We've also tried to see luminescence with an really good non-EM
non-intensified camera (Orca AG) on a widefield 'scope and saw
nothing, so I agree that a confocal would be much worse.  An
intensified camera worked though.

-Esteban


On Fri, Apr 3, 2009 at 8:39 AM, Keith J Morris <[log in to unmask]> wrote:
> 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]
>



-- 
G. Esteban Fernandez, Ph.D.
Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO  65211

http://www.biotech.missouri.edu/mcc/

573-882-4895
573-884-9676 fax