Re: Quantifying fluorescence help

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quantifying-fluorescence-help-tp593618p593627.html

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How good an idea of photo-bleaching would such a bead give you?  I know it will be different from whatever dye is being used in your tissue, but can you still use it somehow to gauge photo-bleaching rates?

Thanks,

Craig

On Fri, Jul 18, 2008 at 8:03 AM, Rietdorf, Jens <[hidden email]> wrote:

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Dear Maria,

(no commercial interest) Invitrogen sells a product called InSpeck, ie
beads with a calibrated intensity of labelling. They come in different
intensities, like 100%, 50% 25% usf. If you include those in all your
preparations you can normalise your signal to the beads, which may be
sufficient to have a rough idea of the relative brightness of your
labelling.

Cheers, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Maria Mazzillo
Sent: Freitag, 18. Juli 2008 15:26
To: [hidden email]
Subject: Quantifying fluorescence help

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I am a PhD student at Auburn University just getting started into my
thesis work.  I am looking for a reliable method for quantifying
fluorescence.

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in hosts (usually cnidarians).  I am working with
cultures or isolates of only the dinoflagellates for my confocal work.
I have an antibody that was created against the surface secretions of
mucilage (secreted as part of a daily cycle by the alga) from one strain
of zooxanthellae.
I am attempting to use this antibody to label various strains to
identify differences in mucilage between them.  Thus far, I have seen
that the strain the antibody was created against labels around the cell
fairly brightly.  Most samples either show this or a complete lack of
labeling.  However, a few samples show a faint fluorescence lifted off
the cell surface.  I would like to be able to quantify this fluorescence
in comparison to either the control strain that the antibody was created
against or against a known fluorescence.

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo