Mouse brain imaging w/ lightsheet

[Nadia Halidi]

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Dear Lightsheet people,

Someone brought me mouse brain samples cleared in cubic and wants to image
the entire brain. They have scanned the same brain with CT first then went
ahead and cleared it with cubic. They want to image the entire Brain
without out any loss to compare CT vs lightsheet data. They are desperate
to image.

I have a Z1 lightsheet, I would usually need to "hang" the samples. Their
condition is that they want to image the entire brain. The sample is too
wobbly. I have run out of ideas and about to refer them somewhere where
they place the sample down and rely on gravity, no hanging (like LaVision).
But before I do, I thought to ask here if any of you have any suggestions,
or perhaps have already succeeded. Thank you.

Best regards,
Nadia Halidi

[Nikos Ekizoglou - Planelight]

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Dear Nadia,

Our QLS-scope can measure samples from < 1mm to 250mm in XYZ. We just
put the sample in matching refractive index glass tube and that's all.
No hanging, no gluing, no cutting and you can return the sample to your
colleague after measurement. You are welcomed to our demo lab to Madrid
(Spain) to measure your sample, free of charge. If you are interested,
please send me a personal email.

Best regards

--
*Nikos Ekizoglou*

*Plane Light S.L.*
Edificio CTM, O-309
28053, Madrid
Tel. [phone]
/www.planelight.net/

El 21/01/2020 a las 8:33, Nadia Halidi escribió:

> *****
> 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 Lightsheet people,
>
> Someone brought me mouse brain samples cleared in cubic and wants to image
> the entire brain. They have scanned the same brain with CT first then went
> ahead and cleared it with cubic. They want to image the entire Brain
> without out any loss to compare CT vs lightsheet data. They are desperate
> to image.
>
> I have a Z1 lightsheet, I would usually need to "hang" the samples. Their
> condition is that they want to image the entire brain. The sample is too
> wobbly. I have run out of ideas and about to refer them somewhere where
> they place the sample down and rely on gravity, no hanging (like LaVision).
> But before I do, I thought to ask here if any of you have any suggestions,
> or perhaps have already succeeded. Thank you.
>
> Best regards,
> Nadia Halidi

--
*Nikos Ekizoglou*

*Plane Light S.L.*
Edificio CTM, O-309
28053, Madrid
Tel. [phone]
/www.planelight.net
/

[Gary Laevsky]

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*****

We’ve been making cuvettes out of PDMS and PMMA, depending on the reagent. Put the sample in the cuvette and fill with matching reagent.

Then we hang in our Bruker MuVi. Working well.



Best,

Gary

> On Jan 21, 2020, at 3:26 AM, Nikos Ekizoglou - PlaneLight <[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 Nadia,
>
> Our QLS-scope can measure samples from < 1mm to 250mm in XYZ. We just put the sample in matching refractive index glass tube and that's all. No hanging, no gluing, no cutting and you can return the sample to your colleague after measurement. You are welcomed to our demo lab to Madrid (Spain) to measure your sample, free of charge. If you are interested, please send me a personal email.
>
> Best regards
>
> --
> *Nikos Ekizoglou*
>
> *Plane Light S.L.*
> Edificio CTM, O-309
> 28053, Madrid
> Tel. [phone]
> /www.planelight.net/
>
>> El 21/01/2020 a las 8:33, Nadia Halidi escribió:
>> *****
>> 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 Lightsheet people,
>>
>> Someone brought me mouse brain samples cleared in cubic and wants to image
>> the entire brain. They have scanned the same brain with CT first then went
>> ahead and cleared it with cubic. They want to image the entire Brain
>> without out any loss to compare CT vs lightsheet data. They are desperate
>> to image.
>>
>> I have a Z1 lightsheet, I would usually need to "hang" the samples. Their
>> condition is that they want to image the entire brain. The sample is too
>> wobbly. I have run out of ideas and about to refer them somewhere where
>> they place the sample down and rely on gravity, no hanging (like LaVision).
>> But before I do, I thought to ask here if any of you have any suggestions,
>> or perhaps have already succeeded. Thank you.
>>
>> Best regards,
>> Nadia Halidi
> --
> *Nikos Ekizoglou*
>
> *Plane Light S.L.*
> Edificio CTM, O-309
> 28053, Madrid
> Tel. [phone]
> /www.planelight.net
> /

[Sylvie Le Guyader]

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*****

Hi Nadia

I have seen before in some demo somewhere a Z1 with a little hanging device with a platform where the sample could rest with gravity. You could design one and make it with a 3D printer.
Otherwise the MSquared system we have relies on gravity. You can contact them to ask where there is some in the US.

Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
LCI microscopy blog

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Nadia Halidi
Sent: Tuesday, 21 January, 2020 08:34
To: [hidden email]
Subject: Mouse brain imaging w/ lightsheet

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Dear Lightsheet people,

Someone brought me mouse brain samples cleared in cubic and wants to image the entire brain. They have scanned the same brain with CT first then went ahead and cleared it with cubic. They want to image the entire Brain without out any loss to compare CT vs lightsheet data. They are desperate to image.

I have a Z1 lightsheet, I would usually need to "hang" the samples. Their condition is that they want to image the entire brain. The sample is too wobbly. I have run out of ideas and about to refer them somewhere where they place the sample down and rely on gravity, no hanging (like LaVision).
But before I do, I thought to ask here if any of you have any suggestions, or perhaps have already succeeded. Thank you.

Best regards,
Nadia Halidi


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[James Muller]

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The best solution I have found so far is to glue the sample to a magnet. This will not allow for imaging perfectly 100% of the sample, the glue has a different refractive index than the clearing solution. If you really need to image 100% of the sample you first need to clear the sample in an agarose block (I know this works for iDisco, not sure if this works in cubic...) than glue this block to the magnet. This paper describes a few different methods that have been tested for cleared samples on the Z1 ( https://www.frontiersin.org/articles/10.3389/fnana.2019.00026/full).

The next method will require a 3D printed solution we have 3D printed a similar solution to what is used by the LaVision system in which you have a thumb screw on the bottom of the sample holder and a pin on the opposite site and you screw down the thumb screw to tighten the sample. However, I must admit the early version of our prototype cause sample distortion. You can email me and I can share the CAD files for 3D printing.

The last method involves the use of a cuvette and this solution is dependent on the working distance of your objective. Basically, you mount the sample in a cuvette than you clear some wedges of agarose and use these to "secure" your sample in the cuvette. You need todo some research and find cuvettes that have a matched RI to the immersion medium to avoid distortions due to changes in RI.

I should note this is one of the most challenging problems in light sheet microscopy right now.

I hope this was helpful and let me know if I can help in anyway.

[Nadia Halidi]

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Thank you all for your replies. Looks like 3D printing rules! I will go
with that, they need 100% of the sample, I'm not sure how the wobbly brain
will be screwed down, but perhaps if we just slightly hold it in place that
would suffice. I will let people know here how it goes. Thank you all again!

James muller I will email you for the CAP file. Much appreciated!

Best regards,
Nadia

On Tue, Jan 21, 2020 at 11:57 PM James Muller <[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.
> *****
>
> The best solution I have found so far is to glue the sample to a magnet.
> This will not allow for imaging perfectly 100% of the sample, the glue has
> a different refractive index than the clearing solution. If you really need
> to image 100% of the sample you first need to clear the sample in an
> agarose block (I know this works for iDisco, not sure if this works in
> cubic...) than glue this block to the magnet. This paper describes a few
> different methods that have been tested for cleared samples on the Z1 (
> https://www.frontiersin.org/articles/10.3389/fnana.2019.00026/full).
>
> The next method will require a 3D printed solution we have 3D printed a
> similar solution to what is used by the LaVision system in which you have a
> thumb screw on the bottom of the sample holder and a pin on the opposite
> site and you screw down the thumb screw to tighten the sample. However, I
> must admit the early version of our prototype cause sample distortion. You
> can email me and I can share the CAD files for 3D printing.
>
> The last method involves the use of a cuvette and this solution is
> dependent on the working distance of your objective. Basically, you mount
> the sample in a cuvette than you clear some wedges of agarose and use these
> to "secure" your sample in the cuvette. You need todo some research and
> find cuvettes that have a matched RI to the immersion medium to avoid
> distortions due to changes in RI.
>
> I should note this is one of the most challenging problems in light sheet
> microscopy right now.
>
> I hope this was helpful and let me know if I can help in anyway.
>

[daj1u06]

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On LAVision Ultramicroscope with clearing into DBE, we use screw caps eppendorf lids and fill them with clear silicone aquarium sealant and gently tac the cleared brain onto the surface of that, and then grip the cap in a standard LAvision frame (without insert), which holds it well for imaging. Brain easily peels away after imaging, remount and rescan from other side if you want to get 100% and then merge the data in another software (we've not done that step but there must be something out there that can do it). If 3D printing, chemical compatibility may be an issue for some organic solvents - nylon or polypropylene would work but can be difficult to print with (at least on our ultimaker system), I don't know about PLA's compatibility with DBE as I haven't been able to find any data and I have not tested it out personally.

Regards,

Dave Johnston,
Biomedical Imaging Unit, Southampton

[Nadia Halidi]

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*****

Hi Gary,

Thanks for your reply. I can make a PDMS cuvette. Did you use cubic in the
PDMS is it safe and how did it work with the refractive indices? Did you
use a dipping objective or air? And wouldn't the wobbly brain move around
when you would turn to image from different angles? I would love to learn
about this more? Thanks again.

Best regards,
Nadia

On Tue, Jan 21, 2020 at 11:38 AM Gary Laevsky <[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.
> *****
>
> We’ve been making cuvettes out of PDMS and PMMA, depending on the reagent.
> Put the sample in the cuvette and fill with matching reagent.
>
> Then we hang in our Bruker MuVi. Working well.
>
>
>
> Best,
>
> Gary
>
>
>
> > On Jan 21, 2020, at 3:26 AM, Nikos Ekizoglou - PlaneLight <
> [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 Nadia,
> >
> > Our QLS-scope can measure samples from < 1mm to 250mm in XYZ. We just
> put the sample in matching refractive index glass tube and that's all. No
> hanging, no gluing, no cutting and you can return the sample to your
> colleague after measurement. You are welcomed to our demo lab to Madrid
> (Spain) to measure your sample, free of charge. If you are interested,
> please send me a personal email.
> >
> > Best regards
> >
> > --
> > *Nikos Ekizoglou*
> >
> > *Plane Light S.L.*
> > Edificio CTM, O-309
> > 28053, Madrid
> > Tel. [phone]
> > /www.planelight.net/
> >
> >> El 21/01/2020 a las 8:33, Nadia Halidi escribió:
> >> *****
> >> 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 Lightsheet people,
> >>
> >> Someone brought me mouse brain samples cleared in cubic and wants to
> image
> >> the entire brain. They have scanned the same brain with CT first then
> went
> >> ahead and cleared it with cubic. They want to image the entire Brain
> >> without out any loss to compare CT vs lightsheet data. They are
> desperate
> >> to image.
> >>
> >> I have a Z1 lightsheet, I would usually need to "hang" the samples.
> Their
> >> condition is that they want to image the entire brain. The sample is too
> >> wobbly. I have run out of ideas and about to refer them somewhere where
> >> they place the sample down and rely on gravity, no hanging (like
> LaVision).
> >> But before I do, I thought to ask here if any of you have any
> suggestions,
> >> or perhaps have already succeeded. Thank you.
> >>
> >> Best regards,
> >> Nadia Halidi
> > --
> > *Nikos Ekizoglou*
> >
> > *Plane Light S.L.*
> > Edificio CTM, O-309
> > 28053, Madrid
> > Tel. [phone]
> > /www.planelight.net
> > /
>