Difference between revisions of "High-resolution respirometry"

Revision as of 14:27, 18 July 2017

high-resolution terminology - matching measurements at high-resolution

High-resolution respirometry

Description

Mitochondrial function and dysfunction have gained an increasing interest over the past years, reflecting growing awareness of the fact that mitochondria play a pivotal role in human health and disease. The most advanced way to analyze mitochondrial function is by means of high-resolution respirometry and High-Resolution FluoRespirometry using the Oroboros O2k. Substrate-uncoupler-inhibitor titration (SUIT) protocols allow the diagnosis of numerous mitochondrial pathway and coupling defects in a single respirometric assay. 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. The most important extension of the O2k-Respirometer is the O2k-FluoRespirometer.

High-resolution respirometry (HRR) is defined on the basis of instrumental features of the O2k. The limit of detection of oxygen flux is as low as 1 pmol O₂.s^-1.ml^-1. Oxygen backdiffusion at zero oxygen is <4 pmol.s^-1.ml^-1. Oxygen consumption by the polarographic oxygen sensor at air saturation and standard barometric pressure (100 kPa) is 2.7 pmol.s^-1.ml^-1 (± 0.9 SD; 114 test runs at 37 °C). These highly standardized instrumental background fluxes are a linear function of oxygen concentration, which is used for automatic background correction of oxygen flux (DatLab). Typical exponential time constants of the oxygen sensors are <4 s, used for dynamic corrections in kinetic studies. Signal noise at zero oxygen concentration is <0.05 µM O₂.

Abbreviation: HRR

Reference: Gnaiger 1995 J Bioenerg Biomembr

MitoPedia methods: Respirometry, Fluorometry, Spectrophotometry

MitoPedia O2k and high-resolution respirometry: DatLab

» Keywords

Instrument

» DatLab; » MitoPedia: DatLab

» Instrumental background oxygen flux »» MiPNet14.06 Instrumental O2 background

» O2k-Core

» O2k-FluoRespirometer

» O2k-MultiSensor

» Oroboros O2k; » MitoPedia: O2k hardware

» Power-O2k

» Static sensor calibration

» TIP2k

Concept

» Concentration

» Coupling control state

» Mitochondrial preparations

» Oxidative phosphorylation

» Oxygen, dioxygen, O₂

» Oxygen flux

» Pathway control state

» Respiration

» Respirometry; » MitoPedia: Respirometry

» Substrate-uncoupler-inhibitor titration (SUIT) protocols; » MitoPedia: SUIT

MitoPedia: O2k

» MitoPedia: O2k hardware

» MitoPedia: DatLab

» MitoPedia: SUIT

Links

» MitoPedia: Respiratory states

-

» Respirometry

» MitoPedia: Respirometry

» Wikipedia: Respirometry

» O2k Applications

» References

O2k-Publications

Selected references

@@ Line 2: / Line 2: @@
 |abbr=HRR
 |description=[[Image:O2k-Fluorometer.JPG|180px|left|link=O2k-FluoRespirometer]]
-Mitochondrial function and dysfunction have gained an increasing interest over the past years, reflecting growing awareness of the fact that mitochondria play a pivotal role in human health and disease. The most advanced way to analyze mitochondrial function is by means of high-resolution respirometry using the [[Oxygraph-2k]] ('''O2k'''). Substrate-uncoupler-inhibitor titration (SUIT) protocols allow the diagnosis of numerous mitochondrial pathway and coupling defects in a single respirometric assay. 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-Module|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. The most important extension to the [[O2k-MultiSensor]] systems is the [[O2k-FluoRespirometer]].
+Mitochondrial function and dysfunction have gained an increasing interest over the past years, reflecting growing awareness of the fact that mitochondria play a pivotal role in human health and disease. The most advanced way to analyze mitochondrial function is by means of '''high-resolution respirometry''' and [[High-Resolution FluoRespirometry]] using the [[Oroboros O2k]]. Substrate-uncoupler-inhibitor titration (SUIT) protocols allow the diagnosis of numerous mitochondrial pathway and coupling defects in a single respirometric assay. 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-Module|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. The most important extension of the [[O2k-Respirometer]] is the [[O2k-FluoRespirometer]].
 '''High-resolution respirometry''' (HRR) is defined on the basis of instrumental features of the O2k. The limit of detection of oxygen flux is as low as 1 pmol O<sub>2</sub>.s<sup>-1</sup>.ml<sup>-1</sup>. Oxygen backdiffusion at zero oxygen is <4 pmol.s<sup>-1</sup>.ml<sup>-1</sup>. Oxygen consumption by the polarographic oxygen sensor at air saturation and standard barometric pressure (100 kPa) is 2.7 pmol.s<sup>-1</sup>.ml<sup>-1</sup> (± 0.9 SD; 114 test runs at 37 °C). These highly standardized instrumental background fluxes are a linear function of oxygen concentration, which is used for automatic background correction of oxygen flux (DatLab). Typical exponential time constants of the oxygen sensors are <4 s, used for dynamic corrections in kinetic studies. Signal noise at zero oxygen concentration is <0.05 µM O<sub>2</sub>.
@@ Line 48: / Line 48: @@
-== What is an Oxygraph? ==
+== MitoPedia: O2k ==
-::::* <big>'''O2'''k:</big> The O2k measures oxygen ([[Oxygen |dioxygen, '''O<sub>2</sub>''']]) as a gas dissolved in aqueous solution.
-::::* With the O2k, experimental data are displayed not only of oxygen concentration over time; but real-time transformation of these data allows the experimenter to follow '''[[respiration]]''' (oxygen consumption, oxygen flux) of biological and biochemical samples while the experiment is still running.
-::::* An Oxygraph is not used to measure oxygen concentration in a tissue, but for high-resolution analysis of respiratory activity of biological or biochemical samples transferred into the experimental chamber.
-[[Image:O2k-Protocols.jpg|right|230px|link=Gnaiger_2014_MitoPathways|MitoPathways]]
-[[File:Pesta 2012 Methods Mol Biol Fig7A.jpg|left|250px|link=Pesta_2012_Methods_Mol_Biol|Pesta and Gnaiger 2012]]
-:::: Specific [[substrate-uncoupler-inhibitor titration]] (SUIT) protocols are applied in studies of cell respiration or mitochondrial activity. In experiments with intact cells, different coupling control states can be studied to evaluate ROUTINE respiratory activity, respiration coupled to ATP production, respiratory excess capacity, residual oxygen consumption, and, by extension of conventional protocols, intactness of cell membranes. In mitochondrial preparations such as isolated mitochondria, permeabilized cells and tissue samples, or homogenized tissue, quantitative information is obtained on selected mitochondrial pathways is obtained, particularly of the process of '''[[oxidative phosphorylation]] (OXPHOS)''' in mitochondria and bacteria. Comprehensive OXPHOS analysis leads us beyond the highly informative process of oxygen consumption, when high-resolution respirometry is combined with O2k-MultiSensor modules to simultaneously measure the production of reactive oxygen species or mitochondrial membrane potential, nitric oxide production, acid-base balance or calcium with O2k-FluoRespirometry or ion selective electrodes. In the O2k, these bioenergetic parameters are always evaluated in combination with the measurement of oxygen concentration and respiration.
-[[File:O2k-universe.jpg|right|240px|]]
-::::* <big>O'''2k''':</big> The O2k is the '''2'''<sup>nd</sup> generation of our high-resolution respirometers. The first model was available as of [[OROBOROS 1992|1992]]. Development of the new O'''2k''' started in '''2k''' (2000) and the first O'''2'''k was delivered in [[OROBOROS 2002|'''2'''00'''2''']].
-::::* <big>O2'''k''':</big> The O2k has two experimental chambers ('''k''': Kammern, German for chambers). [[O2k-Core |Why 2 ml?]]
-::::* <big>'''Power-O2k''':</big> Several O2k units are operated simultaneously ('''P'''1, '''P'''2, ..) to combine high-resolution respirometry with [[Power-O2k |high-output]].
-::::* '''<big>[[MiPNet14.10 O2k-Top 10|Oroboros O2k - tested and trusted]]</big>'''
-::::* '''<big>[[Oroboros O2k |O2k-Concept]]</big>'''
-== MitoPedia: O2k and high-resolution respirometry ==
 ::::» [[MitoPedia: O2k hardware]]
 ::::» [[MitoPedia: DatLab]]