Intracellular molecular imaging using multiphoton-excited microscopy

Detection of the levels of serotonin (5-HT) present in biofluids may be used fordetection of neurological diseases such as Alzheimer's 1 . This project seeks todevelop technologies using multiphoton excitation and resultant luminescenceof serotonin and related pharmacological drugs (e.g. propranolol) to measureconcentrations of serotonin in viable cells and biofluids. Experiments formonitoring real-time serotonin and propranolol (a non-selective beta-blocker)uptake and release have been performed using two-photon (2-PE) 630 nmexcitation with ultraviolet fluorescence (340 nm) emission. Excited statelifetimes have been measured using the time-correlated single-photon countingtechnique adapted for multiphoton microscopy. The solution lifetimesdetermined were different from those in vivo. The lifetime of serotonin reducedfrom 3.8 ns in solution to 1.9 ns in cellular systems while propranolol variesbetween 5 to 10 ns in mammalian cells to 9.8 ns in solution 2 . This techniquehas shown the potential for evaluation of serotonin and propranolol distributionwith high spatial resolution within individual cells as well as to determine itsintracellular concentration. Moreover, the use of 630 nm excitation provides agood ratio between signal intensity and background interference (e.g. autofluorescenceof other cellular components as flavins, tryptophan). Furthermorein comparison with UV excitation, 2-PE at 630 nm significantly reduces thepotential phototoxic effects of exposure to the laser beam.The characteristic hyperluminescence from 5-hydroxyindoles followingmultiphoton photochemistry was investigated to determine its usefulness inintracellular induction of oxidative stress 3 ' 4 . Solutions of tryptophan andmercaptopyridine-N-oxide (MPNO) were irradiated using 750 nm andobservations made on the resultant emission at 500 nm. This emission wasmuch less intense in solutions containing only either MPNO or tryptophan. It issuggested that MPNO generates hydroxyl radicals by 2-photon activation at 750nm. This was confirmed by the scavenging effects of ethanol as well as kineticanalysis of the results