Methods for bioluminescence detection of such important metabolites as ATP and Ca2+ through the use of firefly luciferase or the photoprotein aequorin, respectively, are both well known and documented. Many commercial kits are available and are widely used in cell research for monitoring intracellular metabolites in vitro. The bioluminescent system of bacteria has also been used to measure intracellular NAD(P)H, and, when coupled with NAD(P)H-producing enzymes, it is possible to measure a wide variety of cellular metabolites. Highly sensitive, accurate, and quantitative assays for creatine/creatine phosphate, pyruvate, succinate, and lactate have been described for a variety of cells. These tests, based on the measurement of metabolites following the cell lysis at certain points in time, produce values of average metabolite concentration in the population of cells used (for review, see Ref. 1). With such an approach it is not always possible to monitor fast changes in intracellular concentration, or to monitor the diversity of reaction of single cells on the used treatment. To better understand the connections between different metabolic pathways within the cell, in vivo tests are more advantageous.
5.1 In Vivo Imaging of the Intracellular ATP Dynamic
5.1.1 Bioluminescence in vivo ATP imaging
The first publications describing the monitoring of ATP in vivo in live cells appeared in the early 1990s. It was shown that luciferin and luciferase were not toxic to live cells, and changes in bioluminescence levels correlated well with the intracellular concentration of ATP. However, it was noted that the reaction kinetics of the luciferases present inside the cells differed from those observed for enzyme activity in aqueous solutions. Due to the high pyrophosphate and protein concentrations in the cytoplasm, the bioluminescence signal from the live cell injected with firefly luciferase was greatly depressed and showed remarkably slow decay when compared with in vitro conditions. This 'glowing' type of signal allowed continuous measurement of cytoplasmic ATP in single myocytes by monitoring bioluminescence following the microinjection of the cell with firefly luciferase. When cardiomyocytes metabolically poisoned with cyanide entered rigor, cytoplasmic ATP decreased from 1-2 mM to 20 Î¼M within 30-40 s. After removal of the cyanide, the bioluminescence signal recovered over a period of 2-3 min to a level that was approximately 70% of the original level present in healthy cells, thus indicating that ATP was restored.