You can do this with a true intensified
photon-counting camera, e.g., those from
Hamamatsu. Can't locate on their web site the
model we used about 15 yrs ago, but it was based
on a multi-channel plate that provided about 10E6
amplification. We mounted it on a side port on
our confocal and put the scope in a light box.
The thing was sensitive enough to detect
bacterial luciferase in single cells. As noted,
lots of aluminum foil is recommend. I assume you
are NOT trying to do three-D work, but I've
always been intrigued by that possibility.
At 9:39 AM -0400 4/3/09, Keith J Morris 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]
--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
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