CO2 gassing using Zeiss spinning disc confocal

[Julia Edgar]

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Dear All
I’m using a Zeiss spinning disc confocal microscope with small heated incubation stage and ‘gassing’ lid.
The problem is that the pH of the media changes over time and becomes rather alkaline (turns blue pink). Motor proteins don’t behave normally when pH is not physiological, so this is potentially a big problem.

Do you have any tips to ensure the 5% CO2 reaches the imaging dish (a 35 mm Petri-dish) that sits between the incubation insert and the gassing lid?
The tubing connecting the gassing lid to the water bottle (into which the CO2 is pumped) is rather long. Would it help to shorten it?
Does the volume of water in the water bottle matter?
How can I ensure the little inlet pipes in the gassing lid are not blocked?

Many thanks
Julia
University of Glasgow.

[Douglas Richardson]

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Hi Julia,

I used to have these lines block with condensed water. This is easy to diagnose, if you disconnect the blue line at the CO2 mixer, water will often come out. Some compressed air gets rid of the rest.

Elevating the bottle to a shelf above the stage seems to have prevented this from happening further.

Doug

Sent from my iPhone

> On Apr 3, 2017, at 5:18 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.
> *****
>
> Dear All
> I’m using a Zeiss spinning disc confocal microscope with small heated incubation stage and ‘gassing’ lid.
> The problem is that the pH of the media changes over time and becomes rather alkaline (turns blue pink). Motor proteins don’t behave normally when pH is not physiological, so this is potentially a big problem.
>
> Do you have any tips to ensure the 5% CO2 reaches the imaging dish (a 35 mm Petri-dish) that sits between the incubation insert and the gassing lid?
> The tubing connecting the gassing lid to the water bottle (into which the CO2 is pumped) is rather long. Would it help to shorten it?
> Does the volume of water in the water bottle matter?
> How can I ensure the little inlet pipes in the gassing lid are not blocked?
>
> Many thanks
> Julia
> University of Glasgow.

[Armstrong, Brian]

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Hi Julia, make sure that you see a significant amount of bubbles coming through the water in the humidification chamber. You can clean the small "stone" that the gas goes through. Also, you can get a new and different stone from an aquarium store that will let more gas flow through.

Worked for us,

Brian Armstrong PhD
Associate Research Professor
Developmental and Stem Cell Biology
Diabetes and Metabolic Diseases
Director, Light Microscopy Core
Beckman Research Institute, City of Hope


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julia Edgar
Sent: Monday, April 03, 2017 2:18 AM
To: [hidden email]
Subject: CO2 gassing using Zeiss spinning disc confocal

*****
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
I’m using a Zeiss spinning disc confocal microscope with small heated incubation stage and ‘gassing’ lid.
The problem is that the pH of the media changes over time and becomes rather alkaline (turns blue pink). Motor proteins don’t behave normally when pH is not physiological, so this is potentially a big problem.

Do you have any tips to ensure the 5% CO2 reaches the imaging dish (a 35 mm Petri-dish) that sits between the incubation insert and the gassing lid?
The tubing connecting the gassing lid to the water bottle (into which the CO2 is pumped) is rather long. Would it help to shorten it?
Does the volume of water in the water bottle matter?
How can I ensure the little inlet pipes in the gassing lid are not blocked?

Many thanks
Julia
University of Glasgow.


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[Julia Edgar]

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Hi Brian
Thanks so much. Only very gentle bubbling at the moment. I'll try doing as you suggest.
Best wishes
Julia

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
Sent: 03 April 2017 16:39
To: [hidden email]
Subject: Re: CO2 gassing using Zeiss spinning disc confocal

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Hi Julia, make sure that you see a significant amount of bubbles coming through the water in the humidification chamber. You can clean the small "stone" that the gas goes through. Also, you can get a new and different stone from an aquarium store that will let more gas flow through.

Worked for us,

Brian Armstrong PhD
Associate Research Professor
Developmental and Stem Cell Biology
Diabetes and Metabolic Diseases
Director, Light Microscopy Core
Beckman Research Institute, City of Hope


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julia Edgar
Sent: Monday, April 03, 2017 2:18 AM
To: [hidden email]
Subject: CO2 gassing using Zeiss spinning disc confocal

*****
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
I’m using a Zeiss spinning disc confocal microscope with small heated incubation stage and ‘gassing’ lid.
The problem is that the pH of the media changes over time and becomes rather alkaline (turns blue pink). Motor proteins don’t behave normally when pH is not physiological, so this is potentially a big problem.

Do you have any tips to ensure the 5% CO2 reaches the imaging dish (a 35 mm Petri-dish) that sits between the incubation insert and the gassing lid?
The tubing connecting the gassing lid to the water bottle (into which the CO2 is pumped) is rather long. Would it help to shorten it?
Does the volume of water in the water bottle matter?
How can I ensure the little inlet pipes in the gassing lid are not blocked?

Many thanks
Julia
University of Glasgow.


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[Sven Terclavers-3]

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Hi Julia,

And please remember that in the MTB2011 Configuration software, which acts
as a link between AxioVision/ZEN and your system, you can set the Fan Speed
& Pump Level for the CO2 Module. Also make sure to calibrate the CO2 sensor
(in MTB as well!). I'm sure your local ZEISS representative/specialist can
help you out if necessary!
Best regards,

Sven

On Mon, Apr 3, 2017 at 11:40 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.
> *****
>
> Hi Brian
> Thanks so much. Only very gentle bubbling at the moment. I'll try doing as
> you suggest.
> Best wishes
> Julia
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Armstrong, Brian
> Sent: 03 April 2017 16:39
> To: [hidden email]
> Subject: Re: CO2 gassing using Zeiss spinning disc confocal
>
> *****
> 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.
> *****
>
> Hi Julia, make sure that you see a significant amount of bubbles coming
> through the water in the humidification chamber. You can clean the small
> "stone" that the gas goes through. Also, you can get a new and different
> stone from an aquarium store that will let more gas flow through.
>
> Worked for us,
>
> Brian Armstrong PhD
> Associate Research Professor
> Developmental and Stem Cell Biology
> Diabetes and Metabolic Diseases
> Director, Light Microscopy Core
> Beckman Research Institute, City of Hope
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Julia Edgar
> Sent: Monday, April 03, 2017 2:18 AM
> To: [hidden email]
> Subject: CO2 gassing using Zeiss spinning disc confocal
>
> *****
> 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
> I’m using a Zeiss spinning disc confocal microscope with small heated
> incubation stage and ‘gassing’ lid.
> The problem is that the pH of the media changes over time and becomes
> rather alkaline (turns blue pink). Motor proteins don’t behave normally
> when pH is not physiological, so this is potentially a big problem.
>
> Do you have any tips to ensure the 5% CO2 reaches the imaging dish (a 35
> mm Petri-dish) that sits between the incubation insert and the gassing lid?
> The tubing connecting the gassing lid to the water bottle (into which the
> CO2 is pumped) is rather long. Would it help to shorten it?
> Does the volume of water in the water bottle matter?
> How can I ensure the little inlet pipes in the gassing lid are not blocked?
>
> Many thanks
> Julia
> University of Glasgow.
>
>
> ---------------------------------------------------------------------
> -SECURITY/CONFIDENTIALITY WARNING-
> This message (and any attachments) are intended solely for the individual
> or entity to which they are addressed. This communication may contain
> information that is privileged, confidential, or exempt from disclosure
> under applicable law (e.g., personal health information, research data,
> financial information). Because this e-mail has been sent without
> encryption, individuals other than the intended recipient may be able to
> view the information, forward it to others or tamper with the information
> without the knowledge or consent of the sender. If you are not the intended
> recipient, or the employee or person responsible for delivering the message
> to the intended recipient, any dissemination, distribution or copying of
> the communication is strictly prohibited. If you received the communication
> in error, please notify the sender immediately by replying to this message
> and deleting the message and any accompanying files from your system. If,
> due to the security risks, you do not wish to receive further
> communications via e-mail, please reply to this message and inform the
> sender that you do not wish to receive further e-mail from the sender.
> (LCP301)
> ---------------------------------------------------------------------
>
>