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CO2, humidity, temp sensors

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Paul Herzmark

CO2, humidity, temp sensors

I have a cell dying problem in my microscope when I take pictures of them overnight or longer. I suspect the humidity is failing and I want to monitor that, CO2 and temperature.(It is not photodamage. Cells die in areas away from the objective.)

Can anyone suggest a supplier of recording sensors that I can get next to the well slides on my stage?

Thank you!

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax

Goodhouse, Joseph G.

Re: CO2, humidity, temp sensors

Paul,

Could you describe in detail your heating system. Do you overlay your cells with mineral oil? This prevents evaporation which will alter your salt concentration.

I use a Recordable Temp sensor from Fisher Scientific. Cat No 15-007-26. Inexpensive and works well.

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Visit us at http://www.molbio1.princeton.edu/facility/confocal/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: Thursday, February 12, 2009 2:24 PM
To: [hidden email]
Subject: CO2, humidity, temp sensors

Paul Herzmark

Re: CO2, humidity, temp sensors

I knew I would not get away with out details!

I have an Invivo (company) plexiglass incubator box around my inverted microscope. 5% CO2 is piped inside to a small space (about 50 ml) around the multiwell-Labtech (company)-coverslip. The small space is created by a glass plate with a frame around it that goes over the coverslip.The CO2 passes into the small space via special tubing.The tubing goes under incubator-warmed water where it is supposed to pick up water vapor without bubbling.

1. The whole incubator, with most of the microscope is warmed to 37 degrees
2. Only the small space surrounding the cells is humidified and has CO2.

On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G. <[hidden email]> wrote:

Paul,

Could you describe in detail your heating system. Do you overlay your cells with mineral oil? This prevents evaporation which will alter your salt concentration.

I use a Recordable Temp sensor from Fisher Scientific. Cat No 15-007-26. Inexpensive and works well.

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Visit us at http://www.molbio1.princeton.edu/facility/confocal/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: Thursday, February 12, 2009 2:24 PM
To: [hidden email]
Subject: CO2, humidity, temp sensors

I have a cell dying problem in my microscope when I take pictures of them overnight or longer. I suspect the humidity is failing and I want to monitor that, CO2 and temperature.(It is not photodamage. Cells die in areas away from the objective.)

Can anyone suggest a supplier of recording sensors that I can get next to the well slides on my stage?

Thank you!

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax

Goodhouse, Joseph G.

Re: CO2, humidity, temp sensors

My Guess is still evaporation of media is killing your cells.. Overlay the cell media in each compartment with mineral oil. It stops all evaporation but allows CO2 gas exchange to occur to maintain pH.. I do this with all my chamber systems and it works great. No need to humidify flowing air or gas.

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Visit us at http://www.molbio1.princeton.edu/facility/confocal/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: Thursday, February 12, 2009 4:36 PM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors

On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G. <[hidden email]> wrote:

Paul,

Could you describe in detail your heating system. Do you overlay your cells with mineral oil? This prevents evaporation which will alter your salt concentration.

I use a Recordable Temp sensor from Fisher Scientific. Cat No 15-007-26. Inexpensive and works well.

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Visit us at http://www.molbio1.princeton.edu/facility/confocal/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: Thursday, February 12, 2009 2:24 PM
To: [hidden email]
Subject: CO2, humidity, temp sensors

I have a cell dying problem in my microscope when I take pictures of them overnight or longer. I suspect the humidity is failing and I want to monitor that, CO2 and temperature.(It is not photodamage. Cells die in areas away from the objective.)

Can anyone suggest a supplier of recording sensors that I can get next to the well slides on my stage?

Thank you!

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax

Paul Herzmark

Re: CO2, humidity, temp sensors

Joe,
Thank you for the suggestion. I also suspect the evaporation.

I don't understand how mineral oil will stop water vapor from passing but allow CO2 exchange. Any idea?

Paul

On Thu, Feb 12, 2009 at 2:23 PM, Goodhouse, Joseph G. <[hidden email]> wrote:

My Guess is still evaporation of media is killing your cells.. Overlay the cell media in each compartment with mineral oil. It stops all evaporation but allows CO2 gas exchange to occur to maintain pH.. I do this with all my chamber systems and it works great. No need to humidify flowing air or gas.

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Zoltan

Re: CO2, humidity, temp sensors

In reply to this post by Paul Herzmark

Paul,

We have a similar system and have been using it successfully after spending some time to tune the 5% CO2 flow rate until the pH stayed constant (judged simply by the colour of the medium), and after placing several beakers of water within the 37C area to humidify the warm air and thus keep the evaporation rate low. This way experiments can be done overnight w/o cells dying (appr. 17 hours).

Hope this helps a bit.

Zoltan

On Thu, Feb 12, 2009 at 7:23 PM, Paul Herzmark <[hidden email]> wrote:

I have a cell dying problem in my microscope when I take pictures of them overnight or longer. I suspect the humidity is failing and I want to monitor that, CO2 and temperature.(It is not photodamage. Cells die in areas away from the objective.)

Can anyone suggest a supplier of recording sensors that I can get next to the well slides on my stage?

Thank you!

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

Keith Morris

Re: CO2, humidity, temp sensors

Hi,

Temperature and %rh probes.

It is useful to measure temperature and relative humidity in your incubator
and there is a superb company that produces excellent probes for doing this
on the cheap:

http://www.picotech.com/temperature-humidity.html

Price depends on accuracy: +/-0.3oC or +/-0.01oC, the former being fine for
most. I use the £135 three probe Th-03 system thats accurate to +/-0.3oC,
although theres the more expensive [£400 single] platinum resistance probe
thats accurate to +/- 0.01oC or more. Temp measurement is a complex thing
with probes, e.g. conduction heat loss down the probe etc but they do give
a useful indication of the temp where the cells are rather what an external
control box says. Pico also make a very cheap software oscilloscope and %rh
probes. My PhD is in fuel and combustion, so thermocouples and stuff are
second nature to me, but these Pico data loggers are great value, and simple
to use [although the TH-03 probes are a bit large for some situations].

Evaporation

Yes you can use mineral oil for long term time-lapse, but some worry about
gas exchange and toxic effects [well I do]. A preferred option would be
special gas not water permeable membrane lids/seals for your small Petri
dish to virtually eliminate evaporation: e.g. Zeiss Foil Cover and CultFoil
Foil Covers that extend the incubation time considerably by reducing
evaporation. A special foil (CultFoil), gripped between two thin steel
components, replaces the usual cover of the culture vessel. The foil
effectively reduces the amount of water vapor that is able to escape. Gas
exchange is not affected. But they arent as cheap as mineral oil.

Generally we just humidified the air in the incubators to reduce evaporation
rates. For short term [2-4 hours] I used to just put a beaker filled with
water and overhanging tissues into the incubator [if its a large one],
although you need to change it daily to prevent it getting smelly. However
for long term time-lapse we always used the Zeiss [PeCon] humidifiers that
fitted in-line to air circulation system [cost about £500 each], and these
worked very well. All our systems were Zeiss/Leica and so PeCon friendly.
For CO2 we trusted our fantastically expensive variable% Zeiss CO2
controllers [or the 5% CO2 label on the BOC CO2 cylinder].

Humidifier:
http://www.pe-con.de/pecon/index.htm
search for relative humidity

Regards

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford OX3 7BN,
United Kingdom.

Telephone: +44 (0)1865 287568
Email: [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
________________________________________
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Zoltan Cseresnyes
Sent: 12 February 2009 23:22
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors

Paul,

We have a similar system and have been using it successfully after spending
some time to tune the 5% CO2 flow rate until the pH stayed constant (judged
simply by the colour of the medium), and after placing several beakers of
water within the 37C area to humidify the warm air and thus keep the
evaporation rate low. This way experiments can be done overnight w/o cells
dying (appr. 17 hours).
Hope this helps a bit.

Zoltan

On Thu, Feb 12, 2009 at 7:23 PM, Paul Herzmark <[hidden email]> wrote:
I have a cell dying problem in my microscope when I take pictures of them
overnight or longer. I suspect the humidity is failing and I want to monitor
that, CO2 and temperature.(It is not photodamage. Cells die in areas away
from the objective.)

Can anyone suggest a supplier of recording sensors that I can get next to
the well slides on my stage?

Thank you!

Paul Herzmark
Specialist
[hidden email]

Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

Brotchie, Daniel

Re: CO2, humidity, temp sensors

In reply to this post by Paul Herzmark

Hi,

I guess not applicable to Paul’s micro environment, but SenseAir sell a meter that measures CO2, temperature and humidity simultaneously - cost about 200 USD:

http://www.senseair.se/includes/products/psense_rh.php

Where you have an time-lapse incubator, but have CO2 issues/no CO2 cylinder you can try CO2 independent media:

http://www.invitrogen.com/site/us/en/home/Global/invitrogen-search-results.html?searchTerm=18045&mode=simpleany&searchTypes=meta.collection:cmgtproduct

or use CO2 generating tablets (baking powder):

http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=BR0039&c=UK&lang=EN

NCI

Daniel

------------------------------------------------

Daniel Brotchie

Ophthalmology

School of Clinical Sciences

University Clinical Departments

The Duncan Building

Daulby Street

LIVERPOOL

L69 3GA

Tel: +44 (0) 151 706 4017

Fax: +44 (0) 151 706 5934

E-mail [hidden email]

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Herzmark
Sent: 12 February 2009 19:24
To: [hidden email]
Subject: CO2, humidity, temp sensors

Beat Ludin

Re: CO2, humidity, temp sensors

In reply to this post by Goodhouse, Joseph G.

Over time, the mineral oil overlay will basically void the medium of
all lipophilic components (vitamin E, for instance). This may or may
not be a problem, but you need to be aware of it and do the proper controls.

If you want to check for evaporation, just weigh your sample before
and after the imaging.

Beat

At 23:23 12-02-2009, you wrote:

>My Guess is still evaporation of media is killing your
>cells.. Overlay the cell media in each compartment with mineral
>oil. It stops all evaporation but allows CO2 gas exchange to occur
>to maintain pH.. I do this with all my chamber systems and it works
>great. No need to humidify flowing air or gas.
>
>
>Joe Goodhouse
>Confocal Core Lab Manager
>Dept. of Molecular Biology
>Princeton University
>609-258-5432
>
>Visit us at
><http://www.molbio/>http://www.molbio1.princeton.edu/facility/confocal/
>
>
>
>----------
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Paul Herzmark
>Sent: Thursday, February 12, 2009 4:36 PM
>To: [hidden email]
>Subject: Re: CO2, humidity, temp sensors
>
>
>I knew I would not get away with out details!
>
>I have an Invivo (company) plexiglass incubator box around my
>inverted microscope. 5% CO2 is piped inside to a small space (about
>50 ml) around the multiwell-Labtech (company)-coverslip. The small
>space is created by a glass plate with a frame around it that goes
>over the coverslip.The CO2 passes into the small space via special
>tubing.The tubing goes under incubator-warmed water where it is
>supposed to pick up water vapor without bubbling.
>
>1. The whole incubator, with most of the microscope is warmed to 37 degrees
>2. Only the small space surrounding the cells is humidified and has CO2.
>
>
>
>
>On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G.
><<mailto:[hidden email]>[hidden email]> wrote:
>Paul,
> Could you describe in detail your heating system. Do you
> overlay your cells with mineral oil? This prevents evaporation
> which will alter your salt concentration.
>
> I use a Recordable Temp sensor from Fisher Scientific. Cat No
> 15-007-26. Inexpensive and works well.
>
>
>Joe Goodhouse
>Confocal Core Lab Manager
>Dept. of Molecular Biology
>Princeton University
>609-258-5432
>
>Visit us at
><http://www.molbio/>http://www.molbio1.<http://princeton.edu/facility/confocal/>princeton.edu/facility/confocal/
>
>
>
>
>----------
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Paul Herzmark
>Sent: Thursday, February 12, 2009 2:24 PM
>To: <mailto:[hidden email]>[hidden email]
>Subject: CO2, humidity, temp sensors
>
>I have a cell dying problem in my microscope when I take pictures of
>them overnight or longer. I suspect the humidity is failing and I
>want to monitor that, CO2 and temperature.(It is not photodamage.
>Cells die in areas away from the objective.)
>
>Can anyone suggest a supplier of recording sensors that I can get
>next to the well slides on my stage?
>
>Thank you!
>
>Paul Herzmark
>Specialist
><mailto:[hidden email]>[hidden email]
>
>Department of Molecular and Cell Biology
>479 Life Science Addition
>University of California, Berkeley
>Berkeley, CA 94720-3200
>(510) 643-9603
>(510) 643-9500 fax
>

James Denegre

Re: CO2, humidity, temp sensors and mu\ineral oil

In our lab we equilibrate the mineral oil with the medium to prevent uptake of components, and have successfully imaged under mineral oil for five continuous days.

James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Beat Ludin
Sent: Wednesday, February 25, 2009 5:05 AM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors

Over time, the mineral oil overlay will basically void the medium of
all lipophilic components (vitamin E, for instance). This may or may
not be a problem, but you need to be aware of it and do the proper controls.

If you want to check for evaporation, just weigh your sample before
and after the imaging.

Beat

At 23:23 12-02-2009, you wrote:

Armstrong, Brian

Re: CO2, humidity, temp sensors and mu\ineral oil

Hi James, can you elaborate on your technique " equilibrate the mineral
oil with the medium"?
Thanks,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of James Denegre
Sent: Thursday, February 26, 2009 8:10 AM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors and mu\ineral oil

In our lab we equilibrate the mineral oil with the medium to prevent
uptake of components, and have successfully imaged under mineral oil for
five continuous days.

James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Beat Ludin
Sent: Wednesday, February 25, 2009 5:05 AM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors

Over time, the mineral oil overlay will basically void the medium of
all lipophilic components (vitamin E, for instance). This may or may
not be a problem, but you need to be aware of it and do the proper
controls.

If you want to check for evaporation, just weigh your sample before
and after the imaging.

Beat

At 23:23 12-02-2009, you wrote:

degrees
>2. Only the small space surrounding the cells is humidified and has
CO2.

>
>
>
>
>On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G.
><<mailto:[hidden email]>[hidden email]> wrote:
>Paul,
> Could you describe in detail your heating system. Do you
> overlay your cells with mineral oil? This prevents evaporation
> which will alter your salt concentration.
>
> I use a Recordable Temp sensor from Fisher Scientific. Cat No
> 15-007-26. Inexpensive and works well.
>
>
>Joe Goodhouse
>Confocal Core Lab Manager
>Dept. of Molecular Biology
>Princeton University
>609-258-5432
>
>Visit us at
><http://www.molbio/>http://www.molbio1.<http://princeton.edu/facility/c

onfocal/>princeton.edu/facility/confocal/
>
>
>
>
>----------
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Paul Herzmark
>Sent: Thursday, February 12, 2009 2:24 PM
>To:
<mailto:[hidden email]>[hidden email]
U

>Subject: CO2, humidity, temp sensors
>
>I have a cell dying problem in my microscope when I take pictures of
>them overnight or longer. I suspect the humidity is failing and I
>want to monitor that, CO2 and temperature.(It is not photodamage.
>Cells die in areas away from the objective.)
>
>Can anyone suggest a supplier of recording sensors that I can get
>next to the well slides on my stage?
>
>Thank you!
>
>Paul Herzmark
>Specialist
><mailto:[hidden email]>[hidden email]
>
>Department of Molecular and Cell Biology
>479 Life Science Addition
>University of California, Berkeley
>Berkeley, CA 94720-3200
>(510) 643-9603
>(510) 643-9500 fax
>

---------------------------------------------------------------------

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James Denegre

Re: Mineral Oil

Here is the protocol in use at The Jackson Laboratory for producing mineral oil for use in in vitro fertilizations and embryo culture:

1. In a sterile bottle add 1000mL of oil and 100mL tissue culture medium.
2. Stir oil/medium for two days at room temperature.
3. Let oil/medium settle for one day.
4. Decant medium the next day.
5. Filter the oil using a 0.8цL filter (Fisher #09-740-30), aliquot into 250 ml units.

The oil is stable for two months at room temp.

Use it to layer over cells in medium, to prevent evaporation. It will exchange gases, allowing CO2 to maintain the proper pH (we have verified this using a pH indicator, and moving dishes in and out of CO2 incubators.

James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
Sent: Thursday, February 26, 2009 11:46 AM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors and mu\ineral oil

Hi James, can you elaborate on your technique " equilibrate the mineral
oil with the medium"?
Thanks,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of James Denegre
Sent: Thursday, February 26, 2009 8:10 AM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors and mu\ineral oil

In our lab we equilibrate the mineral oil with the medium to prevent
uptake of components, and have successfully imaged under mineral oil for
five continuous days.

James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Beat Ludin
Sent: Wednesday, February 25, 2009 5:05 AM
To: [hidden email]
Subject: Re: CO2, humidity, temp sensors

Over time, the mineral oil overlay will basically void the medium of
all lipophilic components (vitamin E, for instance). This may or may
not be a problem, but you need to be aware of it and do the proper
controls.

If you want to check for evaporation, just weigh your sample before
and after the imaging.

Beat

At 23:23 12-02-2009, you wrote:

degrees
>2. Only the small space surrounding the cells is humidified and has
CO2.

Beat Ludin

Re: Mineral Oil

James -

Have you verified that this protocol is
sufficient to really equilibrate the mineral oil,
i.e. that the composition of medium is no longer
affect by contact with the equilibrated mineral
oil? Or are you just going by cell
survival/growth, which is certainly a good
indicator but doesn't necessarily mean that the
cells are affected in a non-lethal way?

Also, still need to consider the effects of the
mineral oil if you want to add compounds during
the experiment. BTW, I don't mean to question
that a mineral oil overlay is suitable for many
applications, but as always, it's good to be aware of possible side-effects.

Beat

At 23:17 26-02-2009, you wrote:

>Here is the protocol in use at The Jackson
>Laboratory for producing mineral oil for use in
>in vitro fertilizations and embryo culture:
>
>
>1. In a sterile bottle add 1000mL of oil and 100mL tissue culture medium.
>2. Stir oil/medium for two days at room temperature.
>3. Let oil/medium settle for one day.
>4. Decant medium the next day.
>5. Filter the oil using a 0.8ÃL filter
>(Fisher #09-740-30), aliquot into 250 ml units.
>
>The oil is stable for two months at room temp.
>
>Use it to layer over cells in medium, to prevent
>evaporation. It will exchange gases, allowing
>CO2 to maintain the proper pH (we have verified
>this using a pH indicator, and moving dishes in and out of CO2 incubators.
>
>
>
>James Denegre, Ph.D.
>Associate Research Scientist
>Manager, Light Microscopy
>The Jackson Laboratory
>Bar Harbor, ME 04609
>207.288.6321
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Armstrong, Brian
>Sent: Thursday, February 26, 2009 11:46 AM
>To: [hidden email]
>Subject: Re: CO2, humidity, temp sensors and mu\ineral oil
>
>Hi James, can you elaborate on your technique " equilibrate the mineral
>oil with the medium"?
>Thanks,
>
>Brian D Armstrong PhD
>Light Microscopy Core Manager
>Beckman Research Institute
>City of Hope
>Dept of Neuroscience
>1450 E Duarte Rd
>Duarte, CA 91010
>626-256-4673 x62872
>http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
>ing/Pages/default.aspx
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of James Denegre
>Sent: Thursday, February 26, 2009 8:10 AM
>To: [hidden email]
>Subject: Re: CO2, humidity, temp sensors and mu\ineral oil
>
>In our lab we equilibrate the mineral oil with the medium to prevent
>uptake of components, and have successfully imaged under mineral oil for
>five continuous days.
>
>James Denegre, Ph.D.
>Associate Research Scientist
>Manager, Light Microscopy
>The Jackson Laboratory
>Bar Harbor, ME 04609
>207.288.6321
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Beat Ludin
>Sent: Wednesday, February 25, 2009 5:05 AM
>To: [hidden email]
>Subject: Re: CO2, humidity, temp sensors
>
>Over time, the mineral oil overlay will basically void the medium of
>all lipophilic components (vitamin E, for instance). This may or may
>not be a problem, but you need to be aware of it and do the proper
>controls.
>
>If you want to check for evaporation, just weigh your sample before
>and after the imaging.
>
>Beat
>
>At 23:23 12-02-2009, you wrote:
> >My Guess is still evaporation of media is killing your
> >cells.. Overlay the cell media in each compartment with mineral
> >oil. It stops all evaporation but allows CO2 gas exchange to occur
> >to maintain pH.. I do this with all my chamber systems and it works
> >great. No need to humidify flowing air or gas.
> >
> >
> >Joe Goodhouse
> >Confocal Core Lab Manager
> >Dept. of Molecular Biology
> >Princeton University
> >609-258-5432
> >
> >Visit us at
> ><http://www.molbio/>http://www.molbio1.princeton.edu/facility/confocal/
> >
> >
> >
> >----------
> >From: Confocal Microscopy List
> >[mailto:[hidden email]] On Behalf Of Paul Herzmark
> >Sent: Thursday, February 12, 2009 4:36 PM
> >To: [hidden email]
> >Subject: Re: CO2, humidity, temp sensors
> >
> >
> >I knew I would not get away with out details!
> >
> >I have an Invivo (company) plexiglass incubator box around my
> >inverted microscope. 5% CO2 is piped inside to a small space (about
> >50 ml) around the multiwell-Labtech (company)-coverslip. The small
> >space is created by a glass plate with a frame around it that goes
> >over the coverslip.The CO2 passes into the small space via special
> >tubing.The tubing goes under incubator-warmed water where it is
> >supposed to pick up water vapor without bubbling.
> >
> >1. The whole incubator, with most of the microscope is warmed to 37
>degrees
> >2. Only the small space surrounding the cells is humidified and has
>CO2.
> >
> >
> >
> >
> >On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G.
> ><<mailto:[hidden email]>[hidden email]> wrote:
> >Paul,
> > Could you describe in detail your heating system. Do you
> > overlay your cells with mineral oil? This prevents evaporation
> > which will alter your salt concentration.
> >
> > I use a Recordable Temp sensor from Fisher Scientific. Cat No
> > 15-007-26. Inexpensive and works well.
> >
> >
> >Joe Goodhouse
> >Confocal Core Lab Manager
> >Dept. of Molecular Biology
> >Princeton University
> >609-258-5432
> >
> >Visit us at
> ><http://www.molbio/>http://www.molbio1.<http://princeton.edu/facility/c
>onfocal/>princeton.edu/facility/confocal/
> >
> >
> >
> >
> >----------
> >From: Confocal Microscopy List
> >[mailto:[hidden email]] On Behalf Of Paul Herzmark
> >Sent: Thursday, February 12, 2009 2:24 PM
> >To:
><mailto:[hidden email]>[hidden email]
>U
> >Subject: CO2, humidity, temp sensors
> >
> >I have a cell dying problem in my microscope when I take pictures of
> >them overnight or longer. I suspect the humidity is failing and I
> >want to monitor that, CO2 and temperature.(It is not photodamage.
> >Cells die in areas away from the objective.)
> >
> >Can anyone suggest a supplier of recording sensors that I can get
> >next to the well slides on my stage?
> >
> >Thank you!
> >
> >Paul Herzmark
> >Specialist
> ><mailto:[hidden email]>[hidden email]
> >
> >Department of Molecular and Cell Biology
> >479 Life Science Addition
> >University of California, Berkeley
> >Berkeley, CA 94720-3200
> >(510) 643-9603
> >(510) 643-9500 fax
> >
>
>
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James Denegre

Re: Mineral Oil

Beat,

Your points are well taken.

For us, the test of the mineral oil has been the survival of two-cell mouse embryos to blastocyst stage in culture. The need to equilibrate mineral oil was discovered empirically by John Eppig (O'Brien MJ, Wigglesworth K and Eppig JJ (1993) Mouse oocyte and embryo culture. In Chapin RE and Heudel J (eds) Methods in Reproductive Toxicology, Vol 3B. Academic Press, New York. pp. 128-141).

I am sure some cell lines may not benefit from the mineral oil overlay, and different media may not equilibrate the oil as well; but it is certainly inexpensive enough to give it a try.

James Denegre, Ph.D.
Associate Research Scientist
Manager, Light Microscopy
The Jackson Laboratory
Bar Harbor, ME 04609
207.288.6321

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Beat Ludin
Sent: Friday, February 27, 2009 2:23 AM
To: [hidden email]
Subject: Re: Mineral Oil

James -

Have you verified that this protocol is
sufficient to really equilibrate the mineral oil,
i.e. that the composition of medium is no longer
affect by contact with the equilibrated mineral
oil? Or are you just going by cell
survival/growth, which is certainly a good
indicator but doesn't necessarily mean that the
cells are affected in a non-lethal way?

Also, still need to consider the effects of the
mineral oil if you want to add compounds during
the experiment. BTW, I don't mean to question
that a mineral oil overlay is suitable for many
applications, but as always, it's good to be aware of possible side-effects.

Beat

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