Fasching 2012 Abstract Bioblast
|Fasching M, Sumbalova Z, Gnaiger E (2012) O2k-Fluorometry - a MitoCom project. Mitochondr Physiol Network 17.12.|
Event: Bioblast 2012
High-resolution respiromety  is extended by MultiSensor techniques allowing the simultaneous measurement of oxygen consumption and one additional parameter (mitochondrial membrane potential, pH, Ca2+, or NO concentration [2,3]). Combining optical measurements with high-resolution respirometry further increased the range of analytical opportunities. In particular, fluorometric methods are available for a wide range of analytical parameters of major interest in mitochochondrial physiology: H2O2 production, mitochondrial membrane potential, intracellular pH, Ca2+, Mg2+, and NADH levels.
The simultaneous measurement of additional parameters in a single respirometric chamber under strictly identical conditions offers important advantages: (i) respirometric performance provides a quality control of cells or mitochondrial preparations; (ii) overtitration of uncouplers, incomplete action of inhibitors, or non-saturating substrate concentrations are evaluated by the simultaneous response of multiple parameters, providing objective exclusion criteria; (iii) side effects of TPP+ or fluorophores (inhibition, dyscoupling) are detected by respiration and artifacts are, therefore, excluded; (iv) the direct relationship between the different parameters eliminates artifacts of normalization for a mitochondrial marker; (v) additional information is acquired for a limited amount of sample. In addition, there are practical and economical advantages, saving handling time and money. The glass chamber of the Oroboros O2k (O2k) respirometer allows transmitting optical signals through the chamber wall. This extends MultiSensor applications, obtaining the optical signal in addition to the signals of the oxygen sensor and of other electrodes inserted through the stopper (e.g. respiration, mitochondrial membrane potential and H2O2 production). The O2k-Fluorescence LED2-Module is a LED and filter based fluorometry add-on module to be used together with the O2k. Optical sensors are inserted through the front window of the O2k-glass chambers, for measurement of hydrogen peroxide production (Amplex Ultrared), ATP production (Magnesium green), mt-membrane potential (Safranin), Ca2+ (Calcium green), and numerous other applications open for O2k-user innovation. We describe selected stages form the development of this module. Filter sets were optimized to record a fluorescence signal free from absorption artifacts. The nature of observed drift was studied and performance parameter compared with a traditional spectrofluorometer. We describe an application for simultaneous HRR measurement of oxygen consumption, using Amplex UltraRed®.
- Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352.
- Aguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim Biophys Acta 1797: 557-565.
- O2k-MultiSensor System
• Keywords: O2k-Fluorescence_LED2-Module
Mario Fasching (1), Zuzana Sumbalova (2), Erich Gnaiger (1,3)
(1) Oroboros Instruments GmbH, Innsbruck, Austria
(2) Comenius University, Bratislava, Slovak Republic
(3) Innsbruck Medical University, Austria
Contribution to K-Regio Project MitoCom Tyrol
Shown is a Fuorescence-Sensor: an LED with a specified wavelength, a photodiode, and a filter-cap attached with a specific optical filter for the LED and/or photodiode. Each Fluorescence-Sensor, Green and Blue, is equipped with a removable filter-cap for exchange of optical filters, which is possible independently for optical pathways from the LED and to the photodiode.
Labels: MiParea: Respiration, Instruments;methods
Organism: Mouse Tissue;cell: Nervous system
HRR: Oxygraph-2k, O2k-Fluorometer, Ca