On your widefield microscope with camera, did you try binning of the pixels to image your luciferase?
For BRET imaging tests using luciferase, where very high resolution was not important, we used a CoolSnapHQ binning 4 (then giving a 26 um pixel size) on the side port with 60 to 120 sec exposure time to see where was the signal. Best resolution have been done with a Cascade 512B camera (EMCCD backilluminated) with 30 sec exposure and binning 1. See the Julie Perroy article:
.
Biophys J. 2008 February 1; 94(3): 1001–1009.
Subcellular Imaging of Dynamic Protein Interactions by Bioluminescence Resonance Energy Transfer
Vincent Coulon,*† Martin Audet,‡ Vincent Homburger,* Joël Bockaert,* Laurent Fagni,* Michel Bouvier,‡ and Julie Perroy*
Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
C.P. 6128, succursale Centre-ville
Montréal QC H3C 3J7 Canada
tél. (514) 343-6111 poste 5148
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[log in to unmask]] De la part de Robert J. Palmer Jr.
Envoyé : 3 avril 2009 09:59
À : [log in to unmask]
Objet : Re: Luciferase (Gaussia) activity detection
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
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396
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