A microscope perfusion respirometer for continuous respiration measurement of cultured cells during microscopic observation.

Abstract

The construction of a microscope perfusion respirometer (MPR) for simultaneous recording of cellular respiration and microscopic morphology is described. All light microscope techniques for living cells (e.g. phase contrast, differential interference contrast (DIC), fluorimetry) can be applied to the monolayer cells grown on a coverslip. The main constituents of the MPR are a) a precision operating perfusion pump (constant volume output), b) a modified Dvorak-Stotler perfusion chamber, c) a special holder for the Clark-type oxygen probe, d) gas-tight connections of stainless steel tubing with dead volume-free fittings, and e) a temperature control unit. The cell material, established XTH (Xenopus laevis tadpole heart) cell is characterized. Examples of operation are presented, concerning a) normal respiration, b) respiration during uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and c) lactate production under anoxia. The corresponding mitochondrial in situ-morphology is demonstrated on photomicrographs. Details of construction and application are discussed. This new technique is supposed to extend the use of cell cultures instead of animal experiments in pharmaceutical routine tests.