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

Abstract

Pyrene fluorescence is quenched by oxygen in an inverse and linear manner related to the partial pressure of O 2 in solution. We have developed a microchamber for measuring Q ̇ O 2 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 m m 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 ̇ O 2 of isolated axons was 552 ± 70 × 10 −6 mol O 2/liter tissue × min. Following sequential treatment with 2 m m ouabain and 2 m m NaCN, Q ̇ O 2 decreased by 22 and 82%, respectively. These data are consistent with Q ̇ O 2 measurements of whole nerve cord made with a Clark electrode O 2 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 ̇ O 2 and contraction of single muscle fibers.