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Hi,

I am in the market for an EMCCD for widefield fluorescence 
time-lapse imaging of genetically-labeled cultured cells. I 
am acquiring images frequently (every 10 minutes) for 
several days. The cells seem to be very sensitive to 
phototoxicity and photobleaching (GFP is the fluorophore), 
so I am hoping that replacing my Roper CoolSnapHQ CCD with 
an EMCCD will allow me to use much shorter exposure times.

I realize that this is a confocal list and my application is 
widefield but since EMCCDs are also used with spinning disk 
confocal, I thought I'd ask. Apologies if this question has 
been hashed out before... I searched the archives without 
finding definitive recommendations. (Maybe that's asking for 
too much!) Any help on the following would be much appreciated:

1) What EMCCDs have people found to be the most *sensitive* 
(nice picture in low-light) and the most *reliable* (driver 
doesn't crash, etc.)? I'd especially be interested in any 
experiences or opinions with Roper/Photometrics's CascadeII 
or QuantEM and the Andor iXon cameras.

2) How have people been testing cameras? I've set up demos 
of several cameras so I'd like to do some standard tests. 
I've seen some references on the web for calculating dark 
noise, read noise, and gain, such as:
http://www.qsimaging.com/ccd_noise_measure.html
http://www.mirametrics.com/tech_note_ccdgain.htm
http://www.photomet.com/library/library_encyclopedia/library_enc_gain.php
but I'd love to hear what other tests people think are 
relevant for these cameras. Obviously, I'll try imaging my 
samples (using fiber-coupled LED as a constant illumination 
source)... what else should I do? Fluorescent beads instead 
of my cells perhaps?

3) For coupling the camera to my Olympus IX71, is there 
something like the Zeiss Optovar (which, as I understand it, 
has multiple selectable magnifications of the intermediate 
lens) made by a third party? This would be helpful for 
changing between achieving the full NA of the objective and 
getting less resolution but a bigger field of view for 
different experiments.

Thanks for your help. Best,

- Neville

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Neville Sanjana
Dept. of Brain and Cognitive Science
Massachusetts Institute of Technology