Difference between revisions of "High-resolution respirometry"

Latest revision as of 09:09, 2 April 2024

high-resolution terminology - matching measurements at high-resolution

High-resolution respirometry

Description

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.

Abbreviation: HRR

Reference: Oroboros O2k-technology

»Bioblast links: O2k-technology and HRR - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

O2k-technology

» Oroboros O2k-technology

» High-resolution respirometry

» NextGen-O2k - Category:NextGen-O2k - NextGen-O2k Technical developments

» Oroboros O2k

» Power O2k-Respirometer

» MitoPedia: O2k hardware

» DatLab; » MitoPedia: DatLab

» O2k-MultiSensor, O2k-FluoRespirometer

Oroboros Marketplace

» O2k

Concept

» Concentration

» Coupling-control state

» Mitochondrial preparations

» Oxidative phosphorylation

» Oxygen, dioxygen, O₂

» Oxygen flux

» Electron-transfer-pathway state

» Respiration

» Respirometry; » MitoPedia: Respirometry

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

MitoPedia: O2k and high-resolution respirometry

» MitoPedia: O2k hardware

» MitoPedia: DatLab

» MitoPedia: SUIT

» MitoPedia: Respiratory states

-

» Respirometry

» MitoPedia: Respirometry

» Wikipedia: Respirometry

» O2k Applications

O2k-Publications

Bioblast links: Oxygen signal - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Specific

O2k-Procedures

» High signal at zero oxygen

» POS calibration - static

» POS calibration - dynamic

MiPNet O2k-Procedures

» POS-calibration

» O2k-Manual: POS-service

» O2k-cleaning

» O2k-Series G and DatLab 6: Calibration

General

» Oxygen, dioxygen, O₂

» Oxygen calibration - DatLab

» Oxygen solubility

» Oxygen solubility factor

» Oxygen pressure

» Concentration

» Activity

» Pressure - Pascal

» Barometric pressure

» High-resolution respirometry

» OroboPOS

» Polarographic oxygen sensor

» MitoFit Quality Control System

» Sensitivity

Other keyword lists

» Keywords: Concentration and pressure

Selected references

MitoPedia methods: Respirometry, Fluorometry, Spectrophotometry

MitoPedia O2k and high-resolution respirometry: O2k hardware, O2k-Respirometry, O2k-FluoRespirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=HRR
-|description='''High-resolution respirometry''' ([http://www.oroboros.at/index.php?high-resolution-respirometry HRR]) is based on the [[Oxygraph-2k|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 ([[Titration-Injection microPump|TIP2k]]).  Standardized two-point calibration of the polarographic oxygen sensor ([[POS calibration|static sensor calibration]]), calibration of the sensor response time ([[POS calibration|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.
+|description=[[Image:O2k-Fluorometer.jpg|200px|left|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.
-|info=[[MiPNet06.01]]; [[Gnaiger_2008_POS]]; [[Gnaiger_2001_Respir Physiol]]
-|type=Respiration
-}}
-{{MitoPedia methods
-|mitopedia method=Respirometry, Fluorometry, Spectrophotometry
-|type=Respiration
-}}
-{{MitoPedia topics|type=Respiration
-}}
-== Links ==
-* [[Respirometry]]
+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.
-* [[Glossary: Respirometry]]
-* [http://www.oroboros.at/index.php?respirometry @OROBOROS: Respirometry]
-* [http://en.wikipedia.org/wiki/Respirometry @Wikipedia: Respirometry]
+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-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. HRR can be extended for MultiSensor analysis by using the [[O2k-Fluo Smart-Module]]. [[Smart Fluo-Sensor]]s are integrated into the O2k to measure simultaneously fluorometric signals using specific fluorophores. Potentiometric modules are available with ion-selective electrodes (pH, TPP<sup>+)</sup>. 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.
+|info=[[Oroboros O2k-technology]]
+}}
+{{Template:Keywords: O2k-technology}}
-== Physiological temperature ==
+== MitoPedia: O2k and high-resolution respirometry ==
+::::» [[MitoPedia: O2k hardware]]
+::::» [[MitoPedia: DatLab]]
+::::» [[MitoPedia: SUIT]]
-We recommend determining mitochondrial respiratory activity at the physiologic temperature of a biologic system, no matter whether you work with [[MiPNet14.14 PermeabilizedFibrePreparation|biopsies]], [[MiPNet08.09 CellRespiration|cultivated cells]] or [[MiPNet12.11 MitoRespiration|isolated mitochondria]]. When measuring in an appropriate [[List of media for respirometry|medium]] (protecting and stabilizing mitochondrial function) stability is preserved even in isolated mitochondria.
+::::'''»''' [[MitoPedia: Respiratory states]] [[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ET capacity|ET capacity]] [[Image:L.jpg|link=LEAK respiration|LEAK]]  - [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]]
+::::» [[Respirometry]]
+::::» [[MitoPedia: Respirometry]]
+::::» [http://en.wikipedia.org/wiki/Respirometry Wikipedia: Respirometry]
+::::» [[O2k Applications]]
-== Oxygraphy: Linear versus non-linear slope ==
+[[Image:O2k-Publications.jpg|left|116px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]]
+::: '''O2k-Publications'''
+::::* [[O2k-Publications |O2k-Publications: Chronological]]
+::::* [[O2k-Publications: Topics]]
-=== Oxygen dependence of respiration ===
-In isolated mitochondria and many types of small cells (such as endothelial cells, fibroblasts etc), zero-order kinetics with respect to oxygen pressure (i.e. independence of flux with declining oxygen concentration) applies over a wide range down to very low oxygen levels, where then a hyperbolic oxygen dependence is observed. With a ''p''50 (''c''50, apparent ''K''m) in the order of <1 µM and a hyperbolic oxygen dependence of flux, 99% saturation of flux and hence zero-order kinetics is observed at an oxygen concentration >19 µM (i.e. >2 kPa, >2% oxygen saturation or >10% air saturation). For reviews see [[Gnaiger_1995_J_Bioenerg_Biomembr|Gnaiger et al 1995]], [[Scandurra_2010_Adv_Exp_Med_Biol|Scandurra and Gnaiger 2010]].
+{{Template:Keywords: Oxygen signal}}
-Compared to the difficulties with classical chart recorder tracings, our 'modern' approaches over the past 20 years allowed us to drop the linearity assumption and statistically test for it, frequently rejecting this assumption to describe a non-linear oxygen dependence beyond the low-oxygen range governed by cytochrome c oxidase. For review see [[Gnaiger_2003_Adv_Exp_Med_Biol|Gnaiger 2003]].
+::: '''Selected references'''
+::::* [[Gnaiger 1995 J Bioenerg Biomembr]]
+::::* [[Gnaiger 2001 Respir Physiol]]
+::::* [[Gnaiger 2008 POS]]
+::::* [[Gnaiger 2020 BEC MitoPathways]]
+</div>
+</div>
+<br />
-In permeabilized muscle fibres (30 to 37 °C), oxygen kinetics is shifted 100-fold due to artifially high oxygen gradients, forcing us to apply elevated oxygen levels for obtaining near-zero-order kinetics. For details, see [[Pesta_2012_Methods_Mol_Biol|Pesta and Gnaiger 2012]], [[Gnaiger_2003_Adv_Exp_Med_Biol|Gnaiger 2003]], and [[Permeabilized_muscle_fibres#Oxygen_kinetics_of_permeabilized_fibres|Discussion]].
+{{MitoPedia methods
+|mitopedia method=Respirometry, Fluorometry, Spectrophotometry
-=== ADP dependence of respiration ===
+}}
-The assumption of linearity (linear regression of oxygen concentration over time) is frequently not valid for various reasons other than oxygen kinetics. In classical ‘State 3’, ADP levels are ‘high’ (Chance and Williams 1955), but not necessarily saturating ([[Glossary: Respiratory states]]). Then, an ADP-dependent decline of respiration is observed immediately after titration of a sub-saturating concentration of ADP, which is obscured by any linear regression. This has caused in the past a tremendous underestimation of the apparent Km for ADP, perpetuated even today with the uncritical application of non-adequate software implementing the simple linearity approach only. For a critical approach to ADP kinetics, see [[Gnaiger_2000_Proc_Natl_Acad_Sci_USA|Gnaiger et al 2000]] and [[Gnaiger_2001_Respir_Physiol|Gnaiger 2001]].
+{{MitoPedia O2k and high-resolution respirometry
+|mitopedia O2k and high-resolution respirometry=O2k hardware, O2k-Respirometry, O2k-FluoRespirometry
+}}
-== Titration of chemicals ==
-'''''Do overshoots or short interruptions of the flux have a meaning after injection of chemicals?'''''
-Neither the overshoot nor the short signal disturbance is representing mitochondrial respiration. The disturbance of the signal is due to the injection of the chemical. With the addition of chemicals solved in ethanol a little bit of oxygen is always introduced, since ethanol has higher oxygen solubility than water.
-:>> [[MiPNet09.12 O2k-Titrations]]
-== Various ==
-:>> [[MiPNet19.03 O2k-cleaning and ISS]]
-:>> [[Cleaning the TPP+ electrodes]]
-:>> [[Troubleshooting]]
-{{#set: Technical service=Chamber }}