Figure 6.

Analysis of single cell respiration in the microscope fast exchange chamber. (A) Flow profile in the microscope chamber found by the mathematical model. The flow profile is described by velocity magnitude (shown by color) and streamlines (white lines). At inflow (top left), the velocity is higher than at outflow (bottom right) due to the larger opening of the chamber at outflow. (B) Distribution of ADP (shown by color) predicted by the mathematical model around a cell situated in the middle of a chamber, attached to the glass. The location of the cell is outlined (black wire-frame) on the image. The flow in the box was calculated on basis of the flow distribution in the chamber taking into account the flow around the cell. The flow is indicated by the arrows (left) and streamlines (white lines). Note how flow of solution leads to asymmetric distribution of ADP next to the cell. (C) Influence of ADP mixing mechanisms on respiration rate of a cell in microscope chamber. Assuming that there are no intracellular diffusion restrictions, the calculated affinity of respiration to ADP is very high in the case of infinitely fast diffusion (dashed black line). If ADP is supplied just by diffusion, the affinity drops significantly (dotted black line). Taking into account flow distribution in the chamber, the predicted affinity of respiration to ADP (solid black line) is significantly higher that the one determined from experiments. For comparison, respiration rate dependency determined on the cell suspension in respiration chamber is shown (open dots) as well as respiration rate estimated from NADH (solid squares) and FP (solid triangles). Only by assuming significant intracellular diffusion restrictions modeled by increasing an apparent K_m(ADP) of mitochondrial respiration to 0.15 mM (red line), 0.3 mM (green line), or 0.45 mM (blue line), is it possible to reproduce the measurements if the flow in the chamber is taken into account.