Difference between revisions of "O2k-Specifications"
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{{MitoPedia | {{MitoPedia | ||
|description= | |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 [[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]]. | ||
|info=[http://www.oroboros.at/index.php?hrr-versus-multiwell HRR versus multiwell] | |info=[http://www.oroboros.at/index.php?hrr-versus-multiwell HRR versus multiwell] | ||
|type=Respiration | |type=Respiration | ||
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* [[Talk:Multiwell respirometer|Correspondence]] | * [[Talk:Multiwell respirometer|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. | |||
===Mixing=== | ===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 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 [http://www.oroboros.at/index.php?o2k-specifications 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== | |||
===How is the chamber volume defined?=== | ===How is the chamber volume defined?=== | ||
Revision as of 18:08, 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 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
How is the chamber volume defined?
No information is provided on the accuracy of the chamber volume in a multiwell system. The actual concentrations of titrated substances, therefore, are not known, in contrast to HRR.
Are specifications comparable?
No specifications are given on sensitivity (lower limit of detection of oxygen; non-linearity and restricted linear range; detection limit of oxygen flux) in such multiwell systems. Some advertise that they are so much better than Clark-type electrodes, but no quantitative specifications are provided. There are detailed and unique specifications for the O2k.
How are cell number or mitochondrial protein defined?
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 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?
The number of substances that can be titrated sequentially into a well is limited, such that modern substrate-uncoupler-inhibitor titration (SUIT) protocols cannot be applied in multiwell systems. Not even inhibitor titrations can be applied if only a single injection is possible through each port into the chamber.
How high are the running costs?
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. What is the cost of a plate and the cost of a single measurement? A plate with sensors and injectors can be used once only. The initial costs and the running costs are extremely high (e.g. expensive dischargeable plates and sensor cartridge for single use only). By comparison, the running costs for the OROBOROS O2k are very very low.
The OROBOROS O2k can be considered for a moderately high-throughput approach. 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.
pH measurement
No specifications are given on sensitivity [µpH/s]. The effects of buffers including the bicarbonate system have been ignored. The rate of acidification is 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).
