A pyrene fluorescence technique and microchamber for measurement of oxygen consumption of single isolated axons

Pyrene fluorescence is quenched by oxygen in an inverse and linear manner related to the partial pressure of O₂ in solution. We have developed a microchamber for measuring Q ̇_O2 of a single isolated axon, monitoring the change in fluorescence of a pyrene probe. The probe consists of a Spectra/Por dialysis hollow fiber filled with 2.5 mm pyrene in paraffin oil. The probe is inserted into a 1-mm-i.d. 2-cm-long quartz capillary tube with a freshly isolated crayfish medial giant axon. The capillary is mounted in an apparatus that forms an air- and water-tight seal except for a 0.2-mm-i.d. stainless steel tube at both ends permitting the exchange of solutions. An Olympus inverted microscope, equipped with epifluorescence optics and a 150-W xenon lamp, is used to view the preparation, generate the excitation light, and monitor the emitted fluorescence with a photomultiplier tube placed in the microscope TV port. A dichroic filter unit is utilized to select an excitation wavelength of 350 nm and collect emitted light above 420 nm. The signal is amplified with a Keithley 480 picoammeter and recorded on a strip chart. Q ̇_O2 of isolated axons was 552 ± 70 × 10^-6 mol O₂/liter tissue × min. Following sequential treatment with 2 mm ouabain and 2 mm NaCN, Q ̇_O2 decreased by 22 and 82%, respectively. These data are consistent with Q ̇_O2 measurements of whole nerve cord made with a Clark electrode O₂ monitor. With minimal modification this system could be used for metabolic measurements on small quantities of cells in culture, microgram quantities of biopsy material, and simultaneous measurements of Q ̇_O2 and contraction of single muscle fibers.