Confocal Microscopy List

Re: A1

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FW-A1-tp591961p591967.html

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Somehow I think we've had this discussion about CLEM before - maybe about a year ago? Not sure of the date but I do recall Jim Pawley was involved, and Erik Manders was dragged back into the list to explain it all. this should all be in the archives. I've not used the device in anger but I have seen it demonstrated and it does work. As it was explained to me the various parameters can be controlled (eg at what point you 'give up' and don't collect any more from a region which is giving no useful signal) and yes, of course you can turn it off and you must do so for quantitation.

The collection of light both in and closely around the pinhole at the same time was, I thought, patented by Optiscan. What you can do with it must depend on your sample but at the least it must help in finding thin layers.

Guy

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Tuesday, 19 February 2008 10:25 AM
To: [hidden email]
Subject: Re: A1

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David Knecht wrote

Ø ………Typically, you don't want the laser intensity to vary. …………

My reply is more of a question than a statement, can someone who has used the CLEM device please confirm.

Presumably if you have CLEM fitted to your confocal you can choose to turn it off for quantitative measurements of fluorescence. Turning it on only when required for long time-lapse experiments where reducing bleaching is paramount, and where you are interested primarily in morphology rather than pixel to pixel intensity. Can someone please confirm it is implemented this way on the A1?

Also there has not been in depth discussion of the “VAAS” detector. The conventional confocal detector collects a confocal image after the pinhole, but the light rejected by the pinhole is also collected by a detector. This signal can then be deconvolved with the confocal signal to produce an optical section with more signal compared to conventional confocal.

My question: Is this then equivalent to a deconvolved widefield image? Or is the confocal image used in some way to determine what the resulting deconvolved image should look like?

Cheers

Steve

Stephen H. Cody
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Ludwig Institute for Cancer Research
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht
Sent: Tuesday, 19 February 2008 2:51 AM
To: [hidden email]
Subject: Re: A1

The ability of using this system along with Controlled Light Exposure
Microscopy (CLEM) makes this system for long time live cell imaging and
confocal analysis. CLEM automatically monitors and varying the laser
illumination during time-lapse studies to reduce the risk of laser induced
bleaching, biochemical inconsistency and cell death.

Can you clarify what this means? I don't see how you can monitor the imaging and vary the laser in a way that can avoid cell toxicity. Typically, you don't want the laser intensity to vary. Do you have any quantitative data on cellular toxicity during imaging in comparison to a spinning disk system?

Dr. David Knecht

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

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University of Connecticut

Storrs, CT 06269

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