No it probably isn't possible to use a confocal for luciferase. Just tried
it with our standard wide field inverted microscope, all lights off, lots
of tin foil, Orca ER peltier cooled camera, maximum gain and not a hint of
anything other than background noise. Our Orca ER has a 10 second max
exposure time, and the literature talks of 120 seconds plus exposure times
[and the max camera gain didn't help us]. We concluded we would need a
£25k EMCCD camera or better to use our microscope with our samples, so our
user is simply switching to an immunofluorescence method instead and
giving Luciferase a miss [just not worth spending the time developing the
method without the right microscope kit].

A point scanning confocal will probably be less capable than our Orca ER
camera, and certainly no better. You loose too much light in the optics,
as there's so little to begin with, and so you need a very expensive
low-noise sensitive camera. Normally you use one of those large
light-tight cabinet type microscope imagers, bit like a plate scanner.

Olympus do make the 'boxed' LV200 microscope for this type of work:

http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Bioluminescence_Imaging_System_LV200_Applications.htm

and they talk of 15 minute exposure integration times for image capture.
If you have an expensive cooled B&W fluorescence microscope camera that
integrate for many minutes it might be worth a trying that though -
provided you can get the microscope stage into 'absolute darkness' [you
even have to get down to covering LEDs with insulating tape].

Our user was using a luceriferase kit that reduced light levels a tad to
hopefully keep it going brighter for far longer than the few seconds
Luciferase normally lasts.

Regards

Keith

-----------------------------------------------------------------

Dr Keith J Morris
Molecular Cytogenetics and Microscopy Core
The Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford
OX3 7BN
United Kingdom


> Hi to all,
>
> Is it possible to detect luciferase activity  on cell with a standard
> confocal micriscope (let's say LSM 510 and LSM 710 equipped with  regular
> and spectral detector)?
>
>  Emission of coelenterazine is at 470 nm.
>
> Thanks for you help!
>
> Louis
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [log in to unmask]