Harrison 2012 Abstract Bioblast
|Harrison DK, Fasching M (2012) O2k-Spectrophotometry – A MitoCom Project. Mitochondr Physiol Network 17.12.|
Event: Bioblast 2012
The principal thrust of the technical development of the O2k high-resolution respirometer within the MitoCom project is towards the O2k Fluorometer. However, simultaneously, a parallel project is working towards the integration of spectrophotometry into the O2k in order to measure the redox state of cytochromes – principally cytochrome c and aa3, but also cytochrome b.
The first approach to measuring cytochrome absorption spectra in the O2k respirometer used a specially designed stopper that incorporated two lightguide fibres: one used to conduct light from an external lamp into the cuvette, the other to collect the emergent light scattered by the medium .
However, the development of multiple parameter sensors that can be inserted through the conventional stoppers led to the concept of using the chamber window as the optical port-hole for both fluorometry and spectrophotometry  instead of the special optical stopper (which could not be used in conjunction with other probes). The initial spectrophotometric approach was to use a pair of lightguides: the first to transmit light through the chamber window and the second to receive the light scattered back from the medium. A series of experiments was carried out in order to determine the optimal configuration and spacing for the lightguides. In order to carry out comparisons, it was necessary to develop analytical methods in order to compare the signal-to-noise (S/N) ratios of the difference spectra (reduced minus oxidised cytochrome absorption spectra) obtained using the different configurations and the results will be presented.
The latest approach involves incorporation of a white light emitting diode (LED) into the O2k itself, in addition to the standard illuminating LED. Initial results using this configuration demonstrate an order-of-magnitude increase in S/N ratio compared with the previous configurations. Work is continuing in order to determine the optimal spectral characteristics for the LED.
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- MiPNet17.16 O2k-Spectrophotometry
• O2k-Network Lab: AT Innsbruck Oroboros
Oroboros Instruments, Innsbruck, AT
Contribution to K-Regio Project MitoCom Tyrol
Stress:Cell death Organism: Fungi
Enzyme: Complex IV;cytochrome c oxidase Regulation: Cyt c
HRR: Oxygraph-2k, O2k-Spectrophotometer