Indoleamines: Certain
neurons, pinealocytes, mast cells and enterochromaffin cells, for example,
often contain high concentrations of indoleamines such as serotonin.
Using 3 photon excitation, indoleamine autofluorescence at 350 nm can
be visualized. (See sections VII, VIII,
and IX). The image at left shows the serotonin
containing granules found in the pineal gland (green pseudocolor).
This image was obtained using 720 nm excitation and the emission filters
shown on the previous page. Red pseudocolor
is probably NADH autofluorescence (see below). The specimen consists
of a fresh mouse pineal gland taken during the day; in the dark serotonin
is converted to melatonin which is equally fluorescent.
NAD(P)H
and Flavins: Intracellular
autofluorescence is often dominated by the reduced pyridine nucleotides
(NAD(P)H) and the oxidized flavins (FMN, FAD), both of which are
potentially useful as cellular metabolic indicators. Mitochondrial
NADH autofluorescence can be directly used as an indicator of cellular
respiration (Piston et al.,1995). Since only the reduced form has
an appreciable fluorescence yield, hypoxia, which causes an increase
in the NADH/NAD+ ratio, can be detected as an increase in mitochondrial
autofluorescence. The figure at right is
an autofluorescence image of HeLa cells (700 nm exc; average power
was 10 mW at sample). The spectrum (at right)
was acquired by parking the excitation beam on a cell and collecting
the emission through a fiber optic coupled, LN 2 cooled CCD spectrograph.
The spectrum of NADH is shown for comparison. The blue shift of
the autofluorescence peak is an indication of enzyme-bound NADH
within the cell.
D.W. Piston, B.R. Masters and W.W. Webb. Journal of Microscopy 178:20-27,1995.
