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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
>[log in to unmask]
>Phone: 802-656-1161

Dear Menelaos,

The signal will inevitably be very weak. By magnifying the image, you 
spread a fixed number of photons over more pixels and the signal 
level drops with (mag) squared. Try lower magnification: bigger 
pixels, referred to the specimen, will contain more light emitting 
molecules. Of course, lower mag usually means lower NA, as 
signal/pixel goes with (NA) squared so you need a balance Remember, 
you are actually sampling a volume at each pixel and, as long as your 
specimen has some depth, lower NA will provide more depth of field 
and extends the volume sampled by the pixel on the camera in the z 
direction.

You probably also need a different camera, one with lower read noise 
(like a good EM-CCD), higher raw QE (Back-illuminated), and cooled to 
a low enough temp to allow even longer exposures (minutes).  If this 
is impossible, try binning the pixels in your present camera. Not as 
good but cheaper! Just to see how bad the problem is.

Finally, make a major effort to reduce stray light in the room: No 
operating display screens during image collection, back velvet cap on 
the far side of the specimen from the objective etc.  You may even 
have to put black tape over all the "holes" in your microscope 
between the objective and the camera (to block light from the 
inevitable LED indicator lights). With no exciting light, (turned 
off, not just blocked with a slider) you can  probably do without the 
emission filter because the luciferase reaction should be the only 
light source left.

Good luck
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