Difference between revisions of "O2k-Specifications"
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* Critical selection of materials yielding a nearly [http://www.oroboros.at/index.php?backgroundcorrection diffusion-tight] chamber, | * Critical selection of materials yielding a nearly [http://www.oroboros.at/index.php?backgroundcorrection diffusion-tight] chamber, | ||
* Long-term stability and linearity of the polarographic oxygen sensor ([http://www.oroboros.at/index.php?o2k-pos POS]), | * Long-term stability and linearity of the polarographic oxygen sensor ([http://www.oroboros.at/index.php?o2k-pos POS]), | ||
* Highly automatic but transparent calibration routines and instrumental background correction, | * Highly automatic but transparent [http://www.oroboros.at/index.php?o2k-o2calibration calibration routines] and [http://www.oroboros.at/index.php?o2k-datlab-fluxanalysis instrumental background correction], | ||
* Electronically controlled thermal environment with high temperature stability (+-0.001 °C), | * Electronically controlled thermal environment with high temperature stability (+-0.001 °C), | ||
* Limit of detection of respiratory flux: +-1 pmol.s-1.ml-1. | * Limit of detection of respiratory flux: +-1 pmol.s-1.ml-1. | ||
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===Multi-well=== | ===Multi-well=== | ||
From their basic design, multi-well systems are a tool for qualitative high-throughput screening, particularly for pharmacologic testing. Designed for high-throughput qualitative measurements, they should not be advertised for quantitative measurements. A high standard of quantitative results cannot be obtained with the present technology offered and advertised. Huge oxygen fluxes reported by one group at a mitochondrial physiology meeting (MiPsummer School 2009, Baton Rouge, USA) were explained by a company representative as a "software problem". The absolute oxygen fluxes represent artefacts. More recently, the problems of high oxygen diffusion were recognized, but the large corrections render non-specified errors in the calculation of background-corrected oxygen flux. | From their basic design, multi-well systems are a tool for qualitative high-throughput screening, particularly for pharmacologic testing. Designed for high-throughput qualitative measurements, they should not be advertised for quantitative measurements. A high standard of quantitative results cannot be obtained with the present technology offered and advertised. Huge oxygen fluxes reported by one group at a mitochondrial physiology meeting (MiPsummer School 2009, Baton Rouge, USA) were explained by a company representative as a "software problem". The absolute oxygen fluxes represent artefacts. More recently, the problems of high oxygen diffusion were recognized, but the large corrections render [[Talk:Rogers_2011_PlosOne#Instrumental_specifications_of_the_XF_multi-well_system_are_missing|non-specified errors in the calculation of background-corrected oxygen flux]]. | ||
==Is plastic material compatible with respirometry?== | ==Is plastic material compatible with respirometry?== | ||
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===HRR=== | ===HRR=== | ||
Plastic materials with high oxygen diffusion are eliminated from the chamber in HRR. The durane glass chambers are closed by PVDF or PEEK stoppers which are as diffusion tight as our titanium stoppers (originally used in O2k Series A and B). The magnetic stirrer bars are coated by PVDF or PEEK. Viton O-rings for used for sealing the stoppers, and buyl rubber gaskets provide the seals for the oxygen sensors. | [[http://www.oroboros.at/index.php?backgroundcorrection Plastic materials with high oxygen diffusion are eliminated from the chamber in HRR]]. The durane glass chambers are closed by PVDF or PEEK stoppers which are as diffusion tight as our titanium stoppers (originally used in O2k Series A and B). The magnetic stirrer bars are coated by PVDF or PEEK. Viton O-rings for used for sealing the stoppers, and buyl rubber gaskets provide the seals for the oxygen sensors. | ||
HRR not only minimizes the effect of backdiffusion by avoiding inappropriate plastic materials, but additionally implements automatic correction for instrumental background flux (see [http://www.oroboros.at/index.php?o2k-specifications O2k-specifications]). | HRR not only minimizes the effect of backdiffusion by avoiding inappropriate plastic materials, but additionally implements automatic correction for instrumental background flux (see [http://www.oroboros.at/index.php?o2k-specifications O2k-specifications]). | ||
===Multi-well=== | ===Multi-well=== | ||
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At the high surface-to-volume ratio, the problem is not restricted to oxygen diffusion, but all lipid soluble substances partition between the aqueous and plastic phase, such that the surface-attached biological sample is exposed to undefined effective concentrations. | At the high surface-to-volume ratio, the problem is not restricted to oxygen diffusion, but all lipid soluble substances partition between the aqueous and plastic phase, such that the surface-attached biological sample is exposed to undefined effective concentrations. | ||
==Accuracy of chamber volume and mixing== | ==Accuracy of chamber volume and mixing== | ||
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===HRR=== | ===HRR=== | ||
The O2k is designed for flexible upgrading to the O2k-MultiSensor System, which allows for additional potentiometric measurment of mitochondrial membrane potential (ion sensitive electrode: TPP+), Ca2+ in solution (using the same ion sensitive electrode after mounting a different membrane), pH, and amperometric measurement of NO or H2S, always simultaneously with oxygen flux. The DatLab software provides full flexibility for MultiSensor data monitoring. | The O2k is designed for flexible upgrading to the [http://www.oroboros.at/index.php?mipnetanalyzer O2k-MultiSensor System], which allows for additional potentiometric measurment of mitochondrial membrane potential (ion sensitive electrode: TPP+), Ca2+ in solution (using the same ion sensitive electrode after mounting a different membrane), pH, and amperometric measurement of NO or H2S, always simultaneously with oxygen flux. The DatLab software provides full flexibility for MultiSensor data monitoring. | ||
===Multi-well=== | ===Multi-well=== | ||
The XF system is restricted to the additional measurement of pH. No specifications are given on sensitivity [µpH/s] of the measurement of acidification rate. Until recently, the effects of buffers including the bicarbonate system have been ignored. The rate of acidification was presented in pH units per time, ignoring the log scale of pH. What is the drift of the pH signal?. An oxygen flux of 50 pmol/(s ml) corresponds - at an assumed O2 flux to extracellular H+ flux ratio of 1:1 - to a pH change of about 86 µpH/s in a very weak buffer (2 mM). | The XF system is restricted to the additional measurement of pH. No specifications are given on sensitivity [µpH/s] of the measurement of acidification rate. Until recently, the effects of buffers including the bicarbonate system have been ignored. The rate of acidification was presented in pH units per time, ignoring the log scale of pH. What is the drift of the pH signal?. An oxygen flux of 50 pmol/(s ml) corresponds - at an assumed O2 flux to extracellular H+ flux ratio of 1:1 - to a pH change of about 86 µpH/s in a very weak buffer (2 mM). | ||
==How are cell number or mitochondrial protein defined?== | ==How are cell number or mitochondrial protein defined?== | ||
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How many cells are actually in the closed compartment for measurement of respiration in a well, or which fraction of isolated mitochondria is outside versus inside the effective chamber? How can the recorded change in oxygen concentration be converted to respiration per million cells or per mg protein? Without solving these problems, no quantiative measurements of respiration are possible. | How many cells are actually in the closed compartment for measurement of respiration in a well, or which fraction of isolated mitochondria is outside versus inside the effective chamber? How can the recorded change in oxygen concentration be converted to respiration per million cells or per mg protein? Without solving these problems, no quantiative measurements of respiration are possible. | ||
==Are multiple substrate-uncouplier-inhibitor titrations possible?== | ==Are multiple substrate-uncouplier-inhibitor titrations possible?== | ||
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Elaborating a protocol for starting an experimental series requires a large number of test experiments, such that the cost of discharged wells in an entire experiment approaches the investment into a second HRR system. | Elaborating a protocol for starting an experimental series requires a large number of test experiments, such that the cost of discharged wells in an entire experiment approaches the investment into a second HRR system. | ||
==Are specifications comparable?== | ==Are specifications comparable?== | ||
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There are detailed and [http://www.oroboros.at/index.php?o2k-specifications unique specifications for the O2k]. | There are detailed and [http://www.oroboros.at/index.php?o2k-specifications unique specifications for the O2k]. | ||
===Multi-well=== | ===Multi-well=== | ||
No specifications are given on sensitivity (lower limit of detection of oxygen; non-linearity and restricted linear range; detection limit of oxygen flux) in some multi well systems. | No specifications are given on sensitivity (lower limit of detection of oxygen; non-linearity and restricted linear range; detection limit of oxygen flux) in some multi well systems. | ||
Revision as of 18:30, 4 October 2011
- high-resolution terminology - matching measurements at high-resolution
O2k-Specifications
Description
From their basic design, multi-well systems are a tool for qualitative high-throughput screening, particularly for pharmacologic testing. Designed for high-throughput qualitative measurements, they should not be advertised for quantitative measurements. A high standard of quantitative results cannot be obtained with the present technology offered and advertised. The problems of high oxygen diffusion are well known, and the large corrections render non-specified errors in the calculation of background-corrected oxygen flux.
Reference: HRR versus multiwell
- Discussion: Seahorse XF for isolated mitochondria - High throughput without high output?
- Correspondence
Quantitative respirometry: HRR versus multiwell
The OROBOROS Oxygraph-2k for high-resolution respirometry (HRR) is the gold standard for highly accurate quantitative measurements, using very low amounts of sample. Instrumental and methodological approaches require an adequately advanced level of Quality Assurance in research and clinical applications.
HRR
HRR is the result of long-term expertise in instrumental design, software development, and experimental protocols developed for mitochondrial physiology, clinical and pharmacological applications. Taken together, these developments resulted in new qualitative and quantitative standards summarized as the O2k-Concept.
The [1] specifications of the OROBOROS O2k are based on the many unique instrumental features:
- Critical selection of materials yielding a nearly diffusion-tight chamber,
- Long-term stability and linearity of the polarographic oxygen sensor (POS),
- Highly automatic but transparent calibration routines and instrumental background correction,
- Electronically controlled thermal environment with high temperature stability (+-0.001 °C),
- Limit of detection of respiratory flux: +-1 pmol.s-1.ml-1.
Multi-well
From their basic design, multi-well systems are a tool for qualitative high-throughput screening, particularly for pharmacologic testing. Designed for high-throughput qualitative measurements, they should not be advertised for quantitative measurements. A high standard of quantitative results cannot be obtained with the present technology offered and advertised. Huge oxygen fluxes reported by one group at a mitochondrial physiology meeting (MiPsummer School 2009, Baton Rouge, USA) were explained by a company representative as a "software problem". The absolute oxygen fluxes represent artefacts. More recently, the problems of high oxygen diffusion were recognized, but the large corrections render non-specified errors in the calculation of background-corrected oxygen flux.
Is plastic material compatible with respirometry?
HRR
[Plastic materials with high oxygen diffusion are eliminated from the chamber in HRR]. The durane glass chambers are closed by PVDF or PEEK stoppers which are as diffusion tight as our titanium stoppers (originally used in O2k Series A and B). The magnetic stirrer bars are coated by PVDF or PEEK. Viton O-rings for used for sealing the stoppers, and buyl rubber gaskets provide the seals for the oxygen sensors.
HRR not only minimizes the effect of backdiffusion by avoiding inappropriate plastic materials, but additionally implements automatic correction for instrumental background flux (see O2k-specifications).
Multi-well
Oxygen storage in the plastic material of multiwell plates leads to high oxygen backdiffusion. Since the problems are well known (Gnaiger_1995_JBB), specifications should be provided on backdiffusion, and test protocols applied to enable evaluation of such specifications.
At the high surface-to-volume ratio, the problem is not restricted to oxygen diffusion, but all lipid soluble substances partition between the aqueous and plastic phase, such that the surface-attached biological sample is exposed to undefined effective concentrations.
Accuracy of chamber volume and mixing
HRR
The 2 ml standard chamber volume of the O2k is calibrated at <1% error (depending on calibrated pipettes), when inserting the stopper and filling the capillary at an error of <20 µl.
The entire effective volume (excluding the injection capillaries) is and well controlled stirring is provided in the O2k.
Multi-well
No information is provided on the accuracy of the chamber volume in a multi-well system (~7 µl for the XF24). This inaccuracy translates directly to the calculation of oxyge flux in the closed chamber. Similarly, accurate concentrations of titrated substances are not known.
Mixing by moving the sensor/injector part up and down a few times is inadequate. Undefined diffusion layers develop during a measuring cycle.
Flexibility for MultiSensor applications
HRR
The O2k is designed for flexible upgrading to the O2k-MultiSensor System, which allows for additional potentiometric measurment of mitochondrial membrane potential (ion sensitive electrode: TPP+), Ca2+ in solution (using the same ion sensitive electrode after mounting a different membrane), pH, and amperometric measurement of NO or H2S, always simultaneously with oxygen flux. The DatLab software provides full flexibility for MultiSensor data monitoring.
Multi-well
The XF system is restricted to the additional measurement of pH. No specifications are given on sensitivity [µpH/s] of the measurement of acidification rate. Until recently, the effects of buffers including the bicarbonate system have been ignored. The rate of acidification was presented in pH units per time, ignoring the log scale of pH. What is the drift of the pH signal?. An oxygen flux of 50 pmol/(s ml) corresponds - at an assumed O2 flux to extracellular H+ flux ratio of 1:1 - to a pH change of about 86 µpH/s in a very weak buffer (2 mM).
How are cell number or mitochondrial protein defined?
HRR
In experiments with isolated mitochondria, tissue homogenates or suspended intact or permeabilized cells, the final concentration in the O2k-chamber is either defined by the preparation of the added suspension, and/or determined by taking a subsample from the chamber. In this way, the measured oxygen flux (per volume) can be expressed accurately per unit of biological sample (per mg protein, per million cells, etc.).
In experiments with permeabilized muscle fibers or other tissues, the tissue mass is determined before adding the sample into the O2k-chamber (e.g. 0.7 mg wet weight of mouse heart, 2 mg wet weight of human muscle), and the oxygen flux can then be expressed per tissue mass (mass-specific flux, reflecting mitochondrial density and functional quality).
Multi-well
How many cells are actually in the closed compartment for measurement of respiration in a well, or which fraction of isolated mitochondria is outside versus inside the effective chamber? How can the recorded change in oxygen concentration be converted to respiration per million cells or per mg protein? Without solving these problems, no quantiative measurements of respiration are possible.
Are multiple substrate-uncouplier-inhibitor titrations possible?
HRR
Substrate-uncoupler-inhibitor titration (SUIT) protocols have been developed for diagnostic tests of mitochondrial respiratory function, to study the complex interactions of coupling control and substrate control in a single assay, thus increasing the information obtained per unit sample and per unit time. Up to 20 titration steps may be included in a single SUIT protocol.
Multi-well
The number of different substances that can be titrated into a well is limited, such that modern SUIT protocols cannot be applied in multi-well systems. A 24 well plate with four titrations per well, or a 96 well plate with two titrations per well are insufficient for obtaining the information from a SUIT experiment.
How high are the running costs?
HRR
The running costs for the OROBOROS O2k are very very low.
The OROBOROS O2k is not suited for a high-throughput approach. However, investing the identical amount of money, a large number of O2k chambers provides a unique high-throughput HRR system for quantitative O2k-measurements at low running costs.
Multi-well
The running costs are extremely high, based on expensive dischargeable wells for single use only.
How many of the wells of a dischargeable plate can actually be used for independent measurements? Several wells are required for calibration, edge effects may eliminate the use of wells on the sides.
Elaborating a protocol for starting an experimental series requires a large number of test experiments, such that the cost of discharged wells in an entire experiment approaches the investment into a second HRR system.
Are specifications comparable?
HRR
There are detailed and unique specifications for the O2k.
Multi-well
No specifications are given on sensitivity (lower limit of detection of oxygen; non-linearity and restricted linear range; detection limit of oxygen flux) in some multi well systems.
