Re: Use of a fluorescent organelle in determining a PSF

Posted by Fred Mast on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Coverslip-tp592448p592455.html

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Thank-you for all those who replied...

Yeast don't have a paracrystalline matrix core like other organisms,  
however, it is packed with protein so it would have a higher  
refractive index than it's surroundings. That being said, would it be  
any different than using beads, which have a refractive index closer  
to that of glass? Glen your point about homogenous distribution is a  
good one. While I don't believe it to be an issue here (as the  
peroxisome matrix of yeast is a homogenous compartment), I'm not  
really sure how I would go about evaluating this...

Thanks again for the input,

Fred

On 22-Feb-08, at 5:03 PM, Michael Schell wrote:

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> Since some parts of the peroxisomal lumen are comprised of a  
> "crystalline matrix" of protein, would this affect the refractive  
> index and thus create spherical aberration in the PSF?
>
> Michael J. Schell, Ph.D., CIV, USUHS
> Assist. Professor
> Dept. of Pharmacology
> Uniformed Services University
> 4301 Jones Bridge Rd.
> Bethesda, MD  20814-3220
> tel:  [phone]
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>>>> Glen MacDonald <[hidden email]> 02/22/08 5:47 PM >>>
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> Hi Fred,
> Do you know for certain that the chosen peroxisome has a homogenous
> distribution of fluoropore?  Looking at isolated peroxisomes might
> give a hint and provide a nice control as to whether the distortion
> of the PSF in comparison to plain beads is due to the optical
> properties of the sample. And, as you estimate, presenting a more
> realistic PSF.   I'm assuming you are looking at fixed yeasties, or
> John's concern regarding motion would be very valid.
>
> Regards,
> glen
> Glen MacDonald
> Core for Communication Research
> Virginia Merrill Bloedel Hearing Research Center
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> University of Washington
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>
> On Feb 22, 2008, at 1:54 PM, Fred Mast wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> 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]
>

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]