Super-resolution microscopySuper-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that use the Pendry Superlens and near field scanning optical microscopy) or on the far-field.
Confocal microscopyConfocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), 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 (a process known as optical sectioning) within an object.
MicroscopyMicroscopy 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 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.
Fluorescence microscopeA 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. "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.
Fluorescent tagIn molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.
FluorophoreA fluorophore (or fluorochrome, similarly to a chromophore) 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. Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions).
Total internal reflection fluorescence microscopeA 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. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light.
Green fluorescent proteinThe 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. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.
FluorescenceFluorescence 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 (invisible to the human eye), 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.
Population inversionIn physics, specifically statistical mechanics, a population inversion occurs while a system (such as a group of atoms or molecules) exists in a state in which more members of the system are in higher, excited states than in lower, unexcited energy states. It is called an "inversion" because in many familiar and commonly encountered physical systems, this is not possible. This concept is of fundamental importance in laser science because the production of a population inversion is a necessary step in the workings of a standard laser.
Two-photon excitation microscopyTwo-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where 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. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image.
Optical microscopeThe 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. The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope.
Stimulated emissionStimulated emission is the process by which an incoming photon of a specific frequency can interact with an excited atomic electron (or other excited molecular state), causing it to drop to a lower energy level. The liberated energy transfers to the electromagnetic field, creating a new photon with a frequency, polarization, and direction of travel that are all identical to the photons of the incident wave. This is in contrast to spontaneous emission, which occurs at a characteristic rate for each of the atoms/oscillators in the upper energy state regardless of the external electromagnetic field.
Diffraction-limited systemIn optics, any optical instrument or system a microscope, telescope, or camera has a principal limit to its resolution due to the physics of diffraction. An optical instrument is said to be diffraction-limited if it has reached this limit of resolution performance. Other factors may affect an optical system's performance, such as lens imperfections or aberrations, but these are caused by errors in the manufacture or calculation of a lens, whereas the diffraction limit is the maximum resolution possible for a theoretically perfect, or ideal, optical system.
LaserA laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word laser is an anacronym that originated as 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. A laser differs from other sources of light in that it emits light that is coherent.
Pulsed laserPulsed operation of lasers refers to any laser not classified as continuous wave, so that the optical power appears in pulses of some duration at some repetition rate. This encompasses a wide range of technologies addressing a number of different motivations. Some lasers are pulsed simply because they cannot be run in continuous mode. In other cases the application requires the production of pulses having as large an energy as possible.
MicroscopeA microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope. There are many types of microscopes, and they may be grouped in different ways.
Atomic force microscopyAtomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. Atomic force microscopy (AFM) is a type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.
Spontaneous emissionSpontaneous emission is the process in which a quantum mechanical system (such as a molecule, an atom or a subatomic particle) transits from an excited energy state to a lower energy state (e.g., its ground state) and emits a quantized amount of energy in the form of a photon. Spontaneous emission is ultimately responsible for most of the light we see all around us; it is so ubiquitous that there are many names given to what is essentially the same process.
Ion laserAn ion laser is a gas laser that uses an ionized gas as its lasing medium. Like other gas lasers, ion lasers feature a sealed cavity containing the laser medium and mirrors forming a Fabry–Pérot resonator. Unlike helium–neon lasers, the energy level transitions that contribute to laser action come from ions. Because of the large amount of energy required to excite the ionic transitions used in ion lasers, the required current is much greater, and as a result almost all except for the smallest ion lasers are water-cooled.
