Confocal Microscopy List

Re: microbead calibration to set psf?

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/microbead-calibration-to-set-psf-tp2560403p2565677.html

Dave,
 
       The problem is that a bottle lasts a VERY long time!  My 60nm beads certainly came from Bangs - my 100nm ones were given to me by Carol Cogswell many years ago.  I've not tried anything in the longer wavelength range since I've not had any reason to.  I think Bangs have been taken over so their range may have been reduced since I last dealt with them.  190nm is certainly a bit large for a 1.4NA lens.
 
                                                            Guy
 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
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     http://www.guycox.net

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht
Sent: Wednesday, 1 April 2009 12:31 AM
To: [hidden email]
Subject: Re: microbead calibration to set psf?

Bangs doesn't seem to make alot of colors or sizes of PSF sized beads.  They list 60nm and then the next smallest is 190nm.  Is that too large for PSF applications?  Have you used the Suncoast Yellow, which is the only thing in a yellow/red window they have?   Dave

On Mar 31, 2009, at 10:11 AM, Guy Cox wrote:

Europium chelates are phosphorescent rather than luminescent - that is to say, they have a very long - millisecond - fluorescence lifetime.  That should still work in widefield but would be hopeless for confocal.  Pretty much unfadeable, which is a plus.
 
Tetraspeck bead are for calibrating colocalization rather than PSF - having multiple fluorochromes on one bead must reduce the intensity at any one wavelength.  As I said before, I've used bead from Bangs Labs and I've had good images from beads down to 60nm.  (Though at that scale it requires care and patience).
 
                                                                       Guy
 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
     http://www.guycox.net

 

From: Confocal Microscopy List [[hidden email]] On Behalf Of David Knecht
Sent: Tuesday, 31 March 2009 11:57 PM
To: [hidden email]
Subject: Re: microbead calibration to set psf?

I am looking for some very bright 100nm beads.  I tried to look for the beads you mention and it looks like Duke Scientific no longer exists and is now part of hte Thermo conglomerate.  The closest thing I found to what you describe is the Europium chelate 100nm beads.  Is that what you were referrring to?  THey provide very little data on fluorescence properties and they are unusual in apparently being broad excitation spectrum but there is no emission spectrum in their literature I can find.  Since they fluoresce maximally at 613nm it would seem they would be a poor fit for most FITC, TRITC, CY3 or CY5 type filter sets.  Can you clarify your experience with them?  Dave

On Mar 31, 2009, at 3:36 AM, Mariette P.C. Kemner - van de Corput wrote:

I have good experience with fluorescent beads from Duke scientific. The
100nm beads are very bright and are perfect for determining the PSF. I use
the red, green and blue 100nm beads to determine the PSF in three channels
and to deconvolved my 3 colour FISH images.
I have trouble finding the 100nm and 200nm TetraSpeck beads on my slide
beacuse they are rather weak in intensity. So that's why I switched to the
single coloured 100nm beads from DS. The 500nm TetraSpeck beads are very
good and I use them to determine the chromatic shift in 4 channels. Once
made, bead slides can be used for a long period of time.

Mariette

____________________________________________

Dr. M.P.C. Kemner-van de Corput,
____________________________________________

MGC - Dept. of Cell Biology & Genetics
Erasmus Medical Center
Dr. Molewaterplein 50, 3015 GE Rotterdam
POB 2040, 3000 CA Rotterdam, The Netherlands

Office: H-Ee751; tel: +31 10 704.3949
Lab: H-Ee710; tel lab: +31 10 704.3315
tel secr: +31 10 704.3169
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http://www2.eur.nl/fgg/ch1/cellbiology/
http://www.thesis.kemner.biz/
____________________________________________

Op Di, 31 maart, 2009 1:59 am, schreef Matiar Jafari:
hey again,

I'm trying to find out how and what the best way to setup my own psf
is in AutoQuant.  I know i need microbeads and im wondering what brand
and what product i need and then how i need to shoot it and load it.
Also what diameter does it need to be.  A cat# would be great too also
i don't know if it matters on what im shooting but just to put it out
there im shooting gfp slices.  Sorry if this is vague. Any help would
be greatly appreciated.  Thanks in advance

Thank You
--
Matiar Jafari


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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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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