Highly diffusible dye for filling neurons in vivo

[Peter Rupprecht-2]

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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

[mmodel]

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Hi Peter,

You can obtain cell morphology by adding the dye Acid Blue 9 to the medium at ~7 mg/ml. The sample is placed in a shallow chamber. The dye stays outside the cells and does no harm to them. Then you collect a transmission image at 630 nm, where the dye absorbance is the strongest. Where the cells are thicker the absorbing layer is thinner, and the image brightness quantitatively reflects cell thickness. If you want we can talk more about it, and I can send you some papers.

Mike Model

---

From: Confocal Microscopy List <[hidden email]> on behalf of Peter Rupprecht <[hidden email]>
Sent: Wednesday, December 21, 2016 3:50 AM
To: [hidden email]
Subject: Highly diffusible dye for filling neurons in vivo

 

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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

[Franco Del Principe]

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Hi again

As I supposed my post got rejected.

So you better google the citation and download the PDF yourself.

Cheers
----------------------------------------------------------------

Hi Peter

As an old electrophysiologist of course Lucifer Yellow comes to mind.

Since I don't remember the 2P excitation wavelength I quote this paper
that has also other dyes listed:

"Measurement of two-photon excitation cross sections of
molecular fluorophores with data from 690 to 1050 nm"
C. Xu and W. W. Webb Vol. 13, No. 3/March 1996/J. Opt. Soc. Am. B 481

I have attached a PDF but I'm not sure it will be transmitted by the server.

Best regards

Franco

On 21-Dec-16 09:50, Peter Rupprecht wrote:

> ***** To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
> http://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

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   /______/ /__/  /______/             Dr. Franco Del Principe
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[Iain Johnson]

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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

[Martin Wessendorf-2]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Iain et al.--

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

[Christian Wilms]

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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 on http://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

[Glen MacDonald-2]

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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:
>
> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
> 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

[Peter Rupprecht-2]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

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:
>
> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
> 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