Re: Use of a fluorescent organelle in determining a PSF

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-tp592448p592460.html

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This sounds like a great idea to me, Fred, but how do you account for  
the movement of the peroxisome and the eventual photobleaching of  
some of the fluorescent labels during the PSF acquisition?

John Oreopoulos

On 22-Feb-08, at 4:54 PM, Fred Mast wrote:

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>
> Hello all,
>
> I would like some input into an idea I have for obtaining a PSF. I  
> study peroxisomes using yeast as a model system for understanding  
> how they are created. In yeast, peroxisomes have a well  
> characterized morphology, being spherical organelles with a  
> diameter of 100 to 200nm. Other than this size variability I think  
> they are excellent candidates for obtaining a PSF as they can be  
> easily, fluorescently labelled (by targeting fluorescent protein  
> chimeras to their matrix), are similar in size to what is typically  
> used to obtain PSF's, and are "embedded" in the sample. I do a lot  
> of live cell imaging, using a LSM510 Meta and am always looking for  
> ways to improve my system. As a result most of my images are fairly  
> noisy and I rely on deconvolution to remove the noise, and improve  
> contrast and resolution. My initial attempts at using peroxisomes  
> for this purpose have provided me with a PSF that is slightly  
> different from what I obtain with fluorescent beads (the peroxisome  
> derived PSF is less symmetrical) and provides, in my estimation, a  
> more realistic result. Your thoughts and concerns on this idea  
> would be most welcome.
>
> Fred
>
> Fred D. Mast
> Department of Cell Biology
> Medical Sciences Building Room 5-14
> University of Alberta
> Edmonton, Alberta, T6G 2H7
> Canada
>
> Tel: [phone]
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