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Hi Menelaos,

The first thing I would do is remove the filter cube.  While FITC will line
up with the maximum of your luciferase, you are still losing a lot of light
at the edges as the emission of luciferase is fairly broad relative to most
FITC filters.

Assuming your system is completely light tight (as in, _absolutely_ no
light is getting in, if not, make it so) you can also try integrating
longer.  I had one of the older TEC cooled CoolSnaps, and they have very
low noise even at long integration times.

Mike

On Sun, Dec 14, 2014 at 1:06 PM, Menelaos Symeonides <[log in to unmask]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>