Re: Quantifying fluorescence help

Posted by Jurriaan Zwier on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quantifying-fluorescence-help-tp593618p593623.html

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Hi Maria,

Quite hard to really quantify your confocal images. We've tried and
devoloped a method using ca 100 nm thin fluorescent layers and used them
for calibrating the total intensities of a z-stack of confocal images. Our
efforts were just published here:
'Quantitative image correction and calibration for confocal fluorescence
microscopy using thin reference layers and SIPchart-based calibration
procedures', J.M. Zwier, L. Oomen, L.Brocks, K.Jalink, G.J. Brakenhoff,
Journal of microscopy 231 (2008) p59-69

kind regards,
Jurriaan

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
>