Overlay of the two images is thus performed automatically as a result of the integration of two microscopes.

STED microscopy

STED microscopy

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

RESOLFT

RESOLFT

RESOLFT, an acronym for REversible Saturable OpticaL Fluorescence Transitions, denotes a group of optical fluorescence microscopy techniques with very high resolution. Using standard far field visible light optics a resolution far below the diffraction limit down to molecular scales can be obtained.

Nanostructure

Nanostructure

A nanostructure is a structure of intermediate size between microscopic and molecular structures. Nanostructural detail is microstructure at nanoscale.

Fluorescence intermittency

Fluorescence intermittency

Fluorescence intermittency, or blinking, is the phenomenon of random switching between ON (bright) and OFF (dark) states of the emitter under its continuous excitation. It is a common property of the nanoscale emitters related to the competition between the radiative and non-radiative relaxation pathways. The peculiar feature of such blinking in most cases is the power-law statistics of the ON and OFF time distributions, meaning that the measurements of the time-averaged intensity of a single emitter is not reproducible in different experiments and implying a complex dynamics of the involved process. In other words, in one experiment the emitter can blink frequently, while in another it may stay ON for almost entire length of the experiment.

Green fluorescent protein

Green fluorescent protein

The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.

Photoactivated localization microscopy

Photoactivated localization microscopy

Photo-activated localization microscopy and stochastic optical reconstruction microscopy (STORM) are widefield fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit. The methods were proposed in 2006 in the wake of a general emergence of optical super-resolution microscopy methods, and were featured as Methods of the Year for 2008 by the Nature Methods journal. The development of PALM as a targeted biophysical imaging method was largely prompted by the discovery of new species and the engineering of mutants of fluorescent proteins displaying a controllable photochromism, such as photo-activatible GFP. However, the concomitant development of STORM, sharing the same fundamental principle, originally made use of paired cyanine dyes. One molecule of the pair, when excited near its absorption maximum, serves to reactivate the other molecule to the fluorescent state.

Fluorescence micrograph gallery

  • A z-projection of an osteosarcoma cell, stained with phalloidin to visualise actin filaments. The image was taken on a confocal microscope, and the subsequent deconvolution was done using an experimentally derived point spread function.

    A z-projection of an osteosarcoma cell, stained with phalloidin to visualise actin filaments. The image was taken on a confocal microscope, and the subsequent deconvolution was done using an experimentally derived point spread function.

  • Epifluorescent imaging of the three components in a dividing human cancer cell. DNA is stained blue, a protein called INCENP is green, and the microtubules are red. Each fluorophore is imaged separately using a different combination of excitation and emission filters, and the images are captured sequentially using a digital CCD camera, then overlaid to give a complete image.

    Epifluorescent imaging of the three components in a dividing human cancer cell. DNA is stained blue, a protein called INCENP is green, and the microtubules are red. Each fluorophore is imaged separately using a different combination of excitation and emission filters, and the images are captured sequentially using a digital CCD camera, then overlaid to give a complete image.

  • Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are labeled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial (BPAE) cells

    Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are labeled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial (BPAE) cells

  • 3D dual-color super-resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568. LIMON microscopy

    3D dual-color super-resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568. LIMON microscopy

  • Human lymphocyte nucleus stained with DAPI with chromosome 13 (green) and 21 (red) centromere probes hybridized (Fluorescent in situ hybridization (FISH))

    Human lymphocyte nucleus stained with DAPI with chromosome 13 (green) and 21 (red) centromere probes hybridized (Fluorescent in situ hybridization (FISH))

  • Yeast cell membrane visualized by some membrane proteins fused with RFP and GFP fluorescent markers. Imposition of light from both of markers results in yellow color.

    Yeast cell membrane visualized by some membrane proteins fused with RFP and GFP fluorescent markers. Imposition of light from both of markers results in yellow color.

  • Super-resolution microscopy: Single YFP molecule detection in a human cancer cell. Typical distance measurements in the 15 nm range measured with a Vertico-SMI/SPDMphymod microscope

    Super-resolution microscopy: Single YFP molecule detection in a human cancer cell. Typical distance measurements in the 15 nm range measured with a Vertico-SMI/SPDMphymod microscope

  • Super-resolution microscopy: Co-localization microscopy (2CLM) with GFP and RFP fusion proteins (nucleus of a bone cancer cell) 120.000 localized molecules in a wide-field area (470 µm2) measured with a Vertico-SMI/SPDMphymod microscope

    Super-resolution microscopy: Co-localization microscopy (2CLM) with GFP and RFP fusion proteins (nucleus of a bone cancer cell) 120.000 localized molecules in a wide-field area (470 µm2) measured with a Vertico-SMI/SPDMphymod microscope

  • Fluorescence microscopy of DNA Expression in the Human Wild-Type and P239S Mutant Palladin.

    Fluorescence microscopy of DNA Expression in the Human Wild-Type and P239S Mutant Palladin.

  • Fluorescence microscopy images of sun flares pathology in a blood cell showing the affected areas in red.

    Fluorescence microscopy images of sun flares pathology in a blood cell showing the affected areas in red.

Discover more about Fluorescence micrograph gallery related topics

DNA

DNA

Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Protein

Protein

Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.

INCENP

INCENP

Inner centromere protein is a protein that in humans is encoded by the INCENP gene.

Microtubule

Microtubule

Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nm and have an inner diameter between 11 and 15 nm. They are formed by the polymerization of a dimer of two globular proteins, alpha and beta tubulin into protofilaments that can then associate laterally to form a hollow tube, the microtubule. The most common form of a microtubule consists of 13 protofilaments in the tubular arrangement.

Fluorophore

Fluorophore

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Palladin

Palladin

Palladin is a protein that in humans is encoded by the PALLD gene. Palladin is a component of actin-containing microfilaments that control cell shape, adhesion, and contraction.

Source: "Fluorescence microscope", Wikipedia, Wikimedia Foundation, (2023, February 20th), https://en.wikipedia.org/wiki/Fluorescence_microscope.

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Further Reading

Microscopy

Microscope

Optical microscope

Fluorophore

Immunofluorescence

Confocal microscopy

Two-photon excitation microscopy

Autofluorescence

Vertico spatially modulated illumination

Fluorescence in the life sciences

Light sheet fluorescence microscopy

Live-cell imaging

Fluorescence imaging
See also
References
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