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In the microarray field there were stories a couple of years ago of
ozone killing the fluorophores (Cy5, a not-quite-identical twin of
AF647) in dry air. Apparently typical lab ozone levels is high enough to
be a CyDye-killer.
George
p.s. Protasenko et al 2005 -
http://chemeducator.org/sbibs/s0010004/spapers/1040269mk.htm - obtained
nice TIRF results with a 1.25 NA oil immersion lens under your dry
conditions:
"The purpose of this paper is to describe the assembly, from commercial
off-the-shelf components, of a low-cost (~$3300) optical microscope
capable of single-molecule detection."
On 6/10/2013 11:50 AM, Jill Herschleb wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hello,
>
> I am using single alexa fluor 647 molecules to tag biomolecules of interest. I
> don't have a ton of experience in this field, and was wondering if someone would
> be able to point me in the right direction.
>
> Issue: Imaging the single alexa 647 molecules in a dry environment
> (immobilized on a cover glass) is easy, and the fluors are easy to detect and
> enumerate. However, using the same immobilized A647 probes on the same
> surface, when I add an wet mounting media (I've tried Invitrogen Slowfade -
> glycerol, sodium phosphate buffer with or without b-ME), I can still see the fluor
> molecules, but they are much dimmer than when they are dry.
>
> Has anyone experienced this before? The wet mounting media does not seem to
> affect the brightness of other fluors (Cy3, Alexa594). So far I'm having the most
> trouble with Alexa 647.
>
> Thank you for your advice! Sincerely, Jill
>
>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
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