Judy Trogadis asks:
> Has anyone tried collecting light emission from luciferin using a
> confocal microscope?
 
A gentle reminder: the basic principle of a confocal microscope
is to reject out-of-focus light using a pinhole in a conjugate
image plane. This of course requires that one is only illuminating
a point at a time for either reflection or fluorescence. (Okay,
Nipkow disk machines illuminate many points at once, but in any
case the illumination is not wide-field.) Therefore, it is impossible
to obtain a confocal image of glowing luciferin, since it is not
fluorescent but luminescent.
 
A slow-scan cooled CCD with long integration times (as Scott Fraser
suggests) or even better an intensified camera such as an ICCD or
ISIT, is probably the best choice for such low light levels.
Alternately, if you are looking at fixed specimens, there are quite
good antibodies available for luciferase; you could then go confocal
by using a fluorescent secondary antibody. Luciferase is nice because
you can do a quick assay of enzyme levels using a luminometer, but
for single-cell visualization, immunofluorescence is probably the
best. (Viz., Holt et al. (1990), Neuron 4:203-214.) If you really
want to get z-sectioning using luminescence, you could consider
taking a z-series with a low-light camera, then deconvolving in
software.
 
--Chi-Bin Chien
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