High-resolution is distinguished from conventional approaches by a combination of unique features and specifications. These set a new standard in bioenergetics, mitochondrial physiology, clinical research and diagnosis of mitochondrial pathologies:

Paradigm shift from minimum to optimum chamber volume. MiPNet09.01

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 isolate mitochondria. The surface to volume ratio increases with decreasing chamber volume, whence 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.

DatLab 4.3 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). Under Windows(TM).

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.

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.

Integral electronically controlled thermostat

Built-in Peltier thermostat, replacing the conventional water jacket, for superior temperature stability (<0.01 °C) and accuracy in the range 4 °C to 45 °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.

In contrast to merely offering an oxygen measuring device, the OROBOROS Oxygraph-2k is integrated into a systems approach to high-resolution respirometry. This spans from experimental tools which are invaluable in the lab (e.g. ISS), or little aids that are crucial for tissue preparation (forceps), to information on mitochondrial respration medium (MiR06), titration protocols, and the unique O2k-Workshops.