Re: spinning disk confocal Vs deltavision?. .

[Sam's Mail]

Carl, Zhan and Listmembers,

You are correct that in theory and practice widefield imaging will always be somewhat more sensitive then SD approaches, directly due to the pinholes in play. However, our recent experiences with the newest CSU-X paired with a quality EMCCD, such as an Evolve, will yield a highly sensitive system that most certainly does not struggle with dim samples. I am certainly a big fan of widefield imaging, but if you are interested in the image "quality" available with either decon or a spinning disk, then the phototoxicity issues become non-trivial again. To gain the quality of a SD image, one must acquire multiple z-planes for deconvolution, adding significantly more harmful light into your sample than a single z-plane from a spinning disk approach.

For your 12-hour imaging question, we routinely do multiple fluors (1-5) every few minutes on SD systems without significant phototoxicity issues.  We can and do image more quickly of course, but often times when you are imaging for 12 hours, the biological kinetics of interest do not necessitate running super fast.  This pause between images also allows us to visit multiple stage positions during one 12-hour time-lapse session, enabling multiple experimental controls and test samples to be imaged in parallel. That very same pause can be of biological importance in terms of phototoxicity recovery as well.  Of course, this is all possible on a widefield system, but the decon requirements during image acquistion, not just post processing, are not to be overlooked in terms of the cost benefit analysis.

Importantly, as Carl astutely suggested, if you can execute an experiment successfully with meaningful data extracted, then starting in widefield is a reasonable approach. If you are having challenges extracting "meaningful" data, then spinning disk is certainly one approach that is highly amenable to time-lapse tissue-culture imaging. 3D decon is a real solid choice as well, but has some trade-offs as I've discussed in certain scenarios.

--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office [phone]
Cell [phone]
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carl Boswell
Sent: Tuesday, February 23, 2010 11:25 AM
To: [hidden email]
Subject: Re: spinning disk confocal Vs deltavision?. .

Two points to make.

First, while my only experience with spinning disks have been multiple demos
while looking for high speed imaging, the sensitivity issue seemed
significant.  The SD systems we had to look at struggled mightily when the
samples were dim, while the DV could extract meaningful data from the same
thing.  Second, the pixel size of EMCCD's are going to effect resolution
compared with the CoolSnap HQ (1024x1024).

The photobleaching/toxicity should be similar, so the bonus I've seen from
SD is the speed.  In our hands, the limiting factor for imaging speed with
the DV was the exposure time, again talking about dim samples.  Shutter time
was a trivial issue.

From my view, your choices are resolution and sensitivity vs. speed.

Good luck,
c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "zhan cheng" <[hidden email]>
To: <[hidden email]>
Sent: Monday, February 22, 2010 6:50 PM
Subject: spinning disk confocal Vs deltavision?


Hello everyone,
   Our facility is planning to buy a live cell imaging setup, spinning disk
confocal and deltavision seem both good. We are concering about imaging
quality, imaging speed, phototoxin, and photo bleaching for long term
imaging.
Could you share your experience? some time lapse our imaging maybe last over
12 hours, so  sometimes the data is huge. I know the  deltavision system's
imaging quality is good after deconvolution, but I worry about the 3D
deconvolution's speed, especially for huge data.
    Thanks.
    zhan cheng



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