tiny chambers for Slidescanner / how to keep sections moist during scan

[Tobias Rasse-2]

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Dear All,

a potential user would be interested to

a)       image fresh cyrosections at our slidescanner (GFP,RFP)

b)      then perform a lacz staining

c)       then image the same sections again.

In the current pilot (at a standard wide field microscope) sections are
covered by a cover slip only after b). They are kept moist during a)
manually.

Images from a) and c) shall be registered. Thus, putting a coverslip before
a) and removing it for b) seems no option.

Do you have any advice on how to keep sections moist (but not spilling
everything with water during robot movements)?

e.g. small chambers glued on after recovering the sections from the
cryostat?

(we have a Märzhäuser SlideExpress 2 – Nikon) system.

Thanks a lot & Kind regards

Tobias

[Craig Brideau]

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I had to do extended CARS microscopy on fresh brain sections with no
coverslip, and came up with a couple solutions that may be useful for your
scenario: If the tissue is wandering off, you can use 'crazy glue' to hold
it down if a net doesn't do the job. If you are lucky charged slides may do
the trick as well, no glue required. For hydration, I 3D printed a well
that you place the entire slide in. It has sloped sides that can contain
water/PBS/CSF, and the slope will not obstruct the lens from accessing the
edges of the sample during XY panning of the stage. Finally, you may notice
the water 'slosh' at the end of each movement; this can cause blurring or
XY shift of your image. My method for dealing with this was to simply wait
a fixed amount of time after every stage move before imaging. Hopefully you
slide scanner allows some sort of settling time to be entered for each tile.

Craig

On Tue, Aug 6, 2019 at 1:50 PM Tobias Rasse <
[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,
>
> a potential user would be interested to
>
> a)       image fresh cyrosections at our slidescanner (GFP,RFP)
>
> b)      then perform a lacz staining
>
> c)       then image the same sections again.
>
> In the current pilot (at a standard wide field microscope) sections are
> covered by a cover slip only after b). They are kept moist during a)
> manually.
>
> Images from a) and c) shall be registered. Thus, putting a coverslip before
> a) and removing it for b) seems no option.
>
> Do you have any advice on how to keep sections moist (but not spilling
> everything with water during robot movements)?
>
> e.g. small chambers glued on after recovering the sections from the
> cryostat?
>
> (we have a Märzhäuser SlideExpress 2 – Nikon) system.
>
> Thanks a lot & Kind regards
>
> Tobias
>

[Jeremy Adler-4]

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re

Finally, you may notice
the water 'slosh' at the end of each movement; this can cause blurring or
XY shift of your image.


you may be able to reduce the speed the stage moves at.

which will reduce the magnitude of the 'slosh'


Jeremy Adler

BioVis

Uppsala U


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Craig Brideau <[hidden email]>
Sent: 06 August 2019 22:41:13
To: [hidden email]
Subject: Re: tiny chambers for Slidescanner / how to keep sections moist during scan

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

I had to do extended CARS microscopy on fresh brain sections with no
coverslip, and came up with a couple solutions that may be useful for your
scenario: If the tissue is wandering off, you can use 'crazy glue' to hold
it down if a net doesn't do the job. If you are lucky charged slides may do
the trick as well, no glue required. For hydration, I 3D printed a well
that you place the entire slide in. It has sloped sides that can contain
water/PBS/CSF, and the slope will not obstruct the lens from accessing the
edges of the sample during XY panning of the stage. Finally, you may notice
the water 'slosh' at the end of each movement; this can cause blurring or
XY shift of your image. My method for dealing with this was to simply wait
a fixed amount of time after every stage move before imaging. Hopefully you
slide scanner allows some sort of settling time to be entered for each tile.

Craig

On Tue, Aug 6, 2019 at 1:50 PM Tobias Rasse <
[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,
>
> a potential user would be interested to
>
> a)       image fresh cyrosections at our slidescanner (GFP,RFP)
>
> b)      then perform a lacz staining
>
> c)       then image the same sections again.
>
> In the current pilot (at a standard wide field microscope) sections are
> covered by a cover slip only after b). They are kept moist during a)
> manually.
>
> Images from a) and c) shall be registered. Thus, putting a coverslip before
> a) and removing it for b) seems no option.
>
> Do you have any advice on how to keep sections moist (but not spilling
> everything with water during robot movements)?
>
> e.g. small chambers glued on after recovering the sections from the
> cryostat?
>
> (we have a Märzhäuser SlideExpress 2 – Nikon) system.
>
> Thanks a lot & Kind regards
>
> Tobias
>

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[Konstantín Levitskiy]

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*****

Does it mean that LacZ staining procedure turns off GFP/RFP fluorescence?

See for more
https://www.nature.com/articles/srep02937

Regards,
Konstantin

-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Tobias Rasse
Enviado el: martes, 06 de agosto de 2019 21:39
Para: [hidden email]
Asunto: tiny chambers for Slidescanner / how to keep sections moist during scan

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*****

Dear All,

a potential user would be interested to

a)       image fresh cyrosections at our slidescanner (GFP,RFP)

b)      then perform a lacz staining

c)       then image the same sections again.

In the current pilot (at a standard wide field microscope) sections are
covered by a cover slip only after b). They are kept moist during a)
manually.

Images from a) and c) shall be registered. Thus, putting a coverslip before
a) and removing it for b) seems no option.

Do you have any advice on how to keep sections moist (but not spilling
everything with water during robot movements)?

e.g. small chambers glued on after recovering the sections from the
cryostat?

(we have a Märzhäuser SlideExpress 2 – Nikon) system.

Thanks a lot & Kind regards

Tobias

[Sylvie Le Guyader]

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*****

Hi Tobias

We usually use a ring a silicon. This makes a rather high well around the sample so you can capture your a image without the water spilling. You can also use the well for the lacz stain.
Then you can add the mounting medium of your choice, cover with a coverslip and push on the coverslip to seal.

Good luck! :)

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7C,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tobias Rasse
Sent: 06 August 2019 21:39
To: [hidden email]
Subject: tiny chambers for Slidescanner / how to keep sections moist during scan

*****
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,

a potential user would be interested to

a)       image fresh cyrosections at our slidescanner (GFP,RFP)

b)      then perform a lacz staining

c)       then image the same sections again.

In the current pilot (at a standard wide field microscope) sections are covered by a cover slip only after b). They are kept moist during a) manually.

Images from a) and c) shall be registered. Thus, putting a coverslip before
a) and removing it for b) seems no option.

Do you have any advice on how to keep sections moist (but not spilling everything with water during robot movements)?

e.g. small chambers glued on after recovering the sections from the cryostat?

(we have a Märzhäuser SlideExpress 2 – Nikon) system.

Thanks a lot & Kind regards

Tobias


När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.


Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

[VERMEREN Matthieu]

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Dear all,

Sometimes, LacZ makes large dense deposit that may quench fluorescence.  It’s sample specific.   In my experience, GFP stays bright after LacZ.  It may be a good idea to use a GFP booster (Chromotek's nanobodies are pricey but works very well, no commercial interest) -just in case.  Of course, it depends what you are trying to image that is GFP+, with which level of resolution and whether it is lost in a sea of XGal or not...

As far as the Nature paper cited by Konstantin, it's true that LacZ can be imaged in the far red end of the spectrum.  I've had acceptable images using a Leica SP8, imaging GFP and LacZ in zebrafish skin.  However, good controls are necessary.

Sincerely,

Matthieu


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Konstantín Levitskiy
Sent: Wednesday, 07 August, 2019 10:01
To: [hidden email]
Subject: Re: tiny chambers for Slidescanner / how to keep sections moist during scan

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*****

Does it mean that LacZ staining procedure turns off GFP/RFP fluorescence?

See for more
https://www.nature.com/articles/srep02937

Regards,
Konstantin

-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Tobias Rasse Enviado el: martes, 06 de agosto de 2019 21:39
Para: [hidden email]
Asunto: tiny chambers for Slidescanner / how to keep sections moist during scan

*****
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,

a potential user would be interested to

a)       image fresh cyrosections at our slidescanner (GFP,RFP)

b)      then perform a lacz staining

c)       then image the same sections again.

In the current pilot (at a standard wide field microscope) sections are covered by a cover slip only after b). They are kept moist during a) manually.

Images from a) and c) shall be registered. Thus, putting a coverslip before
a) and removing it for b) seems no option.

Do you have any advice on how to keep sections moist (but not spilling everything with water during robot movements)?

e.g. small chambers glued on after recovering the sections from the cryostat?

(we have a Märzhäuser SlideExpress 2 – Nikon) system.

Thanks a lot & Kind regards

Tobias
The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.

[Deanna Wolfson]

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Hi,
Our group uses PDMS chambers fairly often. Typically we pour it into a petri dish to be the thickness we want (e.g. 100 um), and then use a scalpel to cut out chambers; one 10cm dish gives 20 or so chambers for us. This is a nice alternative to the silicon ring since we don't need to remove it before adding a coverslip (but we can if we want), and we can customize the size of the chamber to fit a sample if needed.
Regards,
Deanna

[Roy Edward-2]

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Dear Tobias
Following up on Sylvie’s suggestion..

I also use Silicone o-rings lightly smeared with silicone grease on the bottom edge to adhere to the well/slide. They’re excellent for creating small wells on slides - I use them with CyGEL.  I got mine from a little specialist business in the UK.

These o-rings are 8.4mm internal diameter and 1.2 or 1.5mm high. If you’d like some I’ll mail some to you. They can be re-used many times.
Kind regards,
Roy

Roy Edward
Biostatus Ltd.
www.Biostatus.com

> On 8 Aug 2019, at 06:00, CONFOCALMICROSCOPY automatic digest system <[hidden email]> wrote:
>
> 1. tiny chambers for Slidescanner / how to keep sections moist during scan

[Sylvie Le Guyader]

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To be clearer than I first was about or setup: we use silicon grease to make the well, not silicon as I wrote. Thus we do not need to remove the silicon grease before mounting for the final round of imaging, we instead leave the grease on and use it to seal the coverslip to the slide.

So in your case I would use a long coverslip, make an open silicon grease well, do the first round of imaging, stain again using the well walls to contain reagents, as the slide and press gently to seal.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7C,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

On 8 Aug 2019 08:20, Roy Edward <[hidden email]> wrote:
*****
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Dear Tobias
Following up on Sylvie’s suggestion..

I also use Silicone o-rings lightly smeared with silicone grease on the bottom edge to adhere to the well/slide. They’re excellent for creating small wells on slides - I use them with CyGEL.  I got mine from a little specialist business in the UK.

These o-rings are 8.4mm internal diameter and 1.2 or 1.5mm high. If you’d like some I’ll mail some to you. They can be re-used many times.
Kind regards,
Roy

Roy Edward
Biostatus Ltd.
www.Biostatus.com<http://www.Biostatus.com>

> On 8 Aug 2019, at 06:00, CONFOCALMICROSCOPY automatic digest system <[hidden email]> wrote:
>
> 1. tiny chambers for Slidescanner / how to keep sections moist during scan



När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.


Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

[Dan Focht]

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Tobias

I don't know if this would fit the protocol but if you can work with a 30mm coverslip, Bioptechs makes a device called an ICD (Interchangeable Coverslip Dish) there is a Heated Lid attachment for it that accommodates a micro-humidification system that provides an atmospherically sealed, humidity controlled microenvironment that will keep a specimen from drying out, provide high N.A. compatibility and you can remove and replace the coverslip.

Additional info available:
(Scroll down from each link for additional info.)

https://bioptechs.com/product/interchangeable-coverglass-dish/

https://bioptechs.com/product/micro-gas-humidifier/

https://bioptechs.com/product/interchangable-coverslip-dish-heated-lid/

Dan

Dan Focht
Bioptechs Inc.
3560 Beck Road
Butler, PA 16002-9259
Office: 724-282-7145
Toll Free: 877-LIVE-CELL (548-3235)
[hidden email]
www.bioptechs.com



On Aug 6, 2019, at 3:39 PM, Tobias Rasse <[hidden email]> wrote:

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Dear All,

a potential user would be interested to

a)       image fresh cyrosections at our slidescanner (GFP,RFP)

b)      then perform a lacz staining

c)       then image the same sections again.

In the current pilot (at a standard wide field microscope) sections are
covered by a cover slip only after b). They are kept moist during a)
manually.

Images from a) and c) shall be registered. Thus, putting a coverslip before
a) and removing it for b) seems no option.

Do you have any advice on how to keep sections moist (but not spilling
everything with water during robot movements)?

e.g. small chambers glued on after recovering the sections from the
cryostat?

(we have a Märzhäuser SlideExpress 2 – Nikon) system.

Thanks a lot & Kind regards

Tobias

[Tobias Rasse-2]

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Dear All,

Thank you very much for the numerous great suggestions!
The testing of these is a bit delayed by the availability of mice.
PDMS is likely the way to go for us.
I aim at a thickness of 100-200 um to then (likely) cover the sections with a coverslip reversibly.

PDMS holds the potential to be up-scaled to a large number of slides and the resulting protocol is independent of the sample. Thus, we would not need to re-establish protocols for each new mouse strain.

Additionally, I will try to image lacZ & fluorescence as suggested by Konstantin:
https://www.nature.com/articles/srep02937

in the very end. If it works it would be a good control to check the alignment of native fluorescent images prior to staining and bright field images after staining (even if the images are not perfect.)

Thanks again & Kind regards

Tobias