Intracellular molecular imaging using multiphoton-excited microscopy

Doctoral thesis English OPEN
Crisostomo, AG

Detection of the levels of serotonin (5-HT) present in biofluids may be used for\ud detection of neurological diseases such as Alzheimer's 1 . This project seeks to\ud develop technologies using multiphoton excitation and resultant luminescence\ud of serotonin and related pharmacological drugs (e.g. propranolol) to measure\ud concentrations of serotonin in viable cells and biofluids. Experiments for\ud monitoring real-time serotonin and propranolol (a non-selective beta-blocker)\ud uptake and release have been performed using two-photon (2-PE) 630 nm\ud excitation with ultraviolet fluorescence (340 nm) emission. Excited state\ud lifetimes have been measured using the time-correlated single-photon counting\ud technique adapted for multiphoton microscopy. The solution lifetimes\ud determined were different from those in vivo. The lifetime of serotonin reduced\ud from 3.8 ns in solution to 1.9 ns in cellular systems while propranolol varies\ud between 5 to 10 ns in mammalian cells to 9.8 ns in solution 2 . This technique\ud has shown the potential for evaluation of serotonin and propranolol distribution\ud with high spatial resolution within individual cells as well as to determine its\ud intracellular concentration. Moreover, the use of 630 nm excitation provides a\ud good ratio between signal intensity and background interference (e.g. autofluorescence\ud of other cellular components as flavins, tryptophan). Furthermore\ud in comparison with UV excitation, 2-PE at 630 nm significantly reduces the\ud potential phototoxic effects of exposure to the laser beam.\ud The characteristic hyperluminescence from 5-hydroxyindoles following\ud multiphoton photochemistry was investigated to determine its usefulness in\ud intracellular induction of oxidative stress 3 ' 4 . Solutions of tryptophan and\ud mercaptopyridine-N-oxide (MPNO) were irradiated using 750 nm and\ud observations made on the resultant emission at 500 nm. This emission was\ud much less intense in solutions containing only either MPNO or tryptophan. It is\ud suggested that MPNO generates hydroxyl radicals by 2-photon activation at 750\ud nm. This was confirmed by the scavenging effects of ethanol as well as kinetic\ud analysis of the results.
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