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Hi Menelaos,

We tried luminescence imaging of cell monolayers a few times a couple of 
years ago.  I moved around between various microscopes to try to get it 
to work.  I remember starting on the DelltaVision but that was not the 
system that finally worked for us - I wish for the life of me I could 
remember why!  We got it to work vaguely using another old Olympus IX71 
with an EMCCD camera on it - the trick was indeed (as others have said) 
to use a low magnification lens with as high an NA as possible.  I think 
the 4x was actually the best on that system.  On most microscopes the 
issue was the internal LEDs that seem to be hidden inside the microscope 
stand and so you can't do anything about them.  In the end, much to our 
surprise, we had the best luck on our Olympus VivaView system, with an 
empty filter cube position (that was essential - we saw nothing through 
the DAPI, CFP or GFP cubes), the 20x objective (which is the only option 
in there), and the Orca R2 camera (which, BTW, is not intensified). The 
researcher was using Nanoluc and I think he only had to use exposures of 
around 30 secs to see a signal.  The nice thing about the VivaView is 
that it is entirely enclosed in the little incubator so there was no 
problem regarding stray light.

Sadly though, the researcher never got enough data to publish on our 
system because of an entirely different limitation - the Nanoluc 
substrate is very unstable under culture conditions (half life ~ 0.5 h) 
so he would have needed to apply fresh substrate before every 
acquisition, which would have been a bit of a pain since he was imaging 
for several days at a time.  He said they got it to work elsewhere with 
a microfluidic chamber, but that chamber wasn't compatible with the 
VivaView system.

So - don't give up yet!  But it certainly is a challenge.
Best,
Alison



On 12/15/2014 1:37 PM, Sam Lord wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> On Mon, 15 Dec 2014 11:31:00 +0000, Guy Cox <[log in to unmask]>
> wrote:
>> Finally, people have been doing luciferin - luciferase imaging successfully long
> before EMCCD and SCMOS and the latest high-brightness probes.  So it can't be
> that hard.
>
> People have been imaging luciferin, but it is known to be very difficult to image on
> the single-cell level. From the papers I've read for single-cell luminescence
> imaging, they used intensified CCDs (cooled have very low background) in
> digital/photon-counting mode and integrate for tens of minutes.
>
> I tried luciferin imaging once for single cells. I gave up before I got it working.
>
> It's worth thinking about the rates. Fluorescence imaging involves approximately
> the same number of probes, but the excitation/fluorescence cycling rate is going to
> be WAY higher than the chemical turnover. And each turnover gives at most one
> photon. It's not surprising that luminescence signal is orders of magnitude dimmer
> than fluorescence.
>
> The real benefit to luminescence imaging is zero background. But to take
> advantage of that, you need to block absolutely all room light (and LED lights on
> the scope and other electronic equipment). Also, you need a way to integrate for
> minutes without filling up your entire CCD well depth with dark counts.
>
> Don't feel ashamed if you're having trouble. :)
>
> (That said, the suggestion from Guy Cox and others to confirm that light is able to
> reach the camera and that there is not a problem with the hardware is an excellent
> point!)

-- 
Alison J. North, Ph.D.,
Senior Director of the Bio-Imaging Resource Center and
Research Associate Professor,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab    ++ 212 327 7486
Fax:        ++ 212 327 7489