Native indoleamine
(a tryptophan based amine such as serotonin and melatonin) 
fluorescence
absorbs at deep UV energies (<300nm).
However with a three photon process it can be conveniently excited
with an infra-red (~700nm), femtosecond laser source (see right diagram
and 3PE cross-section). Using red light
to excite these high energy transitions eliminates the need for deep
UV optics and their associated aberrations and low transmissivities.
We
first demonstrated three photon excitation (3PE) imaging of native monoamine
fluorescence in
serotonin-loaded RBL-2H3 cells (Maiti et al., 1997), a mucosal mast cell
line. The figures below show 3PE 320- 380nm fluorescence images
of a) unloaded and b) loaded RBL-2H3 cells. In general unloaded cells exhibit
a modest and uniform fluorescence excluded from the nuclei and slightly
enhanced in the golgi regions. Loaded cells, those incubated in serotonin-enriched
media, accumulate serotonin in secretion competent granules by means
of
5-HT transporters in their plasma and granule membranes. By comparing
measured fluorescence intensities to those of known concentration standards,
the
granules were found to contain ~50mM serotonin. c) A single and unusual
(<1%) unloaded cell shows granular fluorescence concentrations similar
to those in the loaded cells.
S. Maiti,
J.B. Shear, R.M. Williams, W.R. Zipfel and W.W. We bb. Science,
275:530, 1997.
