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SmFRET Scheme
Single molecule fluorescence (or Förster) resonance energy transfer (or smFRET) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected on a single molecule level. In contrast to "ensemble FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of biomolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are particula ...
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Biophysics
Biophysics is an interdisciplinary science that applies approaches and methods traditionally used in physics to study biological phenomena. Biophysics covers all scales of biological organization, from molecular to organismic and populations. Biophysical research shares significant overlap with biochemistry, molecular biology, physical chemistry, physiology, nanotechnology, bioengineering, computational biology, biomechanics, developmental biology and systems biology. The term ''biophysics'' was originally introduced by Karl Pearson in 1892.Roland Glaser. Biophysics: An Introduction'. Springer; 23 April 2012. . The term ''biophysics'' is also regularly used in academia to indicate the study of the physical quantities (e.g. electric current, temperature, stress, entropy) in biological systems. Other biological sciences also perform research on the biophysical properties of living organisms including molecular biology, cell biology, chemical biology, and biochemistry. Overview ...
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Photobleaching
In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules. Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores. The number of excitation cycles to achieve full bleaching varies. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy. However, photobleaching may also be used prior to applying the (primarily antibody-linked) fluorescent molecules, in an attempt to quench autofluorescence. This can help improve the signal-to-noise ratio. Photobleaching may also be exploited to study ...
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Poisson Distribution
In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time or space if these events occur with a known constant mean rate and Statistical independence, independently of the time since the last event. It is named after France, French mathematician Siméon Denis Poisson (; ). The Poisson distribution can also be used for the number of events in other specified interval types such as distance, area, or volume. For instance, a call center receives an average of 180 calls per hour, 24 hours a day. The calls are independent; receiving one does not change the probability of when the next one will arrive. The number of calls received during any minute has a Poisson probability distribution with mean 3: the most likely numbers are 2 and 3 but 1 and 4 are also likely and there is a small probability of it being as low as zero and a very smal ...
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White Noise
In signal processing, white noise is a random signal having equal intensity at different frequencies, giving it a constant power spectral density. The term is used, with this or similar meanings, in many scientific and technical disciplines, including physics, acoustical engineering, telecommunications, and statistical forecasting. White noise refers to a statistical model for signals and signal sources, rather than to any specific signal. White noise draws its name from white light, although light that appears white generally does not have a flat power spectral density over the visible band. In discrete time, white noise is a discrete signal whose samples are regarded as a sequence of serially uncorrelated random variables with zero mean and finite variance; a single realization of white noise is a random shock. Depending on the context, one may also require that the samples be independent and have identical probability distribution (in other words independent and iden ...
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Shot Noise
Shot noise or Poisson noise is a type of noise which can be modeled by a Poisson process. In electronics shot noise originates from the discrete nature of electric charge. Shot noise also occurs in photon counting in optical devices, where shot noise is associated with the particle nature of light. Origin In a statistical experiment such as tossing a fair coin and counting the occurrences of heads and tails, the numbers of heads and tails after many throws will differ by only a tiny percentage, while after only a few throws outcomes with a significant excess of heads over tails or vice versa are common; if an experiment with a few throws is repeated over and over, the outcomes will fluctuate a lot. From the law of large numbers, one can show that the relative fluctuations reduce as the reciprocal square root of the number of throws, a result valid for all statistical fluctuations, including shot noise. Shot noise exists because phenomena such as light and electric current co ...
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Motion Blur
Motion blur is the apparent streaking of moving objects in a photograph or a sequence of frames, such as a film or animation. It results when the image being recorded changes during the recording of a single exposure, due to rapid movement or long exposure. Usages / Effects of motion blur Photography When a camera creates an image, that image does not represent a single instant of time. Because of technological constraints or artistic requirements, the image may represent the scene over a period of time. Most often this exposure time is brief enough that the image captured by the camera appears to capture an instantaneous moment, but this is not always so, and a fast moving object or a longer exposure time may result in blurring artifacts which make this apparent. As objects in a scene move, an image of that scene must represent an integration of all positions of those objects, as well as the camera's viewpoint, over the period of exposure determined by the shutter speed. In ...
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SmFRET Trajectory
Single molecule fluorescence (or Förster) resonance energy transfer (or smFRET) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected on a single molecule level. In contrast to "ensemble FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of biomolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are parti ...
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Quantum Yield
The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system. Applications Fluorescence spectroscopy The fluorescence quantum yield is defined as the ratio of the number of photons emitted to the number of photons absorbed.Lakowicz, Joseph R. ''Principles of Fluorescence Spectroscopy'' (Kluwer Academic / Plenum Publishers 1999) p.10. Fluorescence quantum yield is measured on a scale from 0 to 1.0, but is often represented as a percentage. A quantum yield of 1.0 (100%) describes a process where each photon absorbed results in a photon emitted. Substances with the largest quantum yields, such as rhodamines, display the brightest emissions; however, compounds with quantum yields of 0.10 are still considered quite fluorescent. Quantum yield is defined by the fraction of excited state fluorophores that decay through fluorescence: where \Phi_ is the fluorescence quantum yield, k_ is the rate constant f ...
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Charge-coupled Device
A charge-coupled device (CCD) is an integrated circuit containing an array of linked, or coupled, capacitors. Under the control of an external circuit, each capacitor can transfer its electric charge to a neighboring capacitor. CCD sensors are a major technology used in digital imaging. In a CCD image sensor, pixels are represented by p-doped metal–oxide–semiconductor (MOS) capacitors. These MOS capacitors, the basic building blocks of a CCD, are biased above the threshold for inversion when image acquisition begins, allowing the conversion of incoming photons into electron charges at the semiconductor-oxide interface; the CCD is then used to read out these charges. Although CCDs are not the only technology to allow for light detection, CCD image sensors are widely used in professional, medical, and scientific applications where high-quality image data are required. In applications with less exacting quality demands, such as consumer and professional digital cameras, act ...
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Time-correlated Single Photon Counting
Ultrafast laser spectroscopy is a spectroscopic technique that uses ultrashort pulse lasers for the study of dynamics on extremely short time scales ( attoseconds to nanoseconds). 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. Attosecond-to-picosecond spectroscopy Dynamics on the as to fs time scale are in general too fast to be measured electronically. Most measurements are done by employing a sequence of ultrashort light pulses to initiate a process and record its dynamics. The temporal width (duration) of the light pulses has to be on the same scale as the dynamics that are to be measured or even shorter. Light sources Titanium-sapphire laser Ti-sapphire lasers are tunable lasers that emit red and near-infrared light (700 nm- 1100 nm).Ti-sapphire laser oscillators use Ti doped-sapphi ...
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Laser
A 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 acronym for "light amplification by stimulated emission of radiation". The first laser was built in 1960 by Theodore H. Maiman at Hughes Research Laboratories, based on theoretical work by Charles Hard Townes and Arthur Leonard Schawlow. A laser differs from other sources of light in that it emits light which is ''coherent''. Spatial coherence allows a laser to be focused to a tight spot, enabling applications such as laser cutting and lithography. Spatial coherence also allows a laser beam to stay narrow over great distances (collimation), enabling applications such as laser pointers and lidar (light detection and ranging). Lasers can also have high temporal coherence, which allows them to emit light with a very narrow spectrum. Alternatively, temporal coherence can be used to produce ultrashort pulses of ligh ...
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