Re: Use of a fluorescent organelle in determining a PSF

Posted by mmodel on
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Image-Adaptive Deconvolution for Three-Dimensional Deep Biological Imaging by Monvel et al

http://www.biophysj.org/cgi/content/abstract/85/6/3991

 

They used naturally occuring fluorescent spots to find real PSF

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From: Confocal Microscopy List on behalf of Fred Mast
Sent: Fri 2/22/2008 4:54 PM
To: [hidden email]
Subject: Use of a fluorescent organelle in determining a PSF

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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]
[hidden email]