How to preserve endogenous TdTomato signal after whole mouse brain tissue clearing?

[Gu, Tingting]

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Dear all,

We tried Ethanol/ECi-based tissue clearing on the whole mouse brain with endogenous TdTomato fluorescent signal. After the clearing, no TdTomato-specific signal left, but high autofluorescent background in RFP range. ECi cleared brain has some yellow color but it is clear.

Do anyone have experience of using ECi-based tissue clearing method to clear mouse brain with endogenous TdTomato signals? Or any recommendations on which clearing methods can preserve endogenous TdTomato signals. In addition, how to reduce autofluorescent background?

Any suggestions would be greatly appreciated.
Best,
Tingting

[Douglas Richardson]

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Hello Tingting,

Most often it is the dehydration step that causes the loss of fluorescence.
FPs require water molecules to maintain their confirmation, once these are
removed, the chromophore falls apart and you lose fluorescence. Methanol is
the worst for this, the emission will be lost almost immediately.
Ethanol is a little better and THF will give you a day or two of weak
fluorescence. The final clearing solution (DBE, ECi, etc.) also plays a
role, but the dehydration step does the most damage.

The most recent solvent-based clearing protocols have found some tricks to
better protect the FPs and preserve fluorescence for weeks or months. These
include:

1) dehydration with 1-propanol or tert-butanol (or a mixture of both)
2) Making sure all solutions have a basic pH (~9.0)
3) Keeping the sample at 4C during clearing
4) Supplementing the solutions with Quadrol or Polyethylene Glycol

For more info see:
•uDISCO –Pan et al., Nature Methods, 2016
•a-uDISCO – Liet al., Frontiers in Neuroanatomy, 2018
•PEGASOS– Cell Research, 2018

-Doug


On Mon, May 24, 2021 at 2:22 AM Gu, Tingting <[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.
> *****
>
> Dear all,
>
> We tried Ethanol/ECi-based tissue clearing on the whole mouse brain with
> endogenous TdTomato fluorescent signal. After the clearing, no
> TdTomato-specific signal left, but high autofluorescent background in RFP
> range. ECi cleared brain has some yellow color but it is clear.
>
> Do anyone have experience of using ECi-based tissue clearing method to
> clear mouse brain with endogenous TdTomato signals? Or any recommendations
> on which clearing methods can preserve endogenous TdTomato signals. In
> addition, how to reduce autofluorescent background?
>
> Any suggestions would be greatly appreciated.
> Best,
> Tingting
>

[Sverre grødem-3]

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Hi Tingting,

We've had brain samples where tdTomato is expressed cleared using CLARITY at the Wyss center in geneva, the signal is very bright after clearing.

https://imgur.com/a/eNbqVEz
[https://i.imgur.com/ewweZex.png?fb]<https://imgur.com/a/eNbqVEz>
TdTomato CLARITY cleared mouse brain<https://imgur.com/a/eNbqVEz>
Imgur: The magic of the Internet
imgur.com


Best,

Sverre Grødem, University of Oslo
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Douglas Richardson <[hidden email]>
Sent: Monday, May 24, 2021 3:23 PM
To: [hidden email] <[hidden email]>
Subject: Re: How to preserve endogenous TdTomato signal after whole mouse brain tissue clearing?

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hello Tingting,

Most often it is the dehydration step that causes the loss of fluorescence.
FPs require water molecules to maintain their confirmation, once these are
removed, the chromophore falls apart and you lose fluorescence. Methanol is
the worst for this, the emission will be lost almost immediately.
Ethanol is a little better and THF will give you a day or two of weak
fluorescence. The final clearing solution (DBE, ECi, etc.) also plays a
role, but the dehydration step does the most damage.

The most recent solvent-based clearing protocols have found some tricks to
better protect the FPs and preserve fluorescence for weeks or months. These
include:

1) dehydration with 1-propanol or tert-butanol (or a mixture of both)
2) Making sure all solutions have a basic pH (~9.0)
3) Keeping the sample at 4C during clearing
4) Supplementing the solutions with Quadrol or Polyethylene Glycol

For more info see:
•uDISCO –Pan et al., Nature Methods, 2016
•a-uDISCO – Liet al., Frontiers in Neuroanatomy, 2018
•PEGASOS– Cell Research, 2018

-Doug


On Mon, May 24, 2021 at 2:22 AM Gu, Tingting <[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.
> *****
>
> Dear all,
>
> We tried Ethanol/ECi-based tissue clearing on the whole mouse brain with
> endogenous TdTomato fluorescent signal. After the clearing, no
> TdTomato-specific signal left, but high autofluorescent background in RFP
> range. ECi cleared brain has some yellow color but it is clear.
>
> Do anyone have experience of using ECi-based tissue clearing method to
> clear mouse brain with endogenous TdTomato signals? Or any recommendations
> on which clearing methods can preserve endogenous TdTomato signals. In
> addition, how to reduce autofluorescent background?
>
> Any suggestions would be greatly appreciated.
> Best,
> Tingting
>

[Gu, Tingting]

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*****

Sverre,

Thank you for letting me know your experience. The brain image is stunning!

Tingting Gu Ph.D.
Associate Director of Advance Light Microscopy
Samuel Robert Noble Microscopy Laboratory
University of Oklahoma

730 Can Vleet Oval, Room 9

Norman, OK 73019

E-mail: [hidden email]<mailto:[hidden email]>

Phone: [phone]

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Sverre grødem <[hidden email]>
Sent: Tuesday, May 25, 2021 8:42 AM
To: [hidden email] <[hidden email]>
Subject: Re: How to preserve endogenous TdTomato signal after whole mouse brain tissue clearing?

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

Hi Tingting,

We've had brain samples where tdTomato is expressed cleared using CLARITY at the Wyss center in geneva, the signal is very bright after clearing.

https://imgur.com/a/eNbqVEz
[https://i.imgur.com/ewweZex.png?fb]<https://imgur.com/a/eNbqVEz>
TdTomato CLARITY cleared mouse brain<https://imgur.com/a/eNbqVEz>
Imgur: The magic of the Internet
imgur.com


Best,

Sverre Grødem, University of Oslo
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Douglas Richardson <[hidden email]>
Sent: Monday, May 24, 2021 3:23 PM
To: [hidden email] <[hidden email]>
Subject: Re: How to preserve endogenous TdTomato signal after whole mouse brain tissue clearing?

*****
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.
*****

Hello Tingting,

Most often it is the dehydration step that causes the loss of fluorescence.
FPs require water molecules to maintain their confirmation, once these are
removed, the chromophore falls apart and you lose fluorescence. Methanol is
the worst for this, the emission will be lost almost immediately.
Ethanol is a little better and THF will give you a day or two of weak
fluorescence. The final clearing solution (DBE, ECi, etc.) also plays a
role, but the dehydration step does the most damage.

The most recent solvent-based clearing protocols have found some tricks to
better protect the FPs and preserve fluorescence for weeks or months. These
include:

1) dehydration with 1-propanol or tert-butanol (or a mixture of both)
2) Making sure all solutions have a basic pH (~9.0)
3) Keeping the sample at 4C during clearing
4) Supplementing the solutions with Quadrol or Polyethylene Glycol

For more info see:
•uDISCO –Pan et al., Nature Methods, 2016
•a-uDISCO – Liet al., Frontiers in Neuroanatomy, 2018
•PEGASOS– Cell Research, 2018

-Doug


On Mon, May 24, 2021 at 2:22 AM Gu, Tingting <[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.
> *****
>
> Dear all,
>
> We tried Ethanol/ECi-based tissue clearing on the whole mouse brain with
> endogenous TdTomato fluorescent signal. After the clearing, no
> TdTomato-specific signal left, but high autofluorescent background in RFP
> range. ECi cleared brain has some yellow color but it is clear.
>
> Do anyone have experience of using ECi-based tissue clearing method to
> clear mouse brain with endogenous TdTomato signals? Or any recommendations
> on which clearing methods can preserve endogenous TdTomato signals. In
> addition, how to reduce autofluorescent background?
>
> Any suggestions would be greatly appreciated.
> Best,
> Tingting
>

[Kurt Weiss]

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Hi Tingting,

I second the advice from Doug and Sverre and I would also note that it is important to have a strong promoter with high expression if you want to preserve endogenous expression for clearing. Some protein loss is inevitable, so if you're starting with low levels and have high autofluorescence (which is common), things will be even harder. Most publications I've seen with good retention seem to use Thy-1 or a viral promoter.

We have recently published some guidance for navigating these common difficulties with clearing and imaging experiments in a Nature Protocols tutorial style review: https://rdcu.be/ck4g1.

Since there are so many individual factors that go into any such experiment, we aim to give general advice and warnings, similar to the recent Jonkman Confocal tutorial. Among other topics, we include notes on autofluorescence and why the yellowish hue in your samples is evidence of protein retention (a good thing!) that shouldn't interfere with imaging. Hopefully you and others will find it helpful. Good luck!

Regards,
Kurt

________________
Kurt Weiss, Ph.D.
Assistant Scientist
Morgridge Institute for Research
Medical Engineering | Huisken Group
330 N Orchard St.
Madison, WI 53715