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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!)