Full environmental chamber vs stage top incubator

[Alex Paredez]

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Hello,
I am looking into a DeltaVision microscope.  The sales rep wants to sell me an
environmental chamber which controls temp and CO2.  I work on an intestinal
parasite (Giardia) that needs a hypoxic environment.  The sales rep would like me
to put a smaller chamber inside their larger cabinet and control atmospheric
conditions with a gas mixer.  He argues that by heating the entire stage and all
the objectives there will be less thermal drift and better temperature control.  I
don't know what the gas system is going to cost yet but list on the Environmental
chamber is around 11K.  I can get a complete stage top system from Pathology
Devices for around 5K more than the chamber.  I like the idea of not having a
giant box in the way when we are imaging fixed samples. Also I am not sure how
easy it is going to be to load a smaller chamber with samples when it is inside of a
larger cabinet.  I could use some advice.  I am also not set on the DV or any
particular environmental system.  That said my department has plenty of confocal
microscopes and I think deconvolution is probably the best solution for my small
cells.
Thanks,
Alex

[Pascal Weber]

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It's always better to use a big chamber, everythings are at the same temp.No
heating point (cold point) between objectiv  and specimen. And if you want to
do time-acquisition (overnight) it is more stable. Infact more expansive to.

[Straatman, Kees (Dr.)]

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Dear Alex,

It depends on the duration of your time series and if the DV system has a fast hardware autofocus system which finds the bottom of your sample. If you run long time series with minutes intervals between time points it will be very difficult to keep the samples in focus if you have no environmental chamber on the microscope and no hardware autofocus system. Because of changes in room temperature and air flow (you might have air-conditioning in the room?) the whole microscope is shrinking and expanding with as result loss of focus. Using an environmental chamber will keep the whole system at a constant temperature, including your sample as the "cold" environment is far away from it. I personally like to use an environmental chamber + an stage insert which we fill with a gas mixtures. When I worked with inserts in the past we always had thermal drift but hardware autofocus systems have improved a lot since then so that might solve the problem. Will be interesting to hear if somebody has positive experience with only an heated insert and hardware autofocus.

Good luck

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alex Paredez
Sent: 24 July 2012 22:35
To: [hidden email]
Subject: Full environmental chamber vs stage top incubator

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Hello,
I am looking into a DeltaVision microscope.  The sales rep wants to sell me an
environmental chamber which controls temp and CO2.  I work on an intestinal
parasite (Giardia) that needs a hypoxic environment.  The sales rep would like me
to put a smaller chamber inside their larger cabinet and control atmospheric
conditions with a gas mixer.  He argues that by heating the entire stage and all
the objectives there will be less thermal drift and better temperature control.  I
don't know what the gas system is going to cost yet but list on the Environmental
chamber is around 11K.  I can get a complete stage top system from Pathology
Devices for around 5K more than the chamber.  I like the idea of not having a
giant box in the way when we are imaging fixed samples. Also I am not sure how
easy it is going to be to load a smaller chamber with samples when it is inside of a
larger cabinet.  I could use some advice.  I am also not set on the DV or any
particular environmental system.  That said my department has plenty of confocal
microscopes and I think deconvolution is probably the best solution for my small
cells.
Thanks,
Alex

[WILDE Geraint]

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Dear Alex,

For long term imaging having a heated cage incubator is certainly the most stable option, especially if your cells are sensitive to changes in temperature. Hypoxic physiology can obviously be temperature sensitive depending on what you are interested in.  You then have a stage-top incubator for localised gaseous and humidity control.  Sometimes people even like to have this heated as well for added thermal stability.

If your purchase includes a microscope, then you can opt for one with a hardware autofocus, all 4 main companies have this, and that removes any issue of focal drift attributable to thermal changes.  This means that you could then potentially go with just a stage-top incubator, and drop the cage.  However, please note.  If using immersion objectives in combination with a stage top incubator (no cage), your objective will be at room-ish temperature, and so acts as a heat sink.  If you are concerned about the actual temperature your cells are at, then you need to be aware of this.  Stage-top incubators can be set to account for the offset, but I think you should still double check the actual temperature at the coverslip.  The added available component to compensate for this is an objective heater.  Also note that some immersion objectives are temperature corrected when investigating your purchase.

Finally.  If running hypoxic experiments, be sure of the oxygen tensions you want to drop down to.  I used to work in a lab where they found available solutions a challenge to drop below a certain value.  Sorry, cannot remember the finer details at this moment in time.  They did find a solution in the end.

Not sure this really answers any questions, but hopefully provides more information with which to make a decision.

I have run stage top incubators with hardware autofocus in overnight experiments and held focus, though was less certain about temperature stability on the cells themselves! Personally, I would go with a cage incubator.

Best

Geraint

Slightly commercial. Have you considered spinning disk confocal technology?

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alex Paredez
Sent: 24 July 2012 22:35
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] Full environmental chamber vs stage top incubator

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Hello,
I am looking into a DeltaVision microscope.  The sales rep wants to sell me an environmental chamber which controls temp and CO2.  I work on an intestinal parasite (Giardia) that needs a hypoxic environment.  The sales rep would like me to put a smaller chamber inside their larger cabinet and control atmospheric conditions with a gas mixer.  He argues that by heating the entire stage and all the objectives there will be less thermal drift and better temperature control.  I don't know what the gas system is going to cost yet but list on the Environmental chamber is around 11K.  I can get a complete stage top system from Pathology Devices for around 5K more than the chamber.  I like the idea of not having a giant box in the way when we are imaging fixed samples. Also I am not sure how easy it is going to be to load a smaller chamber with samples when it is inside of a larger cabinet.  I could use some advice.  I am also not set on the DV or any particular environmental system.  That said my department has plenty of confocal microscopes and I think deconvolution is probably the best solution for my small cells.
Thanks,
Alex


[http://www.andor.com/newsletter/footer/sig.jpg]<http://www.andor.com/newsletter/footer>

[Carol Norris]

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Hi Alex,

We have both a Pathology Devices stage top incubator (which we love!)  
for temp, humidity and CO2 control and an OKOLabs microscope  
enclosure on our confocal. We had problems with condensation on the  
objectives (both dry and oil) during time lapse experiments at 37C  
using just the stage top incubator.  In addition to being bad for  
imaging, it also caused the autofocus to fail.   Our policy now is  
that any 37C imaging must be done with both the microscope enclosure  
(set at 35C) and the stage top incubator (set at 37C).  I also feel  
better that people using oil objectives are now actually imaging at  
37C!  Although it's not too difficult to work with the OKOLabs box,  
we remove the front panel to make it easier for folks who are imaging  
at room temp.  The only other thing you have to keep an eye on is the  
room temp - the microscope enclosure pumps a fair amount of heat out  
into the room.

Regards,
Carol


On Jul 24, 2012, at 5:34 PM, Alex Paredez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
> I am looking into a DeltaVision microscope.  The sales rep wants to  
> sell me an
> environmental chamber which controls temp and CO2.  I work on an  
> intestinal
> parasite (Giardia) that needs a hypoxic environment.  The sales rep  
> would like me
> to put a smaller chamber inside their larger cabinet and control  
> atmospheric
> conditions with a gas mixer.  He argues that by heating the entire  
> stage and all
> the objectives there will be less thermal drift and better  
> temperature control.  I
> don't know what the gas system is going to cost yet but list on the  
> Environmental
> chamber is around 11K.  I can get a complete stage top system from  
> Pathology
> Devices for around 5K more than the chamber.  I like the idea of  
> not having a
> giant box in the way when we are imaging fixed samples. Also I am  
> not sure how
> easy it is going to be to load a smaller chamber with samples when  
> it is inside of a
> larger cabinet.  I could use some advice.  I am also not set on the  
> DV or any
> particular environmental system.  That said my department has  
> plenty of confocal
> microscopes and I think deconvolution is probably the best solution  
> for my small
> cells.
> Thanks,
> Alex

[Alex Paredez]

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Thank You,
   The advice here has been very helpful.  It is clear that going with a full cage
and smaller chamber is the best solution.  That said it is also more expensive
and less convenient than just a stage top solution.  Some commercial
responses have indicated that stage top incubators combined with autofocus
work well together.  It seems like it would be reasonable start with a stage top
incubator as long as I have autofocus.  If the results are unsatisfactory I could
add the full cage.  I should point out that my cells will grow at room temp but
they will die if exposed to normoxia.  So clearly getting the proper atmospheric
control is my priority.  

To Geraint,
Yes I considered a spinning disk setup.  I spent about two years of my life on a
Leica with a Yokogawa head during my PhD and got great results (plant
cytoskeleton).  I am still working on the cytoskeleton but now my cells are the
size of yeast and under 5um thick.  It is my understanding that a decon
microscope is going to have more efficient light gathering than a spinning disc.  
A systems with solid state illumination (no shutter) coupled with an SCMOS
camera should be very fast, have high sensitivity, and a large dynamic range.  
Perhaps I am buying into the hype, but I think I am going to get nicer images.  
I hope I didn’t just step into something….  Another factor is that the Biology
department at UW has several confocal microscopes but they don’t have any
decon scopes.  The chair was generous enough to provide funds for my own
scope and I think the rest of the department is relieved that I won’t be bringing
my parasites into the shared imaging facility : ).