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MitoPedia: Fluorometry

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List of fluorometry terms

High-resolution terminology - matching measurements at high-resolution >>> Fluori/ometry, Respirometry, Spectrophotometry


TermAbbreviationDescription
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)
Amp calibration - DatLabF5Amp calibration indicates the calibration of the amperometric O2k-channel.
Amplex UltraRedAmRAmplex® UltraRed (AmR) is used as an extrinsic fluorophore for measurement of hydrogen peroxide production (ROS) by cells or mitochondrial preparations. The reaction of H2O2 and AmR is catalyzed by horseradish peroxidase to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm; the fluorescent product according to the supplier is called UltroxRed in the case of Amplex® UltraRed which has a similar structure to resorufin). The change of emitted fluorescence intensity is directly proportional to the concentration of H2O2 added, whereby the H2O2 is consumed.
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.
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.
BlankIn fluorometry and transmission spectrophotometry blank cuvettes (with no samples in them) are used to carry out the balance.
CalciumCaCa2+ is a major signaling molecule in both prokaryotes and eukaryotes. Its cytoplasmic concentration is tightly regulated by transporters in the plasma membrane and in the membranes of various organelles. For this purpose, it is either extruded from the cell through exchangers and pumps or stored in organelles such as the endoplasmic reticulum and the mitochondria. Changes in the concentration of the cation regulate numerous enzymes including many involved in ATP utilizing and in ATP generating pathways and thus ultimately control metabolic activity of mitochondria and of the entire cell. Measuring changes in Ca2+ levels is thus of considerable interest in the context of high-resolution respirometry.
Calcium GreenCaGCalcium GreenTM (CaG) denotes a family of extrinsic fluorophores applied for measurement of Ca2+ concentration with mitochondrial preparations. This dye fluoresces when bound to Ca2+. When measuring mitochondrial calcium uptake it is possible to observe the increase of the CaG signal upon calcium titration, followed by the decrease of CaG signal due to the uptake.
Carboxy SNARF 1SNARFCarboxy SNARF® 1 is a cell-impermeant pH indicator dye. The pKa of ~7.5 makes it useful for measuring pH in the range of pH 7 to pH 8. The emission shifts from yellow-orange at low pH to deep red fluorescence at high pH. Ratiometric fluorometry, therefore, is applied at two emission wavelengths,such as 580 nm and 640 nm. Relative molecular mass: Mr = 453.45
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.
DTPADTPADTPA (Diethylenetriamine-N,N,N',N,N-pentaacetic acid, pentetic acid,(Carboxymethyl)imino]bis(ethylenenitrilo)-tetra-acetic acid) is a polyaminopolycarboxylic acid (like EDTA) chelator of metal cations. DTPA wraps around a metal ion by forming up to eight bounds, because each COO- group and and N-center serves a center for chelation. With transition metals the number of bounds is less than eight. The compound is not cell membrane permeable. In general, it chelates multivalent ions stronger than EDTA.
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.
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.
Electron leakElectrons that escape the electron transfer pathway without completing the reduction of oxygen to water at cytochrome c oxidase, causing the production of ROS. The rate of electron leak depends on the topology of the complex, the redox state of the moiety responsible of electron leakiness and usually on the protonmotive force (Δp). In some cases, the Δp dependance relies more on the ∆pH component than in the ∆Ψ.
Extrinsic fluorophoresExtrinsic fluorophores are molecules labelled with a fluorescent dye (as opposed to intrinsic fluorescence or autofluorescence of molecules which does not require such labelling). They are available for a wide range of parameters including ROS (H2O2, Amplex red) (HOO-, MitoSOX) , mitochondrial membrane potential (Safranin, JC1, TMRM, Rhodamine 123), Ca2+ (Fura2, Indo 1, Calcium Green), pH (Fluorescein, HPTS, SNAFL-1), Mg2+ (Magnesium Green) and redox state (roGFP).
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.
FluorescenceFluorescence 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. Fluorescence is one form of Luminescence, especially Photoluminescence.
Fluorescent markerSee Extrinsic fluorophores
Fluorometric dyesExtrinsic fluorophores; fluorescent markers.
FluorometryFluorometry (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.
FluorophoreA 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.
Flux / SlopeJFlux / Slope is the time derivative of the signal. In DatLab, Flux / Slope is the name of the pull-down menu for (1) normalization of flux (chamber volume-specific flux, sample-specific flux or flow, or flux control ratios), (2) flux baseline correction, (3) Instrumental background oxygen flux, and (4) flux smoothing, selection of the scaling factor, and stoichiometric normalization using a stoichiometric coefficient. Before changing the normalization of flux from volume-specific flux to sample-specific flux or flow, or flux control ratios, please be sure to use the standard Layout 04a (Flux per volume) or 04b (Flux per volume overlay). When starting with the instrumental standard Layouts 1-3, which display the O2 slope negative, the sample-specific flux or flow, or flux control ratios will not be automatically background corrected. To obtain the background corrected specific flux or flux control ratios, it is needed to tick the background correction in the lower part of the slope configuration window. Background correction is especially critical when performing measurements in a high oxygen regime or using samples with a low respiratory flux or flow.
Flux baseline correctionbcFlux baseline correction provides the option to display the plot and all values of the flux (or flow, or flux control ratio) as the total flux, J, minus a baseline flux, J0.
JV(bc) = JV - JV0
JV = (dc/dt) · ν-1 · SF - V
For the oxygen channel, JV is O2 flux per volume [pmol/(s·ml)] (or volume-specific O2 flux), c is the oxygen concentration [nmol/ml = µmol/l = µM], dc/dt is the (positive) slope of oxygen concentration over time [nmol/(s · ml)], ν-1 = -1 is the stoichiometric coefficient for the reaction of oxygen consumption (oxygen is removed in the chemical reaction, thus the stoichiometric coefficient is negative, expressing oxygen flux as the negative slope), SF=1,000 is the scaling factor (converting units for the amount of oxygen from nmol to pmol), and V is the volume-specific background oxygen flux (Instrumental background oxygen flux). Further details: Flux / Slope.
Fura2Fura2 is a ratiometric fluorescence probe for the measurement of calcium. Its derivative Fura-2-acetoxymethyl ester (Fura2-AM) is membrane permable and can thus be used to measure intracellular free calcium concentration (Grynkiewicz et al., 1985). For this purpose, cells are incubated with Fura2-AM, which crosses the cell membrane by diffusion and is cleaved into free Fura2 and acetoxymethyl groups by cellular esterases. Intracellular free calcium is measured by exciting the dye at 340 nm and 380 nm, which are the excitation optima of calcium-bound and free Fura2, respectively, and emission detection above 500 nm. Through the ratiometric detection unequal distribution of the dye within the cell and other potential disturbances are largely cancelled out, making this a widely used and relatively reliable tool for calcium measurements.
HPTSHPTS8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) is a ratiometric pH fluorophore; pKa = 7.3. Relative molecular mass: Mr = 524.39
High-resolution respirometryHRR
O2k-FluoRespirometer
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 in the Oroboros O2k. 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 is a sole source technology, with no other available instrument meeting its specifications for high-resolution respirometry. Technologically, HRR is based on the Oroboros O2k, 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-J 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.
Horseradish peroxidaseHRPHorseradish peroxidase readily combines with hydrogen peroxide (H2O2) and the resultant [HRP-H2O2] complex can oxidize a wide variety of hydrogen donors.
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.
Intrinsic fluorophoresAn Intrinsic flourophore is a naturally occurring fluorophore of which NADH, aromatic amino acids and flavins are examples.
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.
LuminescenceLuminescence is spontaneous emission of radiation from an electronically or vibrationally excited species not in thermal equilibrium with its environment (IUPC definition). An alternative definition is "Luminescence is emission of light by a substance not resulting from heat." Luminescence comprises many different pehnomena. Luminescence from direct photoexcitation of the emitting species is called photoluminescence. Both fluorescence and phosphorescence are forms of photoluminescence. In biomedical research also forms of chemiluminescence (e.g.the luciferin reaction) are used. In chemiluminescence the emission of radiation results from a chemical reaction. For other forms of luminescence see the IUPAC Gold Book.
Magnesium GreenMgGMagnesium Green (MgG) is an extrinsic fluorophore that fluoresces when bound to Mg2+ and is used for measuring mitochondrial ATP production by mitochondrial preparations. Determination of mitochondrial ATP production is based on the different dissociation constants of Mg2+ for ADP and ATP, and the exchange of one ATP for one ADP across the mitochondrial inner membrane by the adenine nucleotide translocase (ANT). Using the dissociation constants for ADP-Mg2+ and ATP-Mg2+ and initial concentrations of ADP, ATP and Mg2+, the change in ATP concentration in the medium is calculated, which reflects mitochondrial ATP production.
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 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.
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. » MiPNet article
Mitochondrial membrane potentialmtMP, ΔΨp+, ΔelFep+ [V]The mitochondrial membrane potential difference, mtMP or ΔΨp+ = ΔelFep+, is the electric part of the protonmotive force, Δp = ΔmFeH+.
ΔelFep+ = ΔmFeH+ - ΔdFeH+
ΔΨp+ = Δp - ΔµH+·(zH+·F)-1
ΔΨp+ is the potential difference across the mitochondrial inner membrane (mtIM), expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ charge or per charge moved across the transmembrane potential difference, with the number of ‘motive’ charges expressed in the unit coulomb [C].
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.
NigericinNigericin is a H+/K+ antiporter, which allows the electroneutral transport of these two ions in opposite directions across the mitochondrial inner membrane following the K+ concentration gradient. In the presence of K+, nigericin decreases pH in the mitchondrial matrix, thus, almost fully collapses the transmembrane ΔpH, which leads to the compensatory increase of the electric mt-membrane potential. Therefore, it is ideal to use to dissect the two components of the protonmotive force, ΔpH and mt-membrane potential. It is recommended to use the lowest possible concentration of nigericin, which creates a maximal mitochondrial hyperpolarization. In the study of Komlodi 2018 J Bioenerg Biomembr, 20 nM was applied on brain mitochondria isolated from guinea-pigs using 5 mM succinate in the LEAK state which caused maximum hyperpolarisation, but did not fully dissipate the transmembrane ΔpH. Other groups (Selivanov et al 2008; Lambert et al 2004), however, used 100 nM nigericin, which in their hands fully collapsed transmembrane ΔpH using succinate as a respiratory substrate on isolated rat brain and skeletal muscle in the LEAK state.
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.
Normalization of rateNormalization of rate (respiratory rate, rate of hydrogen peroxide production, growth rate) is required to report experimental data. Normalization of rates leads to a diversity of formats. Normalization is guided by physicochemical principles, methodological considerations, and conceptual strategies. The challenges of measuring respiratory rate are matched by those of normalization. Normalization of rates for: (1) the number of objects (cells, organisms); (2) the volume or mass of the experimental sample; and (3) the concentration of mitochondrial markers in the instrumental chamber are sample-specific normalizations, which are distinguished from system-specific normalization for the volume of the instrumental chamber (the measuring system). Metabolic flow, I, per countable object increases as the size of the object is increased. This confounding factor is eliminated by expressing rate as sample-mass specific or sample-volume specific flux, J. Flow is an extensive quantity, whereas flux is a specific quantity. If the aim is to find differences in mitochondrial function independent of mitochondrial density, then normalization to a mitochondrial marker is imperative. Flux control ratios and flux control efficiencies are based on internal normalization for rate in a reference state, are independent of externally measured markers and, therefore, are statistically robust.
O2kO2kO2k - Oroboros O2k: the modular system for high-resolution respirometry.
O2k-Fluo LED2-Module
Fluorescence-Control Unit lettered.jpg
The O2k-Fluo LED2-Module is a component of the O2k-Fluorometer (O2k-Series D to G). It is an amperometric add-on module to the O2k-Core (O2k-Series D to G), adding a new dimension to high-resolution respirometry. Optical sensors are inserted through the front window of the O2k-glass chambers, for measurement of hydrogen peroxide production (Amplex® UltraRed), ATP production (Magnesium Green™), mt-membrane potential (Safranin, TMRM, Rhodamine 123), Ca2+ (Calcium Green™), and numerous other applications open for O2k-user innovation.
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