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Glossary: Fluorometry
High-resolution terminology - matching measurements at high-resolution >>> Fluorometry, Respirometry, Spectrophotometry (n.a. = no abbreviation)
| Term | Abbreviation | Description |
|---|---|---|
| Absorbance | A | Also known as attenuation or extinction, absorbance (A) is a measure of the difference between the incident light intensity (I0) and the intensity of light emerging from a sample (I). It is defined as: A = log (I0/I) |
| Accuracy | n.a. | The accuracy of a method is the degree of agreement between an individual test result generated by the method and the true value. |
| Amplex red | AmR | Amplex red (AmR) belongs to the extrinsic fluorophores and makes use of the fact that ROS generate hydrogen peroxide (H2O2) and it is the H2O2 level that Amplex red is used to measure. In the presence of horseradish peroxidase, Amplex red reacts with H2O2 to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm). The emitted fluorescence intensity is directly proportional to the H2O2 concentration. |
| Averaging | n.a. | In order to improve the signal-to-noise ratio a number of sequential spectra may be averaged over time. The number of spectra to be averaged can be set prior to carrying out the measurements, or afterwards during data analysis. |
| Bandwidth | n.a. | Bandwidth is measured in nanometers in terms of the full width half maximum of a peak. This is the portion of the peak that is greater than half of the maximum intensity of that peak. |
| Blank | n.a. | In fluorometry and transmission spectrophotometry blank cuvettes (with no samples in them) are used to carry out the balance. |
| Calcium green | CaG | Calcium green belongs to the extrinsic fluorophores applied for measurement of Ca2+ concentration (KD = 190 nM. |
| Cuvettes | n.a. | Cuvettes, or cells, are used in fluorometry and transmission spectrophotometry to contain the samples. Traditionally they have a square cross-section (10 x 10 mm). For many applications they are made of transparent plastic. Glass cells are used where samples may contain plastic solvents, and for some applications requiring measurements below 300 nm, quartz glass or high purity fused silica cuvettes may be necessary. |
| Detector | n.a. | A detector is a device that converts the light falling upon it into a current or voltage that is proportional to the light intensity. The most common devices in current use for fluorometry and spectrophotometry are photodiodes and photodiode arrays. |
| Diffraction gratings | n.a. | Diffraction gratings are dispersion devices that are made from glass etched with fine grooves, spaced at the same order of magnitude as the wavelength of the light to be dispersed, and then coated with aluminium to reflect the light to the photodiode array. Diffraction gratings reflect the light in different orders and filters need to be incorporated to prevent overlapping. |
| Dispersion devices | n.a. | A dispersion device diffracts light at different angles according to its wavelength. Traditionally, prisms and diffraction gratings are used, the latter now being the most commonly used device in a spectrofluorometer or spectrophotometer. |
| Drift | n.a. | The most common cause of drift is variation in the intensity of the light source. The effect of this can be minimised by carrying out a balance at frequent intervals. |
| Extrinsic fluorophores | n.a. | Extrinsic fluorophores are available for a wide range of parameters including ROS (H2O2, Amplex red) (HOO-, MitoSOX) , mitochondrial membrane potential (Safranin, JC1, TMRM, Rhodamin 123), Ca2+ (Fura2, Indo 1, Calcium Green) , pH (Fluorescein, HPTS, SNAFL-1), Mg2+ (Magnesium orange) and redox state (roGFP). |
| Filters | n.a. | Filters are materials that have wavelength-dependent transmission characteristics. They are can be used to select the wavelength range of the light emerging from a light source, or the range entering the detector, having passed through the sample. In particular they are used in fluorometry to exclude wavelengths greater than the excitation wavelength from reaching the sample, preventing absorption interfering with the emitted fluorescence. Standard filters can also be used for calibrating purposes. |
| Fluorescence | n.a. | Fluorescence is the name given to light emitted by a substance when it is illuminated (excited) by light at a shorter wavelength. The incident light causes an electron transition to a higher energy band in the molecules. The electron then spontaneously returns to its original energy state emitting a photon. The intensity of the emitted light is proportional to the concentration of the substance. |
| Fluorescent marker | n.a. | See Extrinsic fluorophores |
| Fluorimetry | n.a. | Fluorimetry (or fluorometry) is the general term given to the method of measuring the fluorescent emission of a substance following excitation by light at a shorter wavelength. |
| Fluorometric dyes | n.a. | Extrinsic fluorophores; fluorescent markers. |
| Fluorometry | n.a. | Fluorometry (or fluorimetry) is the general term given to the method of measuring the fluorescent emission of a substance following excitation by light at a shorter wavelength. |
| Fluorophore | n.a. | A fluorophore is a fluorescent substance that may occur naturally (intrinsic fluorophores) or that may be added to a sample or preparation whereby the fluorescence intensity is proportional to the concentration of a specific species or parameter within the sample. These are extrinsic fluorophores, also referred to as fluorescent markers. |
| High-resolution respirometry | HRR | High-resolution respirometry (HRR) is based on the OROBOROS Oxygraph-2k, combining chamber design, application of oxygen-tight materials, electrochemical sensors and electronics, Peltier-temperature control and software features (DatLab) to obtain a unique level of quantitative resolution of oxygen concentration and oxygen flux, with a closed-chamber or open-chamber mode of operation (TIP2k). Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the experimental basis for high accuracy of quantitative results and quality control in HRR. |
| Incident light | n.a. | The term incident light is used for a beam of light falling upon a surface. |
| Integration time | n.a. | Integration time is the time taken to scan a single full range spectrum using photodiode arrays. It is equivalent to the exposure time for a camera. The shortest integration time defines the fastest response time of a spectrophotometer. Increasing the integration time increases the sensitivity of the device. The white balance or balance and subsequent measurements must always be carried out at the same integration time. |
| Intrinsic fluorophores | n.a. | An Intrinsic flourophore is a naturally occurring fluorophore of which NADH, aromatic amino acids and flavins are examples. |
| Least squares method | n.a. | This method makes use of all of the data points of the spectrum in order to quantify a measured spectrum with a reference spectrum of known concentration using a least squares method to match the measured spectrum with the reference spectrum. The technique results in improved accuracy compared with the use of only a few characteristic wavelengths. |
| Light source | n.a. | A variety of light sources are available for fluorometry and spectrophotometry. These include deuterium, mercury and xenon arc lamps and quartz halogen bulbs dependent upon the wavelengths required. However, the advent of light emitting diodes has greatly increased the possibilities for the application of fluorometry and spectrophotometry to areas that were previously not practicable, and at a much reduced cost. |
| Light-emitting diode | LED | A light-emitting diode (LED) is a light source (semiconductor), used in many every-day applications and specifically in fluorometry. LEDs are available for specific spectral ranges across wavelengths in the visible, ultraviolet, and infrared range. |
| Lightguides | n.a. | Lightguides consist of optical fibres (either single or in bundles) that can be used to transmit light to a sample from a remote light source and similarly receive light from a sample and transmit it to a remote detector. They have greatly contributed to the range of applications that for which optical methods can be applied. This is particularly true in the fields of medicine and biology. |
| Linearity | n.a. | Linearity is the ability of the method to produce test results that are proportional, either directly or by a well-defined mathematical transformation, to the concentration of the analyte in samples within a given range. This property is inherent in the Beer-Lambert law for absorbance alone, but deviations occur in scattering media. It is also a property of fluorescence, but a fluorophore may not exhibit linearity, particularly over a large range of concentrations. |
| Magnesium green | MgG | Magnesium green belongs to the extrinsic fluorophores applied for measurement of ATP production, based on different dissociation constants for ADP and ATP. |
| Microplates | n.a. | Microplate readers allow large numbers of sample reactions to be assayed in well format microtitre plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 µL per well. a wide range of applications involve the use of fluorescence measurements , although they can also be used in conjunction with absorbance measurements. |
| Multicomponent analysis | n.a. | Similarly to the least squares method, multicomponent analysis makes use of all of the data points of the spectrum in order to analyse the concentration of the component parts of a measured spectrum. To do this, two or more reference spectra are combined using iterative statistical techniques in order to achieve the best fit with the measured spectrum. |
| NADH Fluorescence | n.a | Reduced nicotinamide adenine dinucleotide (NADH) is amongst the intrinsic fluorophores and can be used as a natural indicator of hypoxia. The excitation wavelength is 340nm and emission is at 460nm. |
| Noise | n.a. | In fluorometry and spectrophotometry, noise can be attributed to the statistical nature of the photon emission from a light source and the inherent noise in the instrument’s electronics. The former causes problems in measurements involving samples of analytes with a low extinction coefficient and present only in low concentrations. The latter becomes problematic with high absorbance samples where the light intensity emerging from the sample is very small. |
| O2k | O2k | O2k - OROBOROS Oxygraph-2k: the modular system for high-resolution respirometry. |
| O2k-Fluorescence LED2-Module | The O2k-Fluorescence LED2-Module is an amperometric add-on module to the O2k-Core, adding a new dimension to HRR. Optical sensors are inserted through the front window of the O2k-glass chambers, for measurement of hydrogen peroxide production (Amplex red), ATP production (Magnesium green), mt-membrane potential (Safranin), Ca2+ (Calcium green), and numerous other applications open for O2k-user innovation. The O2k-Fluorescence LED2-Module consists of optical sensors for both O2k-Chambers (LEDs for green and blue excitation), optical filters, Fluorescence-Control Unit for regulation of light intensity, data input into the O2k-Main Unit, and the updated DatLab software. | |
| Optics | n.a. | Optics are the components that are used to relay and focus light through a spectrofluorometer or spectrophotometer. These would normally consist of lenses and/or concave mirrors. The number of such components should be kept to a minimum due to the losses of light (5-10%) that occur at each surface. |
| PEEK | PEEK | Polyether ether ketone (PEEK) is a semicrystalline organic polymer thermoplastic, which is chemically very resistant, with excellent mechanical properties. PEEK is compatible with ultra-high vacuum applications, and its resistance against oxygen diffusion make it an ideal material for high-resolution respirometry (POS insulation; coating of stirrer bars; stoppers for closing the O2k-Chamber). |
| Permeabilized muscle fibres | Pfi | Permeabilized muscle fibres (Pfi) are used as a mitochondrial preparation in respirometry to access mitochondrial function comparable to isolated mitochondria. Pfi are obtained by selectively permeabilizing the plasma membrane mechanically and chemically, for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes. Add MitoPedia topic: Mitochondrial preparations |
| Phosphorescence | n.a. | Phosphorescence is a similar phenomenon to fluorescence. However, instead of the electron returning to its original energy state following excitation, it decays to an intermediate state (with a different spin value) where it can remain for some time (minutes or even hours) before decaying to its original state. |
| Photodiode arrays | n.a. | Photodiode arrays are two dimensional assemblies of photodiodes. They are frequently used in conjunction with charge coupled devices (CCDs) for digital imaging. They can be used in combination with dispersion devices to detect wavelength dependent light intensities in a spectrofluorometer or spectrophotometer. |
| Photodiodes | n.a. | Photodiodes are photodetectors that convert incident light into a current or voltage dependent on their configuration. They have replaced photomultiplier tubes for most applications. For fluorometric measurements that do not require spectral data, a single photodiode with suitable filters can be used. Due to their larger detection area, they are more sensitive than photodiode arrays. |
| Quenching | n.a. | Quenching is the name given to any process that reduces fluorescence intensity. Molecular oxygen is a fluorescence and phosphorescence quencher for some substances – a phenomenon that has been made use of in constructing optical probes for measuring oxygen. |
| Resolution | n.a. | Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths. Two wavelengths are normally considered to be resolved if the minimum detector output signal (trough) between the two peaks is lower than 80% of the maximum. The resolution of a spectrofluorometer or spectrophotometer is dependent on its bandwidth. |
| Safranin | Saf | Safranin is amongst the extrinsic fluorophores used in fluorometry and is one of the most established dyes for measuring mitochondrial membrane potential. The excitation wavelength for this dye is 495 nm and the emission wavelength is 587 nm). |
| Scattering | n.a. | Most biological samples do not consist simply of pigments but also particles (e.g. cells, fibres, mitochondria) which scatter the incident light. The effect of scattering is an apparent increase in absorbance due to an increase in pathlength and the loss of light scattered in directions other than that of the detector. Two types of scattering are encountered. For incident light of wavelength λ, Rayleigh scattering is due to particles of diameter < λ (molecules, sub-cellular particles). The intensity of scatter light is proportional to λ4 and is predominantly backward scattering. Mie scattering is caused by particles of diameter of the order of or greater than λ (tissue cells). The intensity of scatter light is proportional to 1/λ and is predominantly forward scattering. |
| Selectivity | n.a. | Selectivity is the ability of a method to quantify accurately and specifically the analyte or analytes in the presence of other compounds. |
| Sensitivity | n.a. | Sensitivity refers to the response obtained for a given amount of analyte and is often denoted by two factors: the limit of detection and the limit of quantification. |
| Signal-to-noise ratio | S/N | The signal to noise ratio is the ratio of the power of the signal to that of the noise. For example, in fluorimetry it would be the ratio of the square of the fluorescence intensity to the square of the intensity of the background noise. |
| Slit width | n.a. | The slit width determines the amount of light entering the spectrofluorometer or spectrophotometer. A larger slit reduces the signal-to-noise ratio but reduces the wavelength resolution. |
| Smoothing | n.a. | The signal-to-noise ratio can also be improved by spectral ‘’’smoothing’’’. This is achieved by averaging several adjacent data points across the recorded spectrum. For example, if the instrument recorded 5 data points per nm, the average of the 5 points can be taken for each successive nm across the range of the spectrum to give a 5-point smoothing. This method clearly reduces the wavelength resolution. |
| Spectrofluorometer | n.a. | A spectrofluorometer makes use of a spectrophotometer to measure and analyse the fluorescent emission spectra from a fluorophore. It will typically differ from an absorbance spectrophotometer in that it will have a larger slit width (to increase sensitivity) and use a longer integration time. The configuration of the illuminating and receiving optics also differ from spectrophotometry in that the excitation source is directed perpendicularly to the position of the emission detector so that the intensity of the excitation signal reaching the detector is minimised. |
| Spectrophotometer | n.a. | A spectrophotometer is an instrument that consists of an entrance slit, a dispersion device (see dispersion devices and a detector for the purpose of measuring the intensity of light emerging from a sample across a given wavelength range. A light source is also necessary in order for the instrument to function, and this may be located within the instrument or from an external source using lightguides or other optics. |
| Spline | n.a. | Some spectrofluorometer or spectrophotometer software offers the possibility of spline interpolation of the spectral data points. This makes use of a polynomial (the number of spline points is entered by the user) to interpolate the curve between the data points. |
| Stability | n.a. | Stability determines the accuracy of intensity and absorbance measurements as a function of time. Instability (see drift introduces systematic errors in the accuracy of fluorescence and absorbance measurements. |
| Stray light | n.a. | Stray light is defined as the detected light of any wavelength that lies outside the bandwidth of the selected wavelength. In the presence of stray light of intensity Is, the equation for transmittance (T) becomes T = (I + Is)/(I0 + Is) where I0 is the incident light intensity and I is the transmitted light intensity. Clearly, the lower the value of I, the more dominant becomes the stray light term and so can cause errors in the quantification of low fluorescence signals or at high levels of absorbance. |
| Wavelength averaging | n.a. | See smoothing |
