Re: Highly diffusible dye for filling neurons in vivo

Posted by Martin Wessendorf-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Highly-diffusible-dye-for-filling-neurons-in-vivo-tp7586162p7586166.html

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The one time that I tried hydroxystilbamidine with 2-photon, it photobleached really rapidly.  But then I didn't know what I was doing and may have been hitting it too hard. 

The tissue was  fixed with formaldehyde (rather than live) and mounted in DPX, 

Good luck!

Martin Wessendorf

On 12/21/2016 12:46 PM, Iain Johnson wrote:

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Lucifer Yellow has been used with  excitation at 850 nm (https://www.ncbi.nlm.nih.gov/pubmed/?term=14534260).  I think the excitation cross-section is not great, but it obviously works to a usable extent.

Another possibility (untested AFAIK) is hydroxystilbamine (aka FluoroGold).  The structure of the dye suggests that it should have a good two-photon cross section, probably best about 750 nm.

Iain

On Wed, Dec 21, 2016 at 12:50 AM, Peter Rupprecht <[hidden email]> wrote:

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Dear dye experts,

I'm currently patching very small neurons (soma 5-10 um in diameter, dendrites much smaller) in living tissue. In addition to electrophysiological recordings, I'd like to get the morphology without fixing the tissue, so I add a dye (until recently mostly Alexa 594) to the pipette solution.

However, since the neuronal processes are very small, it is very often only the soma and not the small dendrites that get filled by the dye.

Switching from Alexa 594 to Alexa 488 improved things a bit (probably since 488 is smaller? https://en.wikipedia.org/wiki/Alexa_Fluor). But maybe somebody on the list has an idea of an even smaller, highly diffusible, non-toxic dye that I could use for this purpose. Plus, it should of course work with regular 2P excitation (800-950 nm).

Any ideas?

Best,

Peter