Simultaneous monitoring of oxygen consumption and acidification rates of a single zebrafish embryo during embryonic development within a microfluidic device

Abstract

A microfluidic device with a light modulation system was developed to simultaneously measure the oxygen consumption rate (OCR) and acid extrusion rate (AER) of a single zebrafish embryo during embryonic development. The device combines two components: an array of acrylic microwells containing two sensing layers as the dual luminescent sensor for oxygen (O2) and acid (pH) detection, and a microfluidic module with pneumatically actuated glass lids to controllably seal the microwells. The continuous blue LED and modulated UV LED lights were simultaneously used to excite the dual luminescent sensor, with the emission detected by a single photodetector. The detection signals were then split into DC and AC components to measure the time variations in fluorescence intensity and phosphorescence lifetime for pH and O2 detection, respectively. We have successfully measured the OCR and AER of a single developing zebrafish embryo inside a sealed microwell from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage of 48 hpf. We also demonstrated the measurement of the OCR and AER of a single 48 hpf zebrafish that experienced acute hypoxia by using our device to monitor the transition between aerobic and anaerobic metabolism. We observed that the AER began to significantly increase, while the OCR rapidly decreased after 20 min of hypoxia, indicating the time point of transition where the non-mitochondrial metabolism subsequently dominated the energy production. Our proposed methodology provides the potential for studying the bioenergetic metabolism in a developing organism that relates mitochondrial physiology and disease.