Re: Luciferase (Gaussia) activity detection

Posted by Keith Morris on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FW-Cleaning-lens-tp2567899p2583166.html

Hi Monique,

Yes we tried binning to the max 8, Gain to maximum, and exposure to
maximum. Sadly the Hammamatsu Orca 100 [C4742-95]* is limited to a 10
seconds maximum exposure [and we really needed that 120 seconds]. All we
got was camera noise [once we fully light proofed the microscope stage].

From measurements using some sort of luminosity scanner the user did
confirm there was weak light coming from the luciferase, but obviously
nowhere near enough for our Hammatsu Orca 100 camera to image anything. I
see frequent reference to the CoolSnapHQ & CoolSnap2 HQ being used for
imaging Luciferase luminescence, and all saying about 120 seconds exposure
[many modern Hammatsu and Photometrics interline CCD cameras offer similar
long exposure times these days, but our Orca 100 is four+ years old and
wasn't designed for very low light levels anyway]. Plus our inverted
microscope hasn't got the best objectives for fluorescence either [Phase
contrast, long working distance ones mostly], which no doubt put the tin
hat on it, even though we moved as much as possible out of the light path.

Thanks for the advice though.

You can see our setup at
http://www.well.ox.ac.uk/cytogenetics/nikon05.shtml

Keith

* Actually we haven't got an Orca ER - that was at the last place I worked
at, the ER is more sensitive than the 100.
-----------------------------------------------------------------

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

Tel:  +44   ( 0 ) 1865  287568
Email:   [hidden email]
HomePage:  http://www.well.ox.ac.uk/cytogenetics

> 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. [phone] poste 5148
> -----Message d'origine-----
> De : Confocal Microscopy List [mailto:[hidden email]] De
> la part de Robert J. Palmer Jr.
> Envoyé : 3 avril 2009 09:59
> À : [hidden email]
> 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)
>>>
>>>  [hidden email]
>
>
> --
> 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 [phone]
>