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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 lower energy, than the absorbed radiation. The most striking example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, while the emitted light is in the visible region, which gives the fluorescent substance a distinct color that can be seen only when 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.
Fourier-transform spectroscopy is a measurement technique whereby spectra are collected based on measurements of the coherence of a radiative source, using time-domain or space-domain measurements of the electromagnetic radiation or other type of radiation. It can be applied to a variety of types of spectroscopy including optical spectroscopy, infrared spectroscopy, nuclear magnetic resonance (NMR) and magnetic resonance spectroscopic imaging (MRSI), mass spectrometry and electron spin resonance spectroscopy. There are several methods for measuring the temporal coherence of the light, including the continuous wave Michelson or Fourier-transform spectrometer and the pulsed Fourier-transform spectrograph.
Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible spectral regions. This means it uses light in the visible and adjacent ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, atoms and molecules undergo electronic transitions. Absorption spectroscopy is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.
X-ray fluorescence (XRF) is the emission of characteristic "secondary" X-rays from a material that has been excited by being bombarded with high-energy X-rays or gamma rays. The phenomenon is widely used for elemental analysis and chemical analysis, particularly in the investigation of metals, glass, ceramics and building materials, and for research in geochemistry, forensic science, archaeology and art objects such as paintings and murals.
A spectrum analyzer measures the magnitude of an input signal versus frequency within the full frequency range of the instrument. The primary use is to measure the power of the spectrum of known and unknown signals. The input signal that most common spectrum analyzers measure is electrical; however, spectral compositions of other signals, such as acoustic pressure waves and optical light waves, can be considered through the use of an appropriate transducer. Spectrum analyzers for other types of signals also exist, such as optical spectrum analyzers which use direct optical techniques such as a monochromator to make measurements.
In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved spectroscopic techniques.
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.
A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input. The name is from the Greek roots mono-, "single", and chroma, "colour", and the Latin suffix -ator, denoting an agent.
A photometer is an instrument that measures the strength of electromagnetic radiation in the range from ultraviolet to infrared and including the visible spectrum. Most photometers convert light into an electric current using a photoresistor, photodiode, or photomultiplier.
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.
A spectroradiometer is a light measurement tool that is able to measure both the wavelength and amplitude of the light emitted from a light source. Spectrometers discriminate the wavelength based on the position the light hits at the detector array allowing the full spectrum to be obtained with a single acquisition. Most spectrometers have a base measurement of counts which is the un-calibrated reading and is thus impacted by the sensitivity of the detector to each wavelength. By applying a calibration, the spectrometer is then able to provide measurements of spectral irradiance, spectral radiance and/or spectral flux. This data is also then used with built in or PC software and numerous algorithms to provide readings or Irradiance (W/cm2), Illuminance, Radiance (W/sr), Luminance (cd), Flux, Chromaticity, Color Temperature, Peak and Dominant Wavelength. Some more complex spectrometer software packages also allow calculation of PAR µmol/m²/s, Metamerism, and candela calculations based on distance and include features like 2- and 20-degree observer, baseline overlay comparisons, transmission and reflectance.
A nondispersive infrared sensor is a simple spectroscopic sensor often used as a gas detector. It is non-dispersive in the fact that no dispersive element is used to separate out (like a monochromator the broadband light into a narrow spectrum suitable for gas sensing. The majority of NDIR sensors use a broadband lamp source and an optical filter to select a narrow band spectral region that overlaps with the absorption region of the gas of interest. In this context narrow may be 50-300nm bandwidth. Modern NDIR sensors may use Microelectromechanical systems or mid IR LED sources, with or without an optical 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 ].
A fluorometer or fluorimeter is a device used to measure parameters of visible spectrum fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium. Modern fluorometers are capable of detecting fluorescent molecule concentrations as low as 1 part per trillion.
Fellgett's advantage or the multiplex advantage is an improvement in signal to noise ratio that is gained when taking multiplexed measurements rather than direct measurements. The name is derived from P. B. Fellgett, who first made the observation as part of his PhD. When measuring a signal whose noise is dominated by detector noise, a multiplexed measurement such as the signal generated by a Fourier transform spectrometer can produce a relative improvement in signal-to-noise ratio (SNR), compared to an equivalent scanning monochromator, of the order of the square root of m, where m is the number of sample points comprising the spectrum.
Fourier-transform infrared spectroscopy (FTIR) is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid or gas. An FTIR spectrometer simultaneously collects high-spectral-resolution data over a wide spectral range. This confers a significant advantage over a dispersive spectrometer, which measures intensity over a narrow range of wavelengths at a time.
Stray light is light in an optical system, which was not intended in the design. The light may be from the intended source, but follow paths other than intended, or it may be from a source other than the intended source. This light will often set a working limit on the dynamic range of the system; it limits the signal-to-noise ratio or contrast ratio, by limiting how dark the system can be. Ocular straylight is stray light in the human eye.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
Single molecule fluorescence resonance energy transfer 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.
Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.
References
- ↑ Genovesi, Lina. "Spectrofluorometers: Tools for Measuring Fluorescence Signature". Labcompare.