Re: Highly diffusible dye for filling neurons in vivo

Posted by Peter Rupprecht-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Highly-diffusible-dye-for-filling-neurons-in-vivo-tp7586162p7586172.html

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Thanks for all those helpful comments!

I started with Joseph's suggestion (Sulforhodamine), since it was available in the lab. The filling of small dendrites worked better in my hands than with the Alexa dyes I had used before, with the brightness being very high, also with excitation at >900 nm. No effect on spiking activity, resting membrane potential or active components observed. The only downside: Before filling the cell, I'm using the dye for shadowpatching (cf. https://www.ncbi.nlm.nih.gov/pubmed/18157136). The Sulforhodamine dye which is blown into the tissue is not harmful, but it sometimes strongely labels blood vessels. Here is an example of how this looks like in a zebrafish brain: http://imgur.com/gallery/Dbrj5 The data are still useful for me, and maybe I will simply stick to Sulforhodamine, since it works ...

In the next year, I will possibly try out some other dyes (if time allows), starting with Lucifer Yellow or one of the Atto dyes.

@Michael: If I understand it correctly, the method using Acid Blue outside of the cell would not work in my case, since I'm some hundred um deep in the tissue, and I cannot transmit the light from one side of the brain to the other side - there would be too much scattering, outweighing any absorption processes.

@Christian: Maybe I missunderstand your question. I've been patching those cells for ca. 6 months, with no prior electrophysiologist's experience, if this is your question. If you are rather asking about the how long I hold the cells: I typically hold them for 20-40 min, but I do not see any strong improvements of the dye penetration when I wait longer than 10 min.

And thanks for pointing out the chemical properties that affect diffusion - I will keep this in mind.

@Glen: I agree on the phototoxicity issue for lower wavelengths. However, most dyes with the maximum 2P cross section around 850 (like Sulforhodamine) are still bright enough when excited with 920 nm (which I prefer also because I share a laser with a collegue who is using 920-930 nm all the time). This seems to be true also for Lucifer yellow, according to the paper mentioned by Franko.

Glen MacDonald <[hidden email]> schrieb am 15:53 Donnerstag, 22.Dezember 2016:

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The relatively short excitation wavelength of Lucifer Yellow gives some phototoxicity.  Longer wavelength labels, such as Alexa 594,  allowed much longer survival without degraded recordings in our patch clamp experiments.  You might try dyes affixed to short dextrans, which transport fairly well. 

Glen MacDonald
Digital Microscopy Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

> On Dec 22, 2016, at 2:20 AM, Christian Wilms <[hidden email]> wrote:
>
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> How long do you have the cells patched for?
> 
> I doubt that molecular weight is the limiting factor. Charge and the resulting tendency to interact with other cellular elements will also be part of the problem. For labelling cerebellar granule cells (and visualizing their axons, parallel fibers) I used Atto 594 and Atto 488 quite successfully. Try not using hydrazide versions of the dyes, but rather carboxylic acids, as these interact less with proteins, speeding up diffusion.
> 
> Lucifer yellow: small and old-school. It is fairly dim, so requires high concentrations. Which is why people use the lithium salt, as it is more soluble than the potassium salt. Lithium interacts with calcium signalling (IMPase), so depending on what you are looking at, it might not be the best idea.
> 
> Just my thoughts and I am sure others will have additions.
> 
> Cheers, Christian
> 
> 
> From: Peter Rupprecht [mailto:[hidden email]]
> Sent: 21 December 2016 08:51
> Subject: Highly diffusible dye for filling neurons in vivo
> 
> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images onhttp://www.imgur.com and include the link in your posting. *****
> 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