Intracellular molecular imaging using multiphoton-excited microscopy

Crisostomo, AG

Abstract

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

Citation

Crisostomo, A. Intracellular molecular imaging using multiphoton-excited microscopy. (Thesis). Salford : University of Salford

• Thesis Type: Thesis
• Deposit Date: Oct 3, 2012
• Award Date: Jan 1, 2009