CO2 availability during live imaging

[Julia Edgar]

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Dear All
After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
Thank you in advance.
Best wishes
Julia

Julia M Edgar BSc (Hons), PhD, FHEA
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow
Sir Graeme Davies Building
120 University Place
Glasgow
G12 8TA
Tel [phone]

And

Department of Neurogenetics
Max Planck Institute of Experimental Medicine
Herman-Rein-Strasse 3
D-37075 Goettingen
Germany

[Kural, Comert]

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>We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely?

I don't think so. But you can test it. Just place two dishes in the environmental chamber, one clamped tightly the other not and measure the pH (with pH paper) after half an hour or so.

On Nov 16, 2019 8:57 AM, Julia Edgar <[hidden email]> wrote:
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Dear All
After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
Thank you in advance.
Best wishes
Julia

Julia M Edgar BSc (Hons), PhD, FHEA
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow
Sir Graeme Davies Building
120 University Place
Glasgow
G12 8TA
Tel [phone]

And

Department of Neurogenetics
Max Planck Institute of Experimental Medicine
Herman-Rein-Strasse 3
D-37075 Goettingen
Germany

[Sylvie Le Guyader]

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Hi Julia

Sounds unlikely. I would leave a dish in the incubator over night without lid to test.

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
Follow our microscopy blog!

On 16 Nov 2019 14:57, Julia Edgar <[hidden email]> wrote:
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Dear All
After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
Thank you in advance.
Best wishes
Julia

Julia M Edgar BSc (Hons), PhD, FHEA
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow
Sir Graeme Davies Building
120 University Place
Glasgow
G12 8TA
Tel [phone]

And

Department of Neurogenetics
Max Planck Institute of Experimental Medicine
Herman-Rein-Strasse 3
D-37075 Goettingen
Germany



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[Cammer, Michael]

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It would have to truly sealed.  To put a small hole in the lid  off to the side from the imaging area or in side of the dish, take a small nail or wire and heat the tip in a flame until hot enough to melt the plastic and melt a hole in it or use a power drill.


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

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________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Sylvie Le Guyader <[hidden email]>
Sent: Saturday, November 16, 2019 12:17 PM
To: [hidden email]
Subject: Re: CO2 availability during live imaging

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Hi Julia

Sounds unlikely. I would leave a dish in the incubator over night without lid to test.

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
Follow our microscopy blog!

On 16 Nov 2019 14:57, Julia Edgar <[hidden email]> wrote:
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*****

Dear All
After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
Thank you in advance.
Best wishes
Julia

Julia M Edgar BSc (Hons), PhD, FHEA
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow
Sir Graeme Davies Building
120 University Place
Glasgow
G12 8TA
Tel [phone]

And

Department of Neurogenetics
Max Planck Institute of Experimental Medicine
Herman-Rein-Strasse 3
D-37075 Goettingen
Germany



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://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_medarbetare_integritetsskyddspolicy&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=AM2bDlsaxkKcuNhrRmAyjFd_YcSZnyASQjhzODpVoAs&s=8q4EZdYEm1EWuGbH9-Si3fsL_QLpy9cRgJgdJ3FUpmg&e= >.


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[Tim Feinstein]

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I don't think a clamp would do much.  Tissue culture dishes have spacers to prevent the lid from becoming gas-tight regardless of how you press on them.  You said it was a Petri dish, but I'm assuming that you meant a Mattek-style 35mm glass bottom tissue culture dish.  Is that correct?  If it was a TC dish, sometimes medium wicks into the space where the lid and dish overlap and that creates an seal against gas exchange.  If the dish was clean (no medium wicked up) I would start with the assumption that your CO2 delivery is bad.  Best,


T

Timothy Feinstein, Ph.D.


On 11/16/19, 8:56 AM, "Confocal Microscopy List on behalf of Julia Edgar" <[hidden email] on behalf of [hidden email]> wrote:

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    *****
   
    Dear All
    After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
    Thank you in advance.
    Best wishes
    Julia
   
    Julia M Edgar BSc (Hons), PhD, FHEA
    Institute of Infection, Immunity and Inflammation
    College of Medical, Veterinary and Life Sciences
    University of Glasgow
    Sir Graeme Davies Building
    120 University Place
    Glasgow
    G12 8TA
    Tel [phone]
   
    And
   
    Department of Neurogenetics
    Max Planck Institute of Experimental Medicine
    Herman-Rein-Strasse 3
    D-37075 Goettingen
    Germany
   

[Julia Edgar]

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Thanks everybody for all tips. We’ll update you....after we problem-solve.
Definitely no liquid wicks! My student knows better than to risk infection that way. 😉But it’s a good general point.  Thank you.


Sent from my iPhone. Please excuse typos.

> On 17 Nov 2019, at 16:12, Feinstein, Timothy N <[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.
> *****
>
> I don't think a clamp would do much.  Tissue culture dishes have spacers to prevent the lid from becoming gas-tight regardless of how you press on them.  You said it was a Petri dish, but I'm assuming that you meant a Mattek-style 35mm glass bottom tissue culture dish.  Is that correct?  If it was a TC dish, sometimes medium wicks into the space where the lid and dish overlap and that creates an seal against gas exchange.  If the dish was clean (no medium wicked up) I would start with the assumption that your CO2 delivery is bad.  Best,
>
>
> T
>
> Timothy Feinstein, Ph.D.
>
>
> On 11/16/19, 8:56 AM, "Confocal Microscopy List on behalf of Julia Edgar" <[hidden email] on behalf of [hidden email]> wrote:
>
>    *****
>    To join, leave or search the confocal microscopy listserv, go to:
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>    *****
>
>    Dear All
>    After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
>    Thank you in advance.
>    Best wishes
>    Julia
>
>    Julia M Edgar BSc (Hons), PhD, FHEA
>    Institute of Infection, Immunity and Inflammation
>    College of Medical, Veterinary and Life Sciences
>    University of Glasgow
>    Sir Graeme Davies Building
>    120 University Place
>    Glasgow
>    G12 8TA
>    Tel [phone]
>
>    And
>
>    Department of Neurogenetics
>    Max Planck Institute of Experimental Medicine
>    Herman-Rein-Strasse 3
>    D-37075 Goettingen
>    Germany
>
>

[Dan Focht]

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Julia and all interested

To make things easier for anybody faced with this problem, Bioptechs developed and produced the following solutions.

For long term imaging in a dish structure there are several approaches. One involves pre-equilibration of the media then pumping the media through the dish. The other is to create a CO2 microenvironment immediately above the media in the dish. Both techniques require isolation from the ambient atmosphere but different techniques.

In the case you want to have the media in the dish in contact with CO2, we make an O-ring sealed Heated Lid that fits our self heating, high numeric aperture compatible, Delta T dish system. In addition to having a heated window for an unobstructed light path for the condenser, it has a CO2 inlet port and vent on, as well as optional perfusion ports. The result is that you have a micro-incubator on your stage without having to "box" the scope or a box on the stage.

A word of caution, if the dish is heated to physiological temperatures the CO2 going into a dish displaces the humidity that is already at 100% in the dish. Therefore, in order to preserve the osmolarity of the media, the humidity of the incoming gas needs to be matched. We also make an easy to use, inexpensive, CO2 humidification device that mates with the Heated Lid for this purpose.

In cases where the media is pre-equilibrated and perfused through the dish and imaging involves transmitted contrast modes, we also have a Coverglass Lid for the Delta T system. It suspends a coverslip down into the dish to make contact with the media, thereby establishing a uniform optical surface above the specimen so that each contrast image has the same optical path instead of inducing contrast shifts due to fluid motion of an open to air interface.

Here are links to the above mentioned solutions.

Delta T dish system
https://bioptechs.com/product/delta-t-starter-set/

Heated Lid
https://bioptechs.com/product/delta-t-heated-lid/

Coverglass lid
https://bioptechs.com/product/delta-t-coverglass-lid/

CO2 Humidification
https://bioptechs.com/product/micro-gas-humidifier/ 

Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler PA 16002
V724-282-7145
F724-282-0745
Toll Free 877 lIVE-CELL (548-3235)
[hidden email]

> On Nov 17, 2019, at 11:42 AM, Julia Edgar <[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.
> *****
>
> Thanks everybody for all tips. We’ll update you....after we problem-solve.
> Definitely no liquid wicks! My student knows better than to risk infection that way. 😉But it’s a good general point.  Thank you.
>
>
> Sent from my iPhone. Please excuse typos.
>
>> On 17 Nov 2019, at 16:12, Feinstein, Timothy N <[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.
>> *****
>>
>> I don't think a clamp would do much.  Tissue culture dishes have spacers to prevent the lid from becoming gas-tight regardless of how you press on them.  You said it was a Petri dish, but I'm assuming that you meant a Mattek-style 35mm glass bottom tissue culture dish.  Is that correct?  If it was a TC dish, sometimes medium wicks into the space where the lid and dish overlap and that creates an seal against gas exchange.  If the dish was clean (no medium wicked up) I would start with the assumption that your CO2 delivery is bad.  Best,
>>
>>
>> T
>>
>> Timothy Feinstein, Ph.D.
>>
>>
>> On 11/16/19, 8:56 AM, "Confocal Microscopy List on behalf of Julia Edgar" <[hidden email] on behalf of [hidden email]> wrote:
>>
>>   *****
>>   To join, leave or search the confocal microscopy listserv, go to:
>>   https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C03abac0fa70a488a203208d76a9cd08f%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637095094083066978&amp;sdata=GUXizTLRpR%2BimD%2BjCIAFC9IMEqAe%2FXTVdkKAax7mr90%3D&amp;reserved=0
>>   Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C03abac0fa70a488a203208d76a9cd08f%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637095094083066978&amp;sdata=pVCBO1Ushqk8TnUKTwhlf0Vis96OGJZPtWqQBDv%2BaE8%3D&amp;reserved=0 and include the link in your posting.
>>   *****
>>
>>   Dear All
>>   After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
>>   Thank you in advance.
>>   Best wishes
>>   Julia
>>
>>   Julia M Edgar BSc (Hons), PhD, FHEA
>>   Institute of Infection, Immunity and Inflammation
>>   College of Medical, Veterinary and Life Sciences
>>   University of Glasgow
>>   Sir Graeme Davies Building
>>   120 University Place
>>   Glasgow
>>   G12 8TA
>>   Tel [phone]
>>
>>   And
>>
>>   Department of Neurogenetics
>>   Max Planck Institute of Experimental Medicine
>>   Herman-Rein-Strasse 3
>>   D-37075 Goettingen
>>   Germany
>>
>>

[Jacqueline Ross]

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Hi Julia,

Do you have a gas mixer to generate the correct gas mix or do you have a pre-mixed cylinder? We have had issues with a gas mixer where a pump has to be replaced. It wasn't providing the correct 5% CO2.

Also what kind of incubator do you have? A stage-top? You could try using a gas analyzer to check the %.

Cheers,

Jacqui
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Julia Edgar [[hidden email]]
Sent: 17 November 2019 01:53
To: [hidden email]
Subject: CO2 availability during live imaging

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Dear All
After overnight live imaging our media is alkaline and the cells are dead. We think the CO2 gassing is working, but the clips that hold the 35 mm Petri dish in place on the stage are clamping the lid so tightly to the dish that the CO2 cannot enter the dish. Does this seem likely? If so, does anybody use small spacers to lift the lid very slightly and allow the CO2 in?
Thank you in advance.
Best wishes
Julia

Julia M Edgar BSc (Hons), PhD, FHEA
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow
Sir Graeme Davies Building
120 University Place
Glasgow
G12 8TA
Tel [phone]

And

Department of Neurogenetics
Max Planck Institute of Experimental Medicine
Herman-Rein-Strasse 3
D-37075 Goettingen
Germany

[Glyn Nelson]

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We've had problems with condensate building up in the tubing and blocking the flow.  Also, my preference is to replace the petri dish lid with a large circular coverslip- hard to get hold of, but easy to sterilise and reusable. I found there is enough gas exchange and less evaporation if the humidity isn't as high as you would like (paranoia about osmotic shock affecting results!).

[Jeremy Adler-4]

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Evaporation is potential problem - check by weighing the petri dish before and after imaging.
Even a small change in volume will increase the ionic concentrations in the medium which will alter the cells membrane potential - in addition to osmotic effects.

Given the volume of a petri dish and the small number of cells - I would be surprised if metabolic activity changed the gas composition - does anyone have some experimental evidence or calculations ?

When making an air tight seal on a petri dish there is the practical problem of how to seal in a 5% CO2 mix rather than room air - any suggestions - an incubator will glove ports ?

Jeremy Adler
BioVis
UU
Sweden

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Glyn Nelson
Sent: den 18 november 2019 10:20
To: [hidden email]
Subject: Re: CO2 availability during live imaging

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We've had problems with condensate building up in the tubing and blocking the flow.  Also, my preference is to replace the petri dish lid with a large circular coverslip- hard to get hold of, but easy to sterilise and reusable. I found there is enough gas exchange and less evaporation if the humidity isn't as high as you would like (paranoia about osmotic shock affecting results!).








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[Michael Porter]

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I have previously worked with cells that were very sensitive to CO2 levels. Despite CO2 supply in the microscope chamber, it was nowhere near as stable as a TC incubator. I found that supplementing the medium with even more sodium bicarbonate was the key - I filter-sterilised a 7.5% solution and added (I think!) 13ml to a 500ml bottle of medium.

[Steffen Dietzel]

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Hi Julie, hi Jeremy

if the lid were sealed, I don't think you would have that problem.
During our student course we culture Hela cells for several days in a
closed chamber (1 mm high, filled with medium, no air or other gas)
without any trouble. So, Jeremy, instead of trying to seal in 5% CO2 in
the air, I would go for no air, chamber completely filled with 5% CO2
medium straight from the incubator. This also solves the problem of
evaporation.

Cultured cells can go by with very little O2. 20% in the atmosphere is
way more than any mammalian cell will usually experience in the body
(with the exception of those in respiratory system surfaces). Over
night, I only would expect alkaline medium if the CO2 which already is
in the (preconditioned) medium can escape.

Steffen


Am 18.11.2019 um 10:52 schrieb Jeremy Adler:

> *****
> 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.
> *****
>
> Evaporation is potential problem - check by weighing the petri dish
> before and after imaging.
> Even a small change in volume will increase the ionic concentrations
> in the medium which will alter the cells membrane potential - in
> addition to osmotic effects.
>
> Given the volume of a petri dish and the small number of cells - I
> would be surprised if metabolic activity changed the gas composition -
> does anyone have some experimental evidence or calculations ?
>
> When making an air tight seal on a petri dish there is the practical
> problem of how to seal in a 5% CO2 mix rather than room air - any
> suggestions - an incubator will glove ports ?
>
> Jeremy Adler
> BioVis
> UU
> Sweden
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Glyn Nelson
> Sent: den 18 november 2019 10:20
> To: [hidden email]
> Subject: Re: CO2 availability during live imaging
>
> *****
> 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 had problems with condensate building up in the tubing and
> blocking the flow. Also, my preference is to replace the petri dish
> lid with a large circular coverslip- hard to get hold of, but easy to
> sterilise and reusable. I found there is enough gas exchange and less
> evaporation if the humidity isn't as high as you would like (paranoia
> about osmotic shock affecting results!).
>
>
>
>
>
>
>
>
> När du har kontakt med oss på Uppsala universitet med e-post så
> innebär det att vi behandlar dina personuppgifter. För att läsa mer om
> hur vi gör det kan du läsa här:
> http://www.uu.se/om-uu/dataskydd-personuppgifter/
>
> E-mailing Uppsala University means that we will process your personal
> data. For more information on how this is performed, please read here:
> http://www.uu.se/en/about-uu/data-protection-policy

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de