MitoPedia: Spectrophotometry

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high-resolution terminology - matching measurements at high-resolution

MitoPedia: Spectrophotometry

The MitoPedia terminology is developed continuously in the spirit of Gentle Science.

Β»Respirometry, Β»Fluorometry

AbsorbanceAAlso 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 spectrumWhen 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.
AbsorptionAbsWhen 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 spectrumAn absorption spectrum is similar to an absorbance spectrum of a sample, but plotted as a function of absorption against wavelength.
AmplitudeThe 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.
AveragingIn 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.
BalanceIn 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).
BandwidthBandwidth 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 lawB-L lawThis law states that the transmittance (T) of light though a sample is given by: T = e-Ξ΅bc, where Ξ΅ 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 = Ξ΅bc.
BlankIn fluorometry and transmission spectrophotometry blank cuvettes (with no samples in them) are used to carry out the balance.
CuvettesCuvettes are used in fluorometry and transmission spectrophotometry to contain the samples. Use of the term 'cells' for cuvettes is discouraged, to avoid confusion with 'living cells'. Traditionally cuvettes 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 spectroscopyDerivative 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..
DetectorA 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 spectrumA 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 gratingsDiffraction 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.
Dilution effectDilution of the concentration of a compound or sample in the experimental chamber by a titration of another solution into the chamber.
Dispersion devicesA 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.
DriftThe 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 analysisIf 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 540 nm from that at 550nm in order to give a measure of the degree of reduction. Similarly, by subtracting the absorbance at 465 nm from that at 444 nm, an indicator of the redox state of cytochrome aa3 can be obtained.
ExtinctionExtinction is a synonym for absorbance.
Extinction coefficientΞ΅The extinction coefficient (Ξ΅) of a substance is the absorbance of a 1 Β΅molar concentration over a 1 cm pathlength and is wavelength-dependent.
FiltersFilters 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 respirometryHRR
High-resolution respirometry, HRR, is the state-of-the-art approach in mitochondria and cell research to measure respiration in various types of mitochondrial preparations and living cells combined with MultiSensor modules.

Mitochondrial function and dysfunction have gained increasing interest, reflecting growing awareness of the fact that mitochondria play a pivotal role in human health and disease. HRR combines instrumental accuracy and reliability with the versatility of applicable protocols, allowing practically unlimited addition and combination of substrates, inhibitors, and uncouplers using the Oroboros O2k-technology. Substrate-uncoupler-inhibitor titration (SUIT) protocols allow the interrogation of numerous mitochondrial pathway and coupling states in a single respirometric assay. Mitochondrial respiratory pathways may be analyzed in detail to evaluate even minor alterations in respiratory coupling and pathway control patterns.

The O2k-technology provides sole source instruments, with no other available instrument meeting its specifications for high-resolution respirometry. Technologically, HRR is based on the Oroboros O2k-technology, combining optimized chamber design, application of oxygen-tight materials, electrochemical sensors, Peltier-temperature control, and specially developed software features (DatLab) to obtain the unique sensitive and quantitative resolution of oxygen concentration and oxygen flux, with both, a closed-chamber or open-chamber mode of operation (TIP2k). Standardized 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. HRR can be extended for MultiSensor analysis by using the O2k-Fluo Smart-Module. Smart Fluo-Sensors are integrated into the O2k to measure simultaneously fluorometric signals using specific fluorophores. Potentiometric modules are available with ion-selective electrodes (pH, TPP+). The PB-Module extends HRR to PhotoBiology with accurate control of the light intensity and measurement of photosynthesis. The O2k and the NextGen-O2k support all these O2k-Modules. The NextGen-O2k all-in-one, however, is unique in supporting Q-Redox and NADH-Redox Modules.
Incident lightThe term incident light is used for a beam of light falling upon a surface.
Integration timeIntegration 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 methodThis 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 sourceA 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 diodeLEDA 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.
LightguidesLightguides 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.
LinearityLinearity 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.
MicroplatesMicroplate 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 Β΅L per well. a wide range of applications involve the use of fluorescence measurements , although they can also be used in conjunction with absorbance measurements.
Mitochondrial markermt-markerMitochondrial markers are structural or functional properties that are specific for mitochondria. A structural mt-marker is the area of the inner mt-membrane or mt-volume determined stereologically, which has its limitations due to different states of swelling. If mt-area is determined by electron microscopy, the statistical challenge has to be met to convert area into a volume. When fluorescent dyes are used as mt-marker, distinction is necessary between mt-membrane potential dependent and independent dyes. mtDNA or cardiolipin content may be considered as a mt-marker. Mitochondrial marker enzymes may be determined as molecular (amount of protein) or functional properties (enzyme activities). Respiratory capacity in a defined respiratory state of a mt-preparation can be considered as a functional mt-marker, in which case respiration in other respiratory states is expressed as flux control ratios. Β» MiPNet article
Multicomponent analysisSimilarly 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 fluorescenceReduced nicotinamide adenine dinucleotide (NADH) is amongst the intrinsic fluorophores and can be used as an intracellular indicator of hypoxia. The excitation wavelength is 340 nm and emission is at 460 nm.
NoiseIn 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.
O2kO2kO2k - Oroboros O2k: the modular system for high-resolution respirometry.
OpticsOptics 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.
P50p50p50 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%.
PhosphorescencePhosphorescence 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. Phosphorescence is one form of Luminescence, especially Photoluminescence.
Photodiode arraysPhotodiode 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.
PhotodiodesPhotodiodes 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.
Polyether ether ketonePEEKPolyether 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).
Reference spectrumA reference spectrum for a substance is an absorbance spectrum of the same substance at a known concentration and redox state.
Reflectance spectrophotometryIn 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 spectrophotometryIn 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.
ResolutionSpectral 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 stateRespiratory states of mitochondrial preparations and living 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 electron-transfer-pathway state.
ScatteringMost 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 Ξ», Rayleigh scattering is due to particles of diameter < Ξ» (molecules, sub-cellular particles). The intensity of scatter light is proportional to Ξ»4 and is predominantly backward scattering. Mie scattering is caused by particles of diameter of the order of or greater than Ξ» (tissue cells). The intensity of scatter light is proportional to 1/Ξ» and is predominantly forward scattering.
SelectivitySelectivity is the ability of a sensor or method to quantify accurately and specifically the analyte or analytes in the presence of other compounds.
SensitivitySensitivity 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 ratioS/NThe 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 widthThe 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.
SmoothingVarious methods of smoothing can be applied to improve the signal-to-noise ratio. For instance, data points recorded over time [s] or over a range of wavelengths [nm] can be smoothed by averaging n data points per interval. Then the average of the n points per smoothing interval can be taken for each successively recorded data point across the time range or range of the spectrum to give a n-point moving average smoothing. This method decreases the noise of the signal, but clearly reduces the time or wavelength resolution. More advanced methods of smoothing are applied to retain a higher time resolution or wavelength resolution.
SpectrophotometerA 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.
SpectrophotometrySpectrophotometry 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.
SpectroscopySpectroscopy is a broader term than spectrophotometry in that it is concerned with the investigation and measurement of spectra produced when matter interacts with or emits any form electromagnetic radiation.
SplineSome 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.
StabilityStability 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.
Steady stateA system is in a steady state if the state variables of a dynamic system do not change over time due to exchange processes with the environment, which compensate for internal dissipative transformations β€” such as chemical reactions or diffusion β€” and thus prevent any changes of the system and externalize dissipative changes to the environment. The dynamic nature of the steady state differentiates it from the thermodynamic equilibrium state. {Quote} Steady states can be obtained only in open systems, in which changes by internal transformations, e.g., O2 consumption, are instantaneously compensated for by external fluxes across the system boundary, e.g., O2 supply, thus preventing a change of O2 concentration in the system (Gnaiger 1993). Mitochondrial respiratory states monitored in closed systems satisfy the criteria of pseudo-steady states for limited periods of time, when changes in the system (concentrations of O2, fuel substrates, ADP, Pi, H+) do not exert significant effects on metabolic fluxes (respiration, phosphorylation). Such pseudo-steady states require respiratory media with sufficient buffering capacity and substrates maintained at kinetically-saturating concentrations, and thus depend on the kinetics of the processes under investigation. {end of Quote: BEC 2020.1}. Whereas fluxes may change at a steady state over time, concentrations are maintained constant. The 'respiratory steady state' (Chance and Williams 1955) is characterized by constant fluxes (O2 flux, H2O2 flux) and measured variables of state (cytochrome redox states, Q redox state, NADH redox state, mitochondrial membrane potential). High-resolution respirometry allows for the measurement of several parameters (e.g. O2 flux, H2O2 flux, mitochondrial membrane potential) at pseudo-steady states, when changes of concentrations in the closed system do not exert any control on fluxes. Combination with the Titration-Injection microPump (TIP2k) allows operation with programmable titration regimes at steady-state ADP concentration (Gnaiger 2001), oxygen concentration (oxystat mode; Gnaiger et al 2000, Harrison et al 2015) or steady-state pH (pH-stat more), yielding an expanded flexibility in experimental design by combining the technical advantages of closed and open systems approaches.
Stray lightStray 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 spectrophotometryIn 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.
TransmittanceTWhen 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 averagingWavelength averaging is the averaging of several adjacent data points across the recorded spectrum (spectral smoothing), to improve the signal-to-noise ratio. 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.
Wavelength rangeThe 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 balanceIn 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.
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