Re: A1

Posted by Patrick Van Oostveldt on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FW-A1-tp591961p591965.html

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear,

We were using the CLEM set-up developed by Eric Manders, the inventor  
of CLEM, and were able to visualize GFP transformed cells for up to 48  
hours. We could see confocal mitosis and all the nice biology of GFP  
labelled cells.
 From time to time cells escaped our field of view and we had to  
adjust the focal plane so within 48 hours there is gap, due to some  
human shortcomings.

As compared to conventional confocal it really reduce bleaching and  
phototoxicity. More over Eric is a very nice person to collaborate with.

Bye

Patrick Van Oostveldt


Quoting Michael Weber <[hidden email]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi David,
>
> the system measures the amount of fluorescence signal per pixel and
> controls the AOTF for this pixel based on the measured value. So if
> there is no light coming to the detector, laser will be restricted from
> illuminating the sample. Or, if the detector gets close to saturation,
> laser will be restricted as well. This should reduce photobleaching and
> phototoxicity also in out-of-focus areas. That's at least how I
> understand this.
>
> "The CLEM control unit is an optional add-on system for the C1. The  
> CLEM unit calculates the integrated detected signal and exposure  
> time for each pixel in real time, performs AOM high speed shutter  
> control and high speed operation processing based on the amount of  
> acquired fluorescence signal, PMT gain and then outputs the  
> resulting fluorescence signal to the C1 controller’s line grabber  
> electronics circuit."
>
> source: http://www.nikoninstruments.eu/news.php?n=550
>
> I expect the quantification (measuring intensities) to be difficult
> afterwards, since one needs to apply a correction. However, it's one of
> the main things that makes the Nikon system interesting.
>
> Michael
>
>
> David Knecht wrote:
>> Search the CONFOCAL archive at  
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>> 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
>> 91 N. Eagleville Rd.
>> University of Connecticut
>> Storrs, CT 06269
>> 860-486-2200
>> 860-486-4331 (fax)

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