O2k-Core: Oxygraph-2k with Core-Accessories - complete for basic high-resolution respirometry
High-resolution is distinguished from conventional approaches by a combination of HRR features and specifications.
- The unique sole-source O2k-specifications set a new standard in bioenergetics, mitochondrial physiology, clinical research and diagnosis of mitochondrial pathologies.
- The user-friendly DatLab software is an integral component of high-resolution respirometry.
- The stainless-steel housing of the O2k shields the advanced state-of-the-art electronics.
- The OroboPOS is the polarographic oxygen sensor (POS) with the highest dynamic linear range among all available oxygen sensors.
- The Power-O2k turns the O2k into a powerful high-throughput system.
O2k-Chamber: Paradigm shift in HRR
- Oxygraph-2k: Paradigm shift and features of high-resolution respirometry. MiPNet09.01. Including sole source information. download pdf
This chapter (2nd ed) is printed in: Gnaiger E (2007) Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck: 96 pp
Small chamber volumes of 20-50 µl generate problems rather than providing resolution with small amounts of tissue samples, low numbers of cultured cells or few isolated mitochondria. The surface to volume ratio increases with decreasing chamber volume, thus various boundary effects entail larger errors at smaller volume, in particular oxygen diffusion. While the rate of oxygen depletion per unit amount of sample increases linearly with decreasing chamber volume, side effects may increase to a larger degree. Then, sensitivity is lost with decreasing chamber size. We recommend a minimum chamber volume of 1.5-2.0 cm3.
Paradigm shift from mimimum to optimum chamber volume
- Minimization of chamber volume represents a past paradigm, aming at high rates of oxygen consumption per volume. The advantage appears to be obvious, whereas the drawbacks are frequently overlooked (see below).
- Advancements of electronics, data acquisition and analysis, polarographic oxygen sensor specifications and chamber design made possible a superior approach, allowing for respirometric measurements at high dilution, as reviewed by Gnaiger E (2001). In specifically designed mitochondrial respiration media, respiration is stable at high dilution, multiple substrate/inhibitor titrations are possible without oxygen depletion, and a low-oxygen regime may be chosen to prevent elevation of oxidative stress at air-level oxygen saturation. In contrast, micro-chambers are characterized by a high surface-to-volume ratio which hinders optimum stirring, increases unfavourable surface effects and oxygen-backdiffusion, and poses problems with accurate titrations and dilution effects of the sample. These potential - and mostly hidden - artefacts are avoided in high-resolution respirometry, using glass chambers, titanium stoppers, and avoiding teflon-coated stirrers or perspex (yielding high back-diffusion of oxygen).
Assume you have 0.1 mg mitochondrial protein for a respirometric assay. Approach (1) would lead you to search for a 100 µl volume respirometer, to maintain a classical 1 mg/ml protein concentration. In contrast, high-resolution respirometry allows for dilution of mitochondria to 0.02 mg/ml protein. Dilution of 0.1 mg mitochondrial protein in a 2 ml chamber yields an optimum concentration for multiple substrate/inhibitor titrations and kinetic measurements. The high-resolution approach of the OROBOROS Oxygraph-2k offers the unique advantages of a versatile and ready-to-use system for studies in mitochondrial physiology and pathology.
DatLab - the highway to scientific information
DatLab - software for high-resolution respirometry
Software for instrument control, data acquisition, on-line display of oxygen concentration and oxygen flux, recording of local barometric pressure, experimental temperature and Peltier power, on-line and off-line data analysis, multi-channel O2k-MultiSensor monitoring, feedback-control of the Titration-Injection microPump (TIP2k) - running under Windows(TM).
High-resolution respirometry with the OROBOROS Oxygraph-2k established a radically different, integrated systems approach to polarographic measurement of oxygen dynamics, including the DatLab software with on-line display of oxygen concentration and respiration (oxygen flux; see Figure; T-shirt). The mechanics, critical selection of materials (minimum oxygen diffusion), and state-of-the-art electronics provide the basis of signal stability of an optimized polarographic oxygen sensor, in an electronically controlled thermal environment with high temperature stability (0.001 °C). Calibrations and instrumental controls follow standardized HRR protocols, with automatic on-line analysis, including corrections for instrumental background oxygen flux (equation in Figure). Scientific support by the OROBOROS O2k-Team is complementary to the approach of high-resolution respirometry.
The OroboPOS Oxygen Sensor
Choice of the polarographic oxygen sensor (POS)
We developed the polarographic oxygen sensor OroboPOS. This POS is designed with a large cathode (2 mm diameter) since sensitivity is a function of cathode area. The signal to noise ratio increases and the relative signal drift at zero oxygen decreases with cathode diameter. Angular insertion of the POS into the glass chamber places the cathode at an optimum position for stirring, contrary to a centrally inserted POS with the cathode at the point of minimum water current.
Resistant Materials for Minimum Oxygen Diffusion
Oxygen diffusion is minimized by appropriate choice of materials. Avoiding perspex chambers is necessary but not sufficient. We use glass chambers and PVDF stoppers or titanium stoppers or with titanium injection cannulas. PEEK or PVDF coated stirrers exclude oxygen dissolved in the material. In contrast, O2 solubility of Teflon is 106 µM/kPa at 25 °C compared to <11 µM/kPa in physiological salt solutions. A Teflon stirrer bar is an O2 buffer, and oxygen leaks back, at up to -30 pmol O2.s-1.cm-3 when oxygen is lowered quickly. Oxygen diffusion increases by a factor of >10 in our system when choosing the wrong material for an O-ring or sealing. We use Viton O-rings and butyl indian rubber sealings with zero oxygen diffusion. Residual oxygen diffusion (MiPNet14.06) is <2 pmol O2.s-1.cm-3 at zero pO2, 25 °C to 37 °C, and a 2 cm3 chamber volume.
Peltier Temperature Control
Integral electronically controlled thermostat
Built-in Peltier thermostat, replacing the conventional water jacket, for superior temperature stability (±0.001 °C) and accuracy in the range 4 °C to 47 °C (at room temperature). Continuous recording of temperature and Peltier power with DatLab. Temperature change from 20 to 30 °C within 15 min; cooling from 30 to 20 °C within 20 min.