Perfusion double-channel micropipette probes for oxygen flux mapping with single-cell resolution

摘要

Measuring cellular respiration with single-cell spatial resolution is a significant challenge, even with modern tools and techniques. Here, a double-channel micropipette is proposed and investigated as a probe to achieve this goal by sampling fluid near the point of interest. A finite element model (FEM) of this perfusion probe is validated by comparing simulation results with experimental results of hydrodynamically confined fluorescent molecule diffusion. The FEM is then used to investigate the dependence of the oxygen concentration variation and the measurement signal on system parameters, including the pipette’s shape, perfusion velocity, position of the oxygen sensors within the pipette, and proximity of the pipette to the substrate. The work demonstrates that the use of perfusion double-barrel micropipette probes enables the detection of oxygen consumption signals with micrometer spatial resolution, while amplifying the signal, as compared to sensors without the perfusion system. In certain flow velocity ranges (depending on pipette geometry and configuration), the perfusion flow increases oxygen concentration gradients formed due to cellular oxygen consumption. An optimal perfusion velocity for respiratory measurements on single cells can be determined for different system parameters (e.g., proximity of the pipette to the substrate). The optimum perfusion velocities calculated in this paper range from 1.9 to 12.5 μm/s. Finally, the FEM model is used to show that the spatial resolution of the probe may be varied by adjusting the pipette tip diameter, which may allow oxygen consumption mapping of cells within tissue, as well as individual cells at subcellular resolution.