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-tp2560403p2563138.html

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

 

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From: Confocal Microscopy List [mailto:[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

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Erasmus Medical Center
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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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