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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.
Spectroscopy is the field of study that measures and interprets the electromagnetic spectra that result from the interaction between electromagnetic radiation and matter as a function of the wavelength or frequency of the radiation. In simpler terms, spectroscopy is the precise study of color as generalized from visible light to all bands of the electromagnetic spectrum.
Raman spectroscopy is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.
Plasma diagnostics are a pool of methods, instruments, and experimental techniques used to measure properties of a plasma, such as plasma components' density, distribution function over energy (temperature), their spatial profiles and dynamics, which enable to derive plasma parameters.
The term biophotonics denotes a combination of biology and photonics, with photonics being the science and technology of generation, manipulation, and detection of photons, quantum units of light. Photonics is related to electronics and photons. Photons play a central role in information technologies, such as fiber optics, the way electrons do in electronics.
Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.
In physics and physical chemistry, time-resolved spectroscopy is the study of dynamic processes in materials or chemical compounds by means of spectroscopic techniques. Most often, processes are studied after the illumination of a material occurs, but in principle, the technique can be applied to any process that leads to a change in properties of a material. With the help of pulsed lasers, it is possible to study processes that occur on time scales as short as 10−16 seconds. All time-resolved spectra are suitable to be analyzed using the two-dimensional correlation method for a correlation map between the peaks.
Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.
Cavity ring-down spectroscopy (CRDS) is a highly sensitive optical spectroscopic technique that enables measurement of absolute optical extinction by samples that scatter and absorb light. It has been widely used to study gaseous samples which absorb light at specific wavelengths, and in turn to determine mole fractions down to the parts per trillion level. The technique is also known as cavity ring-down laser absorption spectroscopy (CRLAS).
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.
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. "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.
Resonance Raman spectroscopy is a variant of Raman spectroscopy in which the incident photon energy is close in energy to an electronic transition of a compound or material under examination. This similarity in energy (resonance) leads to greatly increased intensity of the Raman scattering of certain vibrational modes, compared to ordinary Raman spectroscopy.
Two-photon excitation microscopy 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. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.
Coherent anti-Stokes Raman spectroscopy, also called Coherent anti-Stokes Raman scattering spectroscopy (CARS), is a form of spectroscopy used primarily in chemistry, physics and related fields. It is sensitive to the same vibrational signatures of molecules as seen in Raman spectroscopy, typically the nuclear vibrations of chemical bonds. Unlike Raman spectroscopy, CARS employs multiple photons to address the molecular vibrations, and produces a coherent signal. As a result, CARS is orders of magnitude stronger than spontaneous Raman emission. CARS is a third-order nonlinear optical process involving three laser beams: a pump beam of frequency ωp, a Stokes beam of frequency ωS and a probe beam at frequency ωpr. These beams interact with the sample and generate a coherent optical signal at the anti-Stokes frequency (ωpr+ωp-ωS). The latter is resonantly enhanced when the frequency difference between the pump and the Stokes beams (ωp-ωS) coincides with the frequency of a Raman resonance, which is the basis of the technique's intrinsic vibrational contrast mechanism.
Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis provides kinetic parameters of the physical processes underlying the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.
Molecular tagging velocimetry (MTV) is a specific form of flow velocimetry, a technique for determining the velocity of currents in fluids such as air and water. In its simplest form, a single "write" laser beam is shot once through the sample space. Along its path an optically induced chemical process is initiated, resulting in the creation of a new chemical species or in changing the internal energy state of an existing one, so that the molecules struck by the laser beam can be distinguished from the rest of the fluid. Such molecules are said to be "tagged".
Ultrafast laser spectroscopy is a spectroscopic technique that uses ultrashort pulse lasers for the study of dynamics on extremely short time scales. Different methods are used to examine the dynamics of charge carriers, atoms, and molecules. Many different procedures have been developed spanning different time scales and photon energy ranges; some common methods are listed below.
Richard Neil Zare is the Marguerite Blake Wilbur Professor in Natural Science and a Professor of Chemistry at Stanford University. Throughout his career, Zare has made a considerable impact in physical chemistry and analytical chemistry, particularly through the development of laser-induced fluorescence (LIF) and the study of chemical reactions at the molecular and nanoscale level. LIF is an extremely sensitive technique with applications ranging from analytical chemistry and molecular biology to astrophysics. One of its applications was the sequencing of the human genome.
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.
Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of time-resolved fluorometry (TRF) with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers. TR-FRET combines the low background aspect of TRF with the homogeneous assay format of FRET. The resulting assay provides an increase in flexibility, reliability and sensitivity in addition to higher throughput and fewer false positive/false negative results. FRET involves two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source produces an energy transfer to the acceptor if the two are within a given proximity to each other. The acceptor in turn emits light at its characteristic wavelength.
References
- 1 2 Kaye, T.G.; Falk, A.R.; Pittman, M.; Sereno, P.C.; Martin, L.D.; Burnham, D.A.; Gong, E.; Xu, X.; Wang, Y. (2015). "Laser-Stimulated Fluorescence in Paleontology". PLOS ONE. 10 (5): e0125923. Bibcode:2015PLoSO..1025923K. doi: 10.1371/journal.pone.0125923 . PMC 4446324 . PMID 26016843.
- ↑ Kinsey, J L (1977). "Laser-Induced Fluorescence". Annual Review of Physical Chemistry. 28 (1): 349–372. Bibcode:1977ARPC...28..349K. doi:10.1146/annurev.pc.28.100177.002025. ISSN 0066-426X.
- ↑ Richard W. Solarz; Jeffrey A. Paisner (29 September 1986). Laser Spectroscopy and its Applications. CRC Press. pp. 623–. ISBN 978-0-8247-7525-4.
- ↑ Tango, William J. (1968). "Spectroscopy of K2 Using Laser-Induced Fluorescence". The Journal of Chemical Physics. 49 (10): 4264–4268. Bibcode:1968JChPh..49.4264T. doi:10.1063/1.1669869. ISSN 0021-9606.
- 1 2 Zare, R. N. (2012). "My Life with LIF: A Personal Account of Developing Laser-Induced Fluorescence". Annual Review of Analytical Chemistry. 5: 1–14. Bibcode:2012ARAC....5....1Z. doi: 10.1146/annurev-anchem-062011-143148 . PMID 22149473. S2CID 44885260.
- ↑ Takeyuki, Tanaka; et al. "The Application of Laser Induced Fluorescence Spectroscopy to Measurement of Purity Level in Textiles". Jido Seigyo Rengo Koenkai. Archived from the original on 13 February 2011. Retrieved 11 October 2008.
- ↑ Lin, Yang-Wei; Chiu, Tai-Chia; Chang, Huan-Tsung (2003). "Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis" (PDF). Journal of Chromatography B. 793 (1): 37–48. doi:10.1016/s1570-0232(03)00363-5. ISSN 1570-0232. PMID 12880853.
- ↑ Skiff, Fred; Bollinger, John (2004). "Mini-conference on laser-induced fluorescence in plasmas". Physics of Plasmas. 11 (5): 2972. Bibcode:2004PhPl...11.2972S. doi:10.1063/1.1668287.
- ↑ Stern, R. A.; Johnson, J. A. (1975). "Plasma Ion Diagnostics Using Resonant Fluorescence". Physical Review Letters. 34 (25): 1548–1551. Bibcode:1975PhRvL..34.1548S. doi:10.1103/PhysRevLett.34.1548.
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