From Bioblast
(Redirected from List of spectrophotometry terms)
| Welcome to MitoPedia | Home | MitoPedia Glossaries | Add Term | www.mitophysiology.org |
Glossary: Spectrophotometry
High-resolution terminology - matching measurements at high-resolution >>> Respirometry, Fluorometry (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) |
| Absorbance spectrum | n.a. | When light enters a sample, the amount of light that it absorbs is dependent upon the wavelength of the incident light. The absorbance spectrum is the curve derived by plotting the measured absorbance against the wavelength of the light emerging from the sample over a given wavelength range. An absorbance spectrum may be characterised by peaks and troughs (absorbance maxima and minima) that can be used to identify, and sometimes quantify, different absorbing substances present in a sample. |
| Absorption | Abs | When light enters a sample and emerges with an intensity (I), absorption (Abs) is the fraction of the light absorbed by the sample compared with the incident light intensity (I0): Abs = 1-I/I0. Absorption can also be expressed as Abs = 1-T, where T is the transmittance. |
| Absorption spectrum | n.a. | An absorption spectrum is similar to an absorbance spectrum of a sample, but plotted as a function of absorption against wavelength. |
| 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. |
| Amplitude | n.a. | The amplitude of the absorbance spectrum can be described in terms of the absorbance differences between the characteristic peaks (absorbance maxima) and troughs (absorbance minima) (see absorbance spectrum) for substances present in the sample. |
| 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. |
| Balance | n.a. | In transmission spectrophotometry blank cuvettes are used to record the incident light intensity (I0) prior to absorbance measurements. (See white balance for reflectance spectrophotometry, remittance spectrophotometry). |
| 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. |
| Beer-Lambert law | B-L law | This law states that the transmittance (T) of light though a sample is given by: T = e-εbc, where ε is the molar extinction coefficient, b is the pathlength of the light through the cuvette (in mm) and c is the concentration of the pigment in the sample (in mM). Transforming this equation, it can be seen that the absorbance of light (A) is simply given by A = εbc. |
| Blank | n.a. | In fluorometry and transmission spectrophotometry blank cuvettes (with no samples in them) are used to carry out the balance. |
| 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. |
| Derivative spectroscopy | n.a. | Derivative spectroscopy can be used to eliminate interfering artefacts or species. A first order derivative will remove a constant background absorbance across the spectral range. A second order derivative spectrum will remove a species whose absorbance is linearly dependent upon the wavelength, etc.. |
| 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. |
| Difference spectrum | n.a. | A difference spectrum is an absorbance spectrum obtained by subtracting that of one substance from that of another. For example, a difference spectrum may be plotted of the absorbance spectrum obtain ed from reduced cytochrome c and subtracting the absorbance spectrum from the same concentration of cytochrome c in its oxidised state. The difference spectrum may be used to quantify the amount to which the cytochrome c is reduced. This can be achieved with the aid of a reference spectrum (or spectra) and the least squares method. |
| 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. |
| Dual wavelength analysis | n.a. | If a sample contains a number of absorbing substances, it is sometimes possible to select discrete pairs of wavelengths at which the change in absorbance of a particular substance (due to oxidation or reduction, for example) is largely independent of changes in the absorbance of other substances present. Dual wavelength analysis can be carried out for cytochrome c by subtracting the absorbance at 540nm from that at 550nm in order to give a measure of the degree of reduction. Similarly, by subtracting the absorbance at 465nm from that at 444nm, an indicator of the redox state of cytochrome aa3 can be obtained. |
| Extinction | n.a. | Extinction is a synonym for absorbance. |
| Extinction coefficient | ε | The extinction coefficient (ε) of a substance is the absorbance of a 1 µmolar concentration over a 1 cm pathlength and is wavelength-dependent. |
| 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. |
| 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. |
| 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. |
| 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. |
| 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. |
| P50 | p50 | p50 is the oxygen partial pressure at which (a) respiratory flux is 50% of maximum oxygen flux, Jmax, at saturating oxygen levels. The oxygen affinity is indirectly proportional to the p50. The p50 depends on metabolic state and rate. (b) p50 is the oxygen partial pressure at which oxygen binding (on myoglobin, haemoglobin) is 50%, or desaturation is 50%. |
| 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). |
| 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. |
| Reference spectrum | n.a. | A reference spectrum for a substance is an absorbance spectrum of the same substance at a known concentration and redox state. |
| Reflectance spectrophotometry | n.a. | In reflectance spectrophotometry the light from the sample is reflected back to the detector using mirrors. Before absorbance measurements can be made, a white balance is carried out. |
| Remittance spectrophotometry | n.a. | In remittance spectrophotometry incident light enters a scattering medium and is scattered back to the receiving optics (usually lightguides) before being directed to the detector. Before absorbance measurements can be made, a white balance is carried out. |
| 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. |
| Respiratory state | n.a. | Respiratory states of mitochondrial preparations and intact cells are defined in the current literature in many ways and with a diversity of terms. Mitochondrial respiratory states must be defined in terms of both, the coupling control state and the substrate control state. |
| 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. |
| Spectrophotometry | n.a. | Spectrophotometry is the use of a spectrophotometer to measure the transmittance, reflectance or remittance of a material as a function of wavelength. See transmission spectrophotometry, reflectance spectrophotometry and remission spectrophotometry. |
| 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. |
| State 1 | n.a. | State 1 is the first respiratory state in an oxygraphic protocol described by Chance and Williams (1955), when isolated mitochondria are added to mitochondrial resipration medium containing oxygen and inorganic phosphate, but no ADP and no reduced respiratory substrates. In State 1, LEAK respiration may be supported to some extent by undefined endogenous substrates, which are oxidized and slowly exhausted. After oxidation of endogenous substrates, only residual oxygen consumption remains (ROX). |
| State 2 | ROXD | Substrate limited state of residual oxygen consumption, after addition of ADP to isolated mitochondria suspended in mitochondrial respiration medium in the absence of reduced substrates (ROXD). Residual endogenous substrates are oxidized during a transient stimulation of oxygen flux by ADP. The peak – supported by endogenous substrates – is, therefore, a pre-steady state phenomenon preceding State 2. Subsequently oxygen flux declines to a low level (or zero) at the steady State 2 (Chance and Williams 1955). ADP concentration (D) remains high during ROXD. |
| State 3 | P | State 3 respiration is the ADP stimulated respiration of isolated coupled mitochondria in the presence of high ADP and Pi concentrations, supported by a defined substrate or substrate combination at saturating oxygen levels (Chance and Williams, 1955). State 3 respiration can also be induced in permeabilized cells, including permeabilized tissue preparations and tissue homogenates. ADP concentrations applied in State 3 are not necessarily saturating, whereas OXPHOS capacity is measured at saturating concentrations of ADP and Pi (state P). For instance, non-saturating ADP concentrations are applied in State 3 in pulse titrations to determine the P/O ratio in State 3→4 (D→T) transitions, when saturating ADP concentrations would deplete the oxygen concentration in the closed oxygraph chamber before State 4 is obtained (Gnaiger et al 2000; Puchowicz et al 2004). Respiration in the OXPHOS state or in State 3 is partially coupled, and partially uncoupled (physiological) or partially dyscoupled (pathological). A high mt-membrane potential provides the driving force for oxidative phosphorylation, to phosphorylate ADP to ATP and to transport ADP and ATP across the inner mt-membrane through the adenine nucleotide translocase (ANT). The mt-membrane potential is reduced, however, in comparison to the LEAK state of respiration, whereas the cytochromes are in a more oxidized redox state. |
| State 4 | LT | State 4 is the respiratory state obtained in isolated mitochondria after State 3, when added ADP is phosphorylated completely to ATP driven by electron transfer from defined respiratory substrates to O2 (Chance and Williams, 1955). State 4 represents LEAK respiration, LT (L for LEAK; T for ATP), or an overestimation of LEAK respiration if ATPase activity prevents final accumulation of ATP and maintains a continuous stimulation of respiration by recycled ADP. This can be tested, by inhibition of ATP synthase by oligomycin (State 4o; LOmy). In the LEAK state (state of non-phosphorylating resting respiration; static head), oxygen flux is decreased to a minimum (correctd for ROX), and the mt-membrane potential is increased to a maximum for a specific substrate or substrate combination. |
| State 5 | n.a. | State 5 is the respiratory state obtained in a protocol with isolated mitochondria after a sequence of State 1 to State 4, when the concentration of O2 is depleted in the closed oxygraph chamber and zero oxygen (the anaerobic state) is reached (Chance and Williams, 1955; Table I). State 5 is defined in the original publication in two ways: State 5 may be obtained by antimycin A treatment or by anaerobiosis (Chance and Williams, 1955; page 414). Antimycin A treatment yields a State 5 equivalent to a state for measurement of residual oxygen consumption, ROX (which may also be induced by rotenone+myxothiazol; Gnaiger 2009). Setting State 5 equivalent to ROX or anoxia (Chance and Williams 1955) can be rationalized only in the context of measurement of cytochrome redox states, whereas in the context of respiration State 5 is usually referred to as 'zero oxygen'. |
| 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. |
| Transmission spectrophotometry | n.a. | In the transmission mode, the incident light passes through the sample cuvettes and the emergent light reaches the detector directly. Before absorbance measurements can be made, a balance is carried out. |
| Transmittance | T | When light enter a sample, transmittance (T) is the fraction of the intensity (I) of the light emerging from the sample compared with the incident light intensity (I0): T = I/I0. |
| Wavelength averaging | n.a. | See smoothing |
| Wavelength range | n.a. | The minimum and the maximum wavelengths over which an absorbance spectrum is measured are described in terms of the wavelength range. It is determined mainly by the specifications of the spectrophotometer and the type of light source used, and the characteristic absorbance spectrum of the sample being investigated. |
| White balance | n.a. | In reflectance spectrophotometry and remission spectrophotometry a white balance is carried out to determine the intensity of the incident light (I0) for the purpose of quantitative absorbance measurements. In reflectance spectrophotometry, a mirror can be used whereas in remission spectrophotometry a standard white tile is more appropriate. |
