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I've obtained samples of some Renilla luciferase substrates from 
NanoLight for the purpose of visualizing bioluminescence in living 
cells. These substrates are formulated for various emission peaks, one 
being V-CTZ which has a peak around 520 nm, which nicely fits in the 
FITC filter set.

I tried transfecting some HEK293T cells with Rluc8 (highly active 
version of the luciferase), then loading them up with the substrate and 
imaging them live on our DeltaVision microscope. We have an Olympus IX70 
base, CoolSnapHQ CCD camera, and I used a 60X Olympus 1.42NA oil 
objective. My strategy was to set up a long exposure (2 to 10 sec) with 
no excitation light (Ex set to BLOCK), collecting emitted light in the 
FITC channel. I started imaging pretty much immediately after I put the 
substrate on the cells. I know that the luciferase signal peaks in the 
first minute and then drops off, but should still be detectable for 
about an hour after that.

I was unable to get any signal whatsoever. The exposure was coming up 
completely negative, I could not even see any autofluorescence, just CCD 
noise at around 100 (of 4095) units, which is the standard background 
noise. I had co-transfected GFP in my cells, and parallel (very short - 
0.05 sec) exposures with 100% excitation light showed very strong GFP 
signal in almost every cell, so I know that the camera was collecting 
light in the FITC channel and that the cells were transfected.

I also tried two other substrates from NanoLight, Prolume Purple and 
Prolume Purple II, which emit close to the DAPI range, and again had 
absolutely no signal. I know for certain that my Rluc8 works in these 
particular cells as it produces very high luminescence using Promega's 
kit, though that assay is designed for lysed cells and uses a plate 
reader, and is not suitable for imaging living cells.

The luciferase substrate was loaded at the upper end of NanoLight's 
recommended concentration for live cells (20 uM). I can try to increase 
the substrate concentration somewhat, though the stock solution (about 
12.5 mM) is dissolved in 10% ethanol and there will be a point beyond 
which I would be adding enough ethanol to the culture medium to affect 
the luciferase reaction. I also don't really expect that I will suddenly 
go from ZERO signal to something substantial/publishable just by 
increasing the concentration.

My conclusion is that the luminescence is far too dim for my camera and 
it seems that I will never be able to see it. Is there anything else I 
can do to increase my ability to see this signal in living cells on my 
microscope? NanoLight suggested that I open the aperture on the 
microscope to maximum, but I don't know how to do that or even if that 
is possible on my setup. I know that there are bioluminescence 
microscopes like the Olympus LV-200, but there does not seem to be 
anything like that on campus. There are plenty of other epi scopes, 
however, and I can look around to see whether there are more sensitive 
CCDs I could take advantage of, if that is the main limiting factor.


-- 
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
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Phone: 802-656-1161