Determination of absolute concentrations can be performed by calibrating the sensor, ideally in the same cell type in which measurements are being made. Full calibration (i.e., determination of a dose-response curve in situ) has been achieved only for the glucose sensor, by blocking glucose consumption while using the glucose transporter or an artificial pore to equilibrate glucose across the plasma membrane.56,62 Detailed protocols have been made available for the use of metabolite sensors in mammalian cells54 and more specifically for the use of the glucose sensor.78 It is not feasible to halt the metabolism of pyruvate, lactate, or NADH without compromising cell viability, but the corresponding sensors have been two-point calibrated by obtaining readouts at extreme values of the parameters in combination with the affinity constants measured in vitro.118,121,122 Thanks to calibration, a glucose gradient was detected across the astrocytic plasma membrane,56,62 consistent with a modulatory role for GLUT transporters, and astrocytes were found to have higher ratios than neurons,118 supporting the shuttling of redox equivalents between astrocytes and neurons.123 Even over a single microscopic field, large differences were detected in glucose concentration and glycolytic flux in adjacent cells, the kind of diversity that may be mined by systems biology approaches, both to extract mechanistic information124 or to serve as readouts for single-cell metabolomics.125 No calibration is possible for the ATP sensors, so they are considered semiquantitative. A more refined use of the sensors is for the determination of flux through specific pathways. For this purpose, the probe needs to be calibrated, so that the fluorescence ratio is converted into absolute concentration. The strategy for flux determination is to halt flux at a known point in the pathway and then to monitor the rate of accumulation of an upstream metabolite or the rate of depletion of a downstream metabolite. With this approach, it has been possible to estimate the rate of glucose consumption (), the rates of lactate production or consumption (Laconic), and the rate of mitochondrial pyruvate consumption (Pyronic), as illustrated in Fig. 2(d). Some biological phenomena that have been identified with flux protocols are the regulation of astrocytic glycolysis by high and glutamate,31,59 feedback control of glycolysis by lactate,65 mitochondrial flux in electrically stimulated neurons,122 and the Warburg effect in T98G glioma cells.121 Using the FRET nanosensors in vivo, Bruno Weber and colleagues have successfully imaged glucose, lactate, and pyruvate in astrocytes and neurons using two-photon microscopy. Practical issues to be addressed for in vivo studies include sensor expression levels, calibration, the metabolic effects of anesthesia, and the adequacy of “inhibitor-stop” protocols for flux determination.