XYZ drifts - giving advice and looking for advice

[Daniel Murphy]

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Hello,

Some advice and a plea for help on XYZ drifts!

We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
capture z-stacks that are typically 10-15 slices thick on a 40X Water with
2.5X optical zoom.  We also use a Warner instruments micro-incubation system
DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
dish cover to maintain a temp of 37C.

We have had persistent issues with drifts in X, Y and Z.  Initially the
problems were sever, with shifts of up to a few microns in all 3 directions.
 We have made progressive adjustments to our protocol with some big
improvements, but the problem is still significant.

First, we found that by surrounding the open area below the stage (where you
can access the objectives) with seran wrap, this provided a good way of
protecting the system from air currents and thermal influence from the
environment.  We also characterized the incubation system and found it works
better to run it without feedback at a constant voltage (the feedback
response was too slow because the incubation system is too much of a heat
sink).  We typically turn on the incubator and let it equilibrate for at
least 15min (with the dish inside as well, whenever possible).

XYZ drift still remains, however.  It seems to come and go.  For one
experiment it will be almost unnoticeable, but for another it will make the
data practically unusable.  Sometimes the drifts are just in one direction.
 Other times the stack shifts in Z up and down several times in one time
sequence.  It seems to be very irregular and so probably due to random
fluctuations in the environment.

XY shifts are not too bad, so long as the area of interest remains in view.
 There are several ways to adjust for the shift post-acquisition --- you
have more wiggle room since there are all those pixels in every direction.
Z shifts are the real issue that plagues us.  Any advice or ideas would be
warmly welcomed.

Daniel Murphy
Albert Einstein College of Medicine
Optical Imaging Manager, Cell and Molecular Neuroimaging Core
1410 Pelham Parkway South
Bronx, NY 10461
Ph 718-430-4027/8985

[Craig Brideau]

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Box in your scope; that is, build an enclosure around it.  The box will
block any air currents or other disturbances.  If temperature drift in the
room is more than 1 degree C you can also add temperature control to the box
to thermally isolate the microscope from the room.  Finally, what sort of
table is the microscope sitting on?

Craig


On Mon, Jan 31, 2011 at 9:49 AM, Daniel Murphy <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Some advice and a plea for help on XYZ drifts!
>
> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert
> 200M
> inverted scope.  Typically these go for 20min durations at 1frame/30sec.
>  We
> capture z-stacks that are typically 10-15 slices thick on a 40X Water with
> 2.5X optical zoom.  We also use a Warner instruments micro-incubation
> system
> DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
> dish cover to maintain a temp of 37C.
>
> We have had persistent issues with drifts in X, Y and Z.  Initially the
> problems were sever, with shifts of up to a few microns in all 3
> directions.
>  We have made progressive adjustments to our protocol with some big
> improvements, but the problem is still significant.
>
> First, we found that by surrounding the open area below the stage (where
> you
> can access the objectives) with seran wrap, this provided a good way of
> protecting the system from air currents and thermal influence from the
> environment.  We also characterized the incubation system and found it
> works
> better to run it without feedback at a constant voltage (the feedback
> response was too slow because the incubation system is too much of a heat
> sink).  We typically turn on the incubator and let it equilibrate for at
> least 15min (with the dish inside as well, whenever possible).
>
> XYZ drift still remains, however.  It seems to come and go.  For one
> experiment it will be almost unnoticeable, but for another it will make the
> data practically unusable.  Sometimes the drifts are just in one direction.
>  Other times the stack shifts in Z up and down several times in one time
> sequence.  It seems to be very irregular and so probably due to random
> fluctuations in the environment.
>
> XY shifts are not too bad, so long as the area of interest remains in view.
>  There are several ways to adjust for the shift post-acquisition --- you
> have more wiggle room since there are all those pixels in every direction.
> Z shifts are the real issue that plagues us.  Any advice or ideas would be
> warmly welcomed.
>
> Daniel Murphy
> Albert Einstein College of Medicine
> Optical Imaging Manager, Cell and Molecular Neuroimaging Core
> 1410 Pelham Parkway South
> Bronx, NY 10461
> Ph 718-430-4027/8985
>

[Craig Brideau]

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From a software side you can also just image volumes and then use image
registration to undo the drifts in the data.  This may not be optimal for
your application though.

Craig


On Mon, Jan 31, 2011 at 10:22 AM, Craig Brideau <[hidden email]>wrote:

> Box in your scope; that is, build an enclosure around it.  The box will
> block any air currents or other disturbances.  If temperature drift in the
> room is more than 1 degree C you can also add temperature control to the box
> to thermally isolate the microscope from the room.  Finally, what sort of
> table is the microscope sitting on?
>
> Craig
>
>
> On Mon, Jan 31, 2011 at 9:49 AM, Daniel Murphy <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> Some advice and a plea for help on XYZ drifts!
>>
>> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert
>> 200M
>> inverted scope.  Typically these go for 20min durations at 1frame/30sec.
>>  We
>> capture z-stacks that are typically 10-15 slices thick on a 40X Water with
>> 2.5X optical zoom.  We also use a Warner instruments micro-incubation
>> system
>> DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
>> dish cover to maintain a temp of 37C.
>>
>> We have had persistent issues with drifts in X, Y and Z.  Initially the
>> problems were sever, with shifts of up to a few microns in all 3
>> directions.
>>  We have made progressive adjustments to our protocol with some big
>> improvements, but the problem is still significant.
>>
>> First, we found that by surrounding the open area below the stage (where
>> you
>> can access the objectives) with seran wrap, this provided a good way of
>> protecting the system from air currents and thermal influence from the
>> environment.  We also characterized the incubation system and found it
>> works
>> better to run it without feedback at a constant voltage (the feedback
>> response was too slow because the incubation system is too much of a heat
>> sink).  We typically turn on the incubator and let it equilibrate for at
>> least 15min (with the dish inside as well, whenever possible).
>>
>> XYZ drift still remains, however.  It seems to come and go.  For one
>> experiment it will be almost unnoticeable, but for another it will make
>> the
>> data practically unusable.  Sometimes the drifts are just in one
>> direction.
>>  Other times the stack shifts in Z up and down several times in one time
>> sequence.  It seems to be very irregular and so probably due to random
>> fluctuations in the environment.
>>
>> XY shifts are not too bad, so long as the area of interest remains in
>> view.
>>  There are several ways to adjust for the shift post-acquisition --- you
>> have more wiggle room since there are all those pixels in every direction.
>> Z shifts are the real issue that plagues us.  Any advice or ideas would be
>> warmly welcomed.
>>
>> Daniel Murphy
>> Albert Einstein College of Medicine
>> Optical Imaging Manager, Cell and Molecular Neuroimaging Core
>> 1410 Pelham Parkway South
>> Bronx, NY 10461
>> Ph 718-430-4027/8985
>>
>
>

[Tim Feinstein-2]

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Hi Daniel,

A while back I solved a similar problem by eliminating vibrations on the table.  We moved the worst offenders (e.g., fans) off the table and putting vibration-absorbing rubber under everything that could not be moved.  Bits of carpet padding work well for that.  It would also be a good idea to find out how stable the climate control is in your room.  A mercury bulb and gas laser can really warm up a room with slow air handling.  Finally, have you checked whether your water miniscus is evaporating?  It could go pretty fast at 37 c. This is basic stuff that you most likely checked already; if not it might help.  

cheers,


Tim Feinstein


On Jan 31, 2011, at 11:49 AM, Daniel Murphy wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Some advice and a plea for help on XYZ drifts!
>
> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
> inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
> capture z-stacks that are typically 10-15 slices thick on a 40X Water with
> 2.5X optical zoom.  We also use a Warner instruments micro-incubation system
> DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
> dish cover to maintain a temp of 37C.
>
> We have had persistent issues with drifts in X, Y and Z.  Initially the
> problems were sever, with shifts of up to a few microns in all 3 directions.
> We have made progressive adjustments to our protocol with some big
> improvements, but the problem is still significant.
>
> First, we found that by surrounding the open area below the stage (where you
> can access the objectives) with seran wrap, this provided a good way of
> protecting the system from air currents and thermal influence from the
> environment.  We also characterized the incubation system and found it works
> better to run it without feedback at a constant voltage (the feedback
> response was too slow because the incubation system is too much of a heat
> sink).  We typically turn on the incubator and let it equilibrate for at
> least 15min (with the dish inside as well, whenever possible).
>
> XYZ drift still remains, however.  It seems to come and go.  For one
> experiment it will be almost unnoticeable, but for another it will make the
> data practically unusable.  Sometimes the drifts are just in one direction.
> Other times the stack shifts in Z up and down several times in one time
> sequence.  It seems to be very irregular and so probably due to random
> fluctuations in the environment.
>
> XY shifts are not too bad, so long as the area of interest remains in view.
> There are several ways to adjust for the shift post-acquisition --- you
> have more wiggle room since there are all those pixels in every direction.
> Z shifts are the real issue that plagues us.  Any advice or ideas would be
> warmly welcomed.
>
> Daniel Murphy
> Albert Einstein College of Medicine
> Optical Imaging Manager, Cell and Molecular Neuroimaging Core
> 1410 Pelham Parkway South
> Bronx, NY 10461
> Ph 718-430-4027/8985

[Dan Focht]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Daniel


I am also very familiar with this problem!
That is why I designed the Bioptechs Stable Z System.
Therefore, I urge you to visit our web site and look at our Stable Z System.
Here is the link.
http://www.bioptechs.com/Products/Stable_Z/Stable_Z.html

You are dealing with problems related to peripheral heat transfer.  
This type of heating is not nearly as efficient as our Delta T System but for those that want to use peripheral heating, it might as well be done right!
The design of the Stable Z will eliminate the Z axis drift and I think that if you eliminate the heat from getting to the microscope stage, another attribute of the Stable Z, it will also help if not eliminate the X,Y drift.

Read the description and cross-sectional drawing and see if you feel it is worth a try.

If you want to try one we make them available for a one week trial.  
That should be ample time to test it.

If this is your choice, please fill out our User Profile Questionnaire available from our home page.
This will give us the additional technical information we need to assure that we provide the most appropriate solution.

Dan


On Jan 31, 2011, at 11:49 AM, Daniel Murphy wrote:

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Hello,

Some advice and a plea for help on XYZ drifts!

We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
capture z-stacks that are typically 10-15 slices thick on a 40X Water with
2.5X optical zoom.  We also use a Warner instruments micro-incubation system
DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
dish cover to maintain a temp of 37C.

We have had persistent issues with drifts in X, Y and Z.  Initially the
problems were sever, with shifts of up to a few microns in all 3 directions.
We have made progressive adjustments to our protocol with some big
improvements, but the problem is still significant.

First, we found that by surrounding the open area below the stage (where you
can access the objectives) with seran wrap, this provided a good way of
protecting the system from air currents and thermal influence from the
environment.  We also characterized the incubation system and found it works
better to run it without feedback at a constant voltage (the feedback
response was too slow because the incubation system is too much of a heat
sink).  We typically turn on the incubator and let it equilibrate for at
least 15min (with the dish inside as well, whenever possible).

XYZ drift still remains, however.  It seems to come and go.  For one
experiment it will be almost unnoticeable, but for another it will make the
data practically unusable.  Sometimes the drifts are just in one direction.
Other times the stack shifts in Z up and down several times in one time
sequence.  It seems to be very irregular and so probably due to random
fluctuations in the environment.

XY shifts are not too bad, so long as the area of interest remains in view.
There are several ways to adjust for the shift post-acquisition --- you
have more wiggle room since there are all those pixels in every direction.
Z shifts are the real issue that plagues us.  Any advice or ideas would be
warmly welcomed.

Daniel Murphy
Albert Einstein College of Medicine
Optical Imaging Manager, Cell and Molecular Neuroimaging Core
1410 Pelham Parkway South
Bronx, NY 10461
Ph 718-430-4027/8985

Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler, PA 16002
www.bioptechs.com
P: [phone]
F: [phone]
[hidden email]

[Ramshesh, Venkat K]

Hi Dan,

  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were caused by the stage controller joystick. We had to replace the controller.  I am not sure if this applies to you but just a thought.
Further as Tim has already pointed out the evaporation of water meniscus also causes drift problems.

Best,
Venkat

Venkat Ramshesh, PhD
Bioengineer/Facility Manager
Cell and Molecular Imaging Core
Hollings Cancer Center and Center for Cell Death, Injury and Regeneration
Medical University of South Carolina
QE302
280 Calhoun Street, MSC 140
Charleston, SC 29425

Ph: 843-792-3530
Fax: [phone]
E-mail: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel Murphy
Sent: Monday, January 31, 2011 11:50 AM
To: [hidden email]
Subject: XYZ drifts - giving advice and looking for advice

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

Hello,

Some advice and a plea for help on XYZ drifts!

We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We capture z-stacks that are typically 10-15 slices thick on a 40X Water with 2.5X optical zoom.  We also use a Warner instruments micro-incubation system DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated dish cover to maintain a temp of 37C.

We have had persistent issues with drifts in X, Y and Z.  Initially the problems were sever, with shifts of up to a few microns in all 3 directions.
 We have made progressive adjustments to our protocol with some big improvements, but the problem is still significant.

First, we found that by surrounding the open area below the stage (where you can access the objectives) with seran wrap, this provided a good way of protecting the system from air currents and thermal influence from the environment.  We also characterized the incubation system and found it works better to run it without feedback at a constant voltage (the feedback response was too slow because the incubation system is too much of a heat sink).  We typically turn on the incubator and let it equilibrate for at least 15min (with the dish inside as well, whenever possible).

XYZ drift still remains, however.  It seems to come and go.  For one experiment it will be almost unnoticeable, but for another it will make the data practically unusable.  Sometimes the drifts are just in one direction.
 Other times the stack shifts in Z up and down several times in one time sequence.  It seems to be very irregular and so probably due to random fluctuations in the environment.

XY shifts are not too bad, so long as the area of interest remains in view.
 There are several ways to adjust for the shift post-acquisition --- you have more wiggle room since there are all those pixels in every direction.
Z shifts are the real issue that plagues us.  Any advice or ideas would be warmly welcomed.

Daniel Murphy
Albert Einstein College of Medicine
Optical Imaging Manager, Cell and Molecular Neuroimaging Core
1410 Pelham Parkway South
Bronx, NY 10461
Ph 718-430-4027/8985

[Danielle Crippen]

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

We've had similar problems and have found that we need to:

A. Box in the system.  We've made several enclosures here...homemade ones are much less expensive than commercial sources (we have both) and work just as well...sometimes better.  Write to me off list and we can talk more about them if you like.  

B. Isolate any fans in the box, so their vibration doesn't interfere with stage movement.

C. Control the temperature and air current in the room. Even with a boxed-in microscope, temperature fluctuations in the surrounding environment have definitely caused Z drift in our experience.  It has also paid off for us to control where the air is blowing in the room (ie. not directly down on the microscope)...we just used some cardboard to re-direct the air current.

Best of luck!!  This is a frustrating issue for sure!

_______________________________
Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Research on Aging
8001 Redwood Blvd
Novato, CA 94945
415-209-2046
TheBuck.org
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ramshesh, Venkat K
Sent: Monday, January 31, 2011 9:41 AM
To: [hidden email]
Subject: Re: XYZ drifts - giving advice and looking for advice

Hi Dan,

  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were caused by the stage controller joystick. We had to replace the controller.  I am not sure if this applies to you but just a thought.
Further as Tim has already pointed out the evaporation of water meniscus also causes drift problems.

Best,
Venkat

Venkat Ramshesh, PhD
Bioengineer/Facility Manager
Cell and Molecular Imaging Core
Hollings Cancer Center and Center for Cell Death, Injury and Regeneration Medical University of South Carolina
QE302
280 Calhoun Street, MSC 140
Charleston, SC 29425

Ph: 843-792-3530
Fax: [phone]
E-mail: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel Murphy
Sent: Monday, January 31, 2011 11:50 AM
To: [hidden email]
Subject: XYZ drifts - giving advice and looking for advice

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

Hello,

Some advice and a plea for help on XYZ drifts!

We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We capture z-stacks that are typically 10-15 slices thick on a 40X Water with 2.5X optical zoom.  We also use a Warner instruments micro-incubation system DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated dish cover to maintain a temp of 37C.

We have had persistent issues with drifts in X, Y and Z.  Initially the problems were sever, with shifts of up to a few microns in all 3 directions.
 We have made progressive adjustments to our protocol with some big improvements, but the problem is still significant.

First, we found that by surrounding the open area below the stage (where you can access the objectives) with seran wrap, this provided a good way of protecting the system from air currents and thermal influence from the environment.  We also characterized the incubation system and found it works better to run it without feedback at a constant voltage (the feedback response was too slow because the incubation system is too much of a heat sink).  We typically turn on the incubator and let it equilibrate for at least 15min (with the dish inside as well, whenever possible).

XYZ drift still remains, however.  It seems to come and go.  For one experiment it will be almost unnoticeable, but for another it will make the data practically unusable.  Sometimes the drifts are just in one direction.
 Other times the stack shifts in Z up and down several times in one time sequence.  It seems to be very irregular and so probably due to random fluctuations in the environment.

XY shifts are not too bad, so long as the area of interest remains in view.
 There are several ways to adjust for the shift post-acquisition --- you have more wiggle room since there are all those pixels in every direction.
Z shifts are the real issue that plagues us.  Any advice or ideas would be warmly welcomed.

Daniel Murphy
Albert Einstein College of Medicine
Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham Parkway South Bronx, NY 10461 Ph 718-430-4027/8985

[Ryan Geil]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Another alternative to this predicament is a complete stage-top incubation system, which is less cumbersome than a whole microscope enclosure.
Quorum Technologies offers complete systems that are equipped for temperature, CO2, humidity control and perfusion.
http://www.quorumtechnologies.com/Environment_Systems.html
That being said, it is critical to isolate mechanical vibration with the use of an anti-vibration platform or table and ensure that there are no air currents directed at the imaging system (as other posters have already pointed out).  It is a good idea to get any controllers off of the table-top if possible.
Cheers,
Ryan


On 2011-01-31, at 12:51 PM, Danielle Crippen wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We've had similar problems and have found that we need to:
>
> A. Box in the system.  We've made several enclosures here...homemade ones are much less expensive than commercial sources (we have both) and work just as well...sometimes better.  Write to me off list and we can talk more about them if you like.  
>
> B. Isolate any fans in the box, so their vibration doesn't interfere with stage movement.
>
> C. Control the temperature and air current in the room. Even with a boxed-in microscope, temperature fluctuations in the surrounding environment have definitely caused Z drift in our experience.  It has also paid off for us to control where the air is blowing in the room (ie. not directly down on the microscope)...we just used some cardboard to re-direct the air current.
>
> Best of luck!!  This is a frustrating issue for sure!
>
> _______________________________
> Danielle Crippen
> Morphology and Imaging Core Manager
> Buck Institute for Research on Aging
> 8001 Redwood Blvd
> Novato, CA 94945
> 415-209-2046
> TheBuck.org
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ramshesh, Venkat K
> Sent: Monday, January 31, 2011 9:41 AM
> To: [hidden email]
> Subject: Re: XYZ drifts - giving advice and looking for advice
>
> Hi Dan,
>
>  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were caused by the stage controller joystick. We had to replace the controller.  I am not sure if this applies to you but just a thought.
> Further as Tim has already pointed out the evaporation of water meniscus also causes drift problems.
>
> Best,
> Venkat
>
> Venkat Ramshesh, PhD
> Bioengineer/Facility Manager
> Cell and Molecular Imaging Core
> Hollings Cancer Center and Center for Cell Death, Injury and Regeneration Medical University of South Carolina
> QE302
> 280 Calhoun Street, MSC 140
> Charleston, SC 29425
>
> Ph: 843-792-3530
> Fax: [phone]
> E-mail: [hidden email]
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel Murphy
> Sent: Monday, January 31, 2011 11:50 AM
> To: [hidden email]
> Subject: XYZ drifts - giving advice and looking for advice
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello,
>
> Some advice and a plea for help on XYZ drifts!
>
> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We capture z-stacks that are typically 10-15 slices thick on a 40X Water with 2.5X optical zoom.  We also use a Warner instruments micro-incubation system DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated dish cover to maintain a temp of 37C.
>
> We have had persistent issues with drifts in X, Y and Z.  Initially the problems were sever, with shifts of up to a few microns in all 3 directions.
> We have made progressive adjustments to our protocol with some big improvements, but the problem is still significant.
>
> First, we found that by surrounding the open area below the stage (where you can access the objectives) with seran wrap, this provided a good way of protecting the system from air currents and thermal influence from the environment.  We also characterized the incubation system and found it works better to run it without feedback at a constant voltage (the feedback response was too slow because the incubation system is too much of a heat sink).  We typically turn on the incubator and let it equilibrate for at least 15min (with the dish inside as well, whenever possible).
>
> XYZ drift still remains, however.  It seems to come and go.  For one experiment it will be almost unnoticeable, but for another it will make the data practically unusable.  Sometimes the drifts are just in one direction.
> Other times the stack shifts in Z up and down several times in one time sequence.  It seems to be very irregular and so probably due to random fluctuations in the environment.
>
> XY shifts are not too bad, so long as the area of interest remains in view.
> There are several ways to adjust for the shift post-acquisition --- you have more wiggle room since there are all those pixels in every direction.
> Z shifts are the real issue that plagues us.  Any advice or ideas would be warmly welcomed.
>
> Daniel Murphy
> Albert Einstein College of Medicine
> Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham Parkway South Bronx, NY 10461 Ph 718-430-4027/8985

[Gitta Hamel]

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

Commercial response

Dear Daniel,

We understand that solving this issue with hardware may be a first
priority as this is where the problem lies and because of the severity
of the drift, especially in the axial direction as you indicated. You
mentioned already the possibility of post-acquisition correction. We
would like to point to the possibility of measuring and correcting drift
or movement (x-y-z translation and axial rotation) with our Huygens
software Object Stabilizer (more info:
http://www.svi.nl/ObjectStabilizer). Both the measurement and subsequent
stabilization can be done at 3D and sub-pixel level. Four different
stabilization methods can be applied for 3D time series, including cross
correlation and multi-object tracking. You're welcome to contact us for
a test version.

Best wishes,

Vincent Schoonderwoert
Gitta Hamel

*****************************************
Scientific Volume Imaging bv
Developers of the *HUYGENS* software
Laapersveld 63
1213 VB Hilversum
The Netherlands
[hidden email]
[hidden email]
phone: ++ 31 35 6 42 16 26
Fax: ++ 31 35 6 83 79 71
*****************************************

^SVI Customer support: mail us your questions [hidden email]
<mailto:[hidden email]>or find answers online in our Huygens
WIKI:http://support.svi.nl/





Ryan Geil wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Another alternative to this predicament is a complete stage-top incubation system, which is less cumbersome than a whole microscope enclosure.
> Quorum Technologies offers complete systems that are equipped for temperature, CO2, humidity control and perfusion.
> http://www.quorumtechnologies.com/Environment_Systems.html
> That being said, it is critical to isolate mechanical vibration with the use of an anti-vibration platform or table and ensure that there are no air currents directed at the imaging system (as other posters have already pointed out).  It is a good idea to get any controllers off of the table-top if possible.
> Cheers,
> Ryan
>
>
> On 2011-01-31, at 12:51 PM, Danielle Crippen wrote:
>
>  
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We've had similar problems and have found that we need to:
>>
>> A. Box in the system.  We've made several enclosures here...homemade ones are much less expensive than commercial sources (we have both) and work just as well...sometimes better.  Write to me off list and we can talk more about them if you like.  
>>
>> B. Isolate any fans in the box, so their vibration doesn't interfere with stage movement.
>>
>> C. Control the temperature and air current in the room. Even with a boxed-in microscope, temperature fluctuations in the surrounding environment have definitely caused Z drift in our experience.  It has also paid off for us to control where the air is blowing in the room (ie. not directly down on the microscope)...we just used some cardboard to re-direct the air current.
>>
>> Best of luck!!  This is a frustrating issue for sure!
>>
>> _______________________________
>> Danielle Crippen
>> Morphology and Imaging Core Manager
>> Buck Institute for Research on Aging
>> 8001 Redwood Blvd
>> Novato, CA 94945
>> 415-209-2046
>> TheBuck.org
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ramshesh, Venkat K
>> Sent: Monday, January 31, 2011 9:41 AM
>> To: [hidden email]
>> Subject: Re: XYZ drifts - giving advice and looking for advice
>>
>> Hi Dan,
>>
>>  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were caused by the stage controller joystick. We had to replace the controller.  I am not sure if this applies to you but just a thought.
>> Further as Tim has already pointed out the evaporation of water meniscus also causes drift problems.
>>
>> Best,
>> Venkat
>>
>> Venkat Ramshesh, PhD
>> Bioengineer/Facility Manager
>> Cell and Molecular Imaging Core
>> Hollings Cancer Center and Center for Cell Death, Injury and Regeneration Medical University of South Carolina
>> QE302
>> 280 Calhoun Street, MSC 140
>> Charleston, SC 29425
>>
>> Ph: 843-792-3530
>> Fax: [phone]
>> E-mail: [hidden email]
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel Murphy
>> Sent: Monday, January 31, 2011 11:50 AM
>> To: [hidden email]
>> Subject: XYZ drifts - giving advice and looking for advice
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello,
>>
>> Some advice and a plea for help on XYZ drifts!
>>
>> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We capture z-stacks that are typically 10-15 slices thick on a 40X Water with 2.5X optical zoom.  We also use a Warner instruments micro-incubation system DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated dish cover to maintain a temp of 37C.
>>
>> We have had persistent issues with drifts in X, Y and Z.  Initially the problems were sever, with shifts of up to a few microns in all 3 directions.
>> We have made progressive adjustments to our protocol with some big improvements, but the problem is still significant.
>>
>> First, we found that by surrounding the open area below the stage (where you can access the objectives) with seran wrap, this provided a good way of protecting the system from air currents and thermal influence from the environment.  We also characterized the incubation system and found it works better to run it without feedback at a constant voltage (the feedback response was too slow because the incubation system is too much of a heat sink).  We typically turn on the incubator and let it equilibrate for at least 15min (with the dish inside as well, whenever possible).
>>
>> XYZ drift still remains, however.  It seems to come and go.  For one experiment it will be almost unnoticeable, but for another it will make the data practically unusable.  Sometimes the drifts are just in one direction.
>> Other times the stack shifts in Z up and down several times in one time sequence.  It seems to be very irregular and so probably due to random fluctuations in the environment.
>>
>> XY shifts are not too bad, so long as the area of interest remains in view.
>> There are several ways to adjust for the shift post-acquisition --- you have more wiggle room since there are all those pixels in every direction.
>> Z shifts are the real issue that plagues us.  Any advice or ideas would be warmly welcomed.
>>
>> Daniel Murphy
>> Albert Einstein College of Medicine
>> Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham Parkway South Bronx, NY 10461 Ph 718-430-4027/8985
>>    
>
>  

[Keith Morris]

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

Hi Daniel,

Danielle is quite right - to reduce Z focus drift significantly a Zeiss Live
cell large XL3 incubator is probably required on your Axiovert 200 to close
in the entire stage, nosepiece and objectives, and even then the stage has
be on for at least 2 hours before the time-lapse and wait 10 minutes before
starting the timelapse after opening the incubator doors [also a bit of
blu-tack can help to hold the Petri-dish in one place, along with any
'mounting screw' adjustments on the heatable Labtek insert P - although you
would probably just put your Warner unit inside the XL3 incubator]. When you
switch on the XL3 incubator and the temperature of the objectives/stage
rises from 22oC to 37oC you get a focus drift of 30um or more [microscopes
are still built largely with metal rather than composites and so suffer from
thermal expansion effects]. We used to use the bijou Zeiss on-stage PeCon
incubator-S units similar to yours, and the Z drift was very bad for
time-lapse [fine for viewing live cells and capturing images as you could
refocus all the time, but not so good for time lapse, where we had to take a
z-stack at each time-point to track the focus point]. Once the large
incubators were fitted things improved considerably [particularly if the
microscope is on an anti-vibration air-table].

Unfortunately a Zeiss XL3 incubator system sets you back £15k+ with the
associated PeCon electronics, precise %CO2 control and heaters. A cheaper
option would be a Zeiss objective heater that fits on the objective under
the stage to minimize thermal expansion effects there [but that’s still
thousands of pounds including TempController, and probably nowhere near as
effective as the XL3 incubator, I've never used one]. Even cheaper still
[and even less effective but better than nothing] is to use the foam
objective insulators Zeiss manufacture specifically for the purpose to put
around each objective. Worse still, with an oil objective and Mattek type
culture dish the heat is conducted away from the culture vessel into the
cold objective. Building you own XL3 type incubator enclosure might be a bit
cheaper if you have a workshop, but you also need the air heater control
sorted out and our in-house incubators were still £5k each for the Perspex
enclosure alone [as our workshop charged an hourly rate]. You also might
need some sort of humidity control for longer timelapses [we get our water
vapour in via the 5% CO2 feed] or some use the PeCon FoilCovers to reduce
media evaporation with culture dishes.

See the XL3 incubator
http://www.well.ox.ac.uk/live-cell-imaging
Zeiss/PeCon incubator options
http://www.pecon.biz/?page_id=55
Foil cover
http://www.pecon.biz/?page_id=245

Antivibration feedback also helps if its just the rubber door-stop feet on
the microscope base and no air-table - but these [Z motorized] focus issues
are mainly thermal [air conditioning and lack of a regulated heated
enclosure surround the entire objective/stage area] - the CRUK in London go
as far as enclosing the entire microscope within an incubator enclosure. For
cheap antivibration control reinforce the bench and add a large heavy slab
of granite [or sealed in concrete if poor] under the microscope and rest the
slab on anti-vibrational pads. It worked for us [we had a workshop to do the
mods]. Vibration can cause XY movement of the manual stage and sample
wobbling during capture. Once these slabs were in place you could tap the
worktop without the specimen dancing on the screen.

Also try and get the room air-conditioning adjusted - the standard office
rated lab air-con is simply balanced to 22oC [knowledge of the Victorian
thermostat now being lost]. You can upgrade the aircon wall temperature
sensor electronics to make it more stable/accurate [cost us £1,000],
assuming you have one, and possibly raise the room balance temperature to
24oC. Simple cardboard deflectors, to make sure the cool air from the aircon
ducting doesnt blow on the microscope, can also be highly effective.

Good luck with the issue, Regards

Keith

No commercial interest

---------------------------------------------------------------------------
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:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Danielle Crippen
Sent: 31 January 2011 17:51
To: [hidden email]
Subject: Re: XYZ drifts - giving advice and looking for advice

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

We've had similar problems and have found that we need to:

A. Box in the system.  We've made several enclosures here...homemade ones
are much less expensive than commercial sources (we have both) and work just
as well...sometimes better.  Write to me off list and we can talk more about
them if you like.  

B. Isolate any fans in the box, so their vibration doesn't interfere with
stage movement.

C. Control the temperature and air current in the room. Even with a boxed-in
microscope, temperature fluctuations in the surrounding environment have
definitely caused Z drift in our experience.  It has also paid off for us to
control where the air is blowing in the room (ie. not directly down on the
microscope)...we just used some cardboard to re-direct the air current.

Best of luck!!  This is a frustrating issue for sure!

_______________________________
Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Research on Aging
8001 Redwood Blvd
Novato, CA 94945
415-209-2046
TheBuck.org
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Ramshesh, Venkat K
Sent: Monday, January 31, 2011 9:41 AM
To: [hidden email]
Subject: Re: XYZ drifts - giving advice and looking for advice

Hi Dan,

  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were
caused by the stage controller joystick. We had to replace the controller.
I am not sure if this applies to you but just a thought.
Further as Tim has already pointed out the evaporation of water meniscus
also causes drift problems.

Best,
Venkat

Venkat Ramshesh, PhD
Bioengineer/Facility Manager
Cell and Molecular Imaging Core
Hollings Cancer Center and Center for Cell Death, Injury and Regeneration
Medical University of South Carolina
QE302
280 Calhoun Street, MSC 140
Charleston, SC 29425

Ph: 843-792-3530
Fax: [phone]
E-mail: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Daniel Murphy
Sent: Monday, January 31, 2011 11:50 AM
To: [hidden email]
Subject: XYZ drifts - giving advice and looking for advice

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

Hello,

Some advice and a plea for help on XYZ drifts!

We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
capture z-stacks that are typically 10-15 slices thick on a 40X Water with
2.5X optical zoom.  We also use a Warner instruments micro-incubation system
DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
dish cover to maintain a temp of 37C.

We have had persistent issues with drifts in X, Y and Z.  Initially the
problems were sever, with shifts of up to a few microns in all 3 directions.
 We have made progressive adjustments to our protocol with some big
improvements, but the problem is still significant.

First, we found that by surrounding the open area below the stage (where you
can access the objectives) with seran wrap, this provided a good way of
protecting the system from air currents and thermal influence from the
environment.  We also characterized the incubation system and found it works
better to run it without feedback at a constant voltage (the feedback
response was too slow because the incubation system is too much of a heat
sink).  We typically turn on the incubator and let it equilibrate for at
least 15min (with the dish inside as well, whenever possible).

XYZ drift still remains, however.  It seems to come and go.  For one
experiment it will be almost unnoticeable, but for another it will make the
data practically unusable.  Sometimes the drifts are just in one direction.
 Other times the stack shifts in Z up and down several times in one time
sequence.  It seems to be very irregular and so probably due to random
fluctuations in the environment.

XY shifts are not too bad, so long as the area of interest remains in view.
 There are several ways to adjust for the shift post-acquisition --- you
have more wiggle room since there are all those pixels in every direction.
Z shifts are the real issue that plagues us.  Any advice or ideas would be
warmly welcomed.

Daniel Murphy
Albert Einstein College of Medicine
Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham
Parkway South Bronx, NY 10461 Ph 718-430-4027/8985

[Daniel Murphy]

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

The scope is sitting on a vibration isolated table stabilized via Nitrogen.

[Glen MacDonald-2]

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

Does it have a motorized XY stage?  They are notorious for adding thermal drift in all dimensions.  The only solution is an enclosure.  You might not need to fully heat the microscope enclosure, it might be enough to block ambient air currents and temp fluctuations. A constant temp control less than 37 might be the next step, with final heating of live mammalian specimens left to a stage incubator.  In the latter case, a commercial incubator is reasonably priced.  1 inch thick styrofoam construction insulation is USD$50 per 4 ft x 4 ft sheet.  Duct tape will not stick, use the heavy aluminum tape with peel-off backing or aliphatic wood glue.  My enclosure lasted several years and I'm now rebuilding it with an aluminum frame.  

Regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]








On Feb 1, 2011, at 8:35 AM, Daniel Murphy wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The scope is sitting on a vibration isolated table stabilized via Nitrogen.

[Daniel Murphy]

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

All:

Thanks to all for the helpful and thorough advice.  From your comments, we
definitely have some ideas to work through to nail down the problem-causing
element(s).  From your comments and after speaking with a tech from Zeiss,
apparently this is an issue that previous users have also had with long
time-lapse imaging of incubated samples on the Axiovert 200M microscope.  He
had done previous studies and found that with a similar setup, except with
40x dry objective (instead of 40x water), there is a Z drift of about 13um
in the first 6 hours of turning the system on, at which point the Z drift is
more or less gone.  So for really stable time-lapse work, the system needs
to be on for at least 6hrs!  This is without an incubator enclosing the
entire scope like the Zeiss XL --- perhaps this would reduce the
equilibration time (any thoughts/experience?).

What solutions are there to the problem of the water evaporating off the
objective?  We use a water substitute with the same n but it is a little
thicker, and so less evaporation.

Thanks!

Dan

On Tue, 1 Feb 2011 13:51:12 -0000, Keith Morris <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Daniel,
>
>Danielle is quite right - to reduce Z focus drift significantly a Zeiss Live
>cell large XL3 incubator is probably required on your Axiovert 200 to close
>in the entire stage, nosepiece and objectives, and even then the stage has
>be on for at least 2 hours before the time-lapse and wait 10 minutes before
>starting the timelapse after opening the incubator doors [also a bit of
>blu-tack can help to hold the Petri-dish in one place, along with any
>'mounting screw' adjustments on the heatable Labtek insert P - although you
>would probably just put your Warner unit inside the XL3 incubator]. When you
>switch on the XL3 incubator and the temperature of the objectives/stage
>rises from 22oC to 37oC you get a focus drift of 30um or more [microscopes
>are still built largely with metal rather than composites and so suffer from
>thermal expansion effects]. We used to use the bijou Zeiss on-stage PeCon
>incubator-S units similar to yours, and the Z drift was very bad for
>time-lapse [fine for viewing live cells and capturing images as you could
>refocus all the time, but not so good for time lapse, where we had to take a
>z-stack at each time-point to track the focus point]. Once the large
>incubators were fitted things improved considerably [particularly if the
>microscope is on an anti-vibration air-table].
>
>Unfortunately a Zeiss XL3 incubator system sets you back �15k+ with the
>associated PeCon electronics, precise %CO2 control and heaters. A cheaper
>option would be a Zeiss objective heater that fits on the objective under
>the stage to minimize thermal expansion effects there [but that�s still
>thousands of pounds including TempController, and probably nowhere near as
>effective as the XL3 incubator, I've never used one]. Even cheaper still
>[and even less effective but better than nothing] is to use the foam
>objective insulators Zeiss manufacture specifically for the purpose to put
>around each objective. Worse still, with an oil objective and Mattek type
>culture dish the heat is conducted away from the culture vessel into the
>cold objective. Building you own XL3 type incubator enclosure might be a bit
>cheaper if you have a workshop, but you also need the air heater control
>sorted out and our in-house incubators were still �5k each for the Perspex
>enclosure alone [as our workshop charged an hourly rate]. You also might
>need some sort of humidity control for longer timelapses [we get our water
>vapour in via the 5% CO2 feed] or some use the PeCon FoilCovers to reduce
>media evaporation with culture dishes.
>
>See the XL3 incubator
>http://www.well.ox.ac.uk/live-cell-imaging
>Zeiss/PeCon incubator options
>http://www.pecon.biz/?page_id=55
>Foil cover
>http://www.pecon.biz/?page_id=245
>
>Antivibration feedback also helps if its just the rubber door-stop feet on
>the microscope base and no air-table - but these [Z motorized] focus issues
>are mainly thermal [air conditioning and lack of a regulated heated
>enclosure surround the entire objective/stage area] - the CRUK in London go
>as far as enclosing the entire microscope within an incubator enclosure. For
>cheap antivibration control reinforce the bench and add a large heavy slab
>of granite [or sealed in concrete if poor] under the microscope and rest the
>slab on anti-vibrational pads. It worked for us [we had a workshop to do the
>mods]. Vibration can cause XY movement of the manual stage and sample
>wobbling during capture. Once these slabs were in place you could tap the
>worktop without the specimen dancing on the screen.
>
>Also try and get the room air-conditioning adjusted - the standard office
>rated lab air-con is simply balanced to 22oC [knowledge of the Victorian
>thermostat now being lost]. You can upgrade the aircon wall temperature
>sensor electronics to make it more stable/accurate [cost us �1,000],
>assuming you have one, and possibly raise the room balance temperature to
>24oC. Simple cardboard deflectors, to make sure the cool air from the aircon
>ducting doesnt blow on the microscope, can also be highly effective.
>
>Good luck with the issue, Regards
>
>Keith
>
>No commercial interest
>
>---------------------------------------------------------------------------
>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:� [phone]
>Email:� [hidden email]
>Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Danielle Crippen
>Sent: 31 January 2011 17:51
>To: [hidden email]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>We've had similar problems and have found that we need to:
>
>A. Box in the system.  We've made several enclosures here...homemade ones
>are much less expensive than commercial sources (we have both) and work just
>as well...sometimes better.  Write to me off list and we can talk more about
>them if you like.  
>
>B. Isolate any fans in the box, so their vibration doesn't interfere with
>stage movement.
>
>C. Control the temperature and air current in the room. Even with a boxed-in
>microscope, temperature fluctuations in the surrounding environment have
>definitely caused Z drift in our experience.  It has also paid off for us to
>control where the air is blowing in the room (ie. not directly down on the
>microscope)...we just used some cardboard to re-direct the air current.
>
>Best of luck!!  This is a frustrating issue for sure!
>
>_______________________________
>Danielle Crippen
>Morphology and Imaging Core Manager
>Buck Institute for Research on Aging
>8001 Redwood Blvd
>Novato, CA 94945
>415-209-2046
>TheBuck.org
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Ramshesh, Venkat K
>Sent: Monday, January 31, 2011 9:41 AM
>To: [hidden email]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
>Hi Dan,
>
>  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were
>caused by the stage controller joystick. We had to replace the controller.
>I am not sure if this applies to you but just a thought.
>Further as Tim has already pointed out the evaporation of water meniscus
>also causes drift problems.
>
>Best,
>Venkat
>
>Venkat Ramshesh, PhD
>Bioengineer/Facility Manager
>Cell and Molecular Imaging Core
>Hollings Cancer Center and Center for Cell Death, Injury and Regeneration
>Medical University of South Carolina
>QE302
>280 Calhoun Street, MSC 140
>Charleston, SC 29425
>
>Ph: 843-792-3530
>Fax: [phone]
>E-mail: [hidden email]
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Daniel Murphy
>Sent: Monday, January 31, 2011 11:50 AM
>To: [hidden email]
>Subject: XYZ drifts - giving advice and looking for advice
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello,
>
>Some advice and a plea for help on XYZ drifts!
>
>We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
>inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
>capture z-stacks that are typically 10-15 slices thick on a 40X Water with
>2.5X optical zoom.  We also use a Warner instruments micro-incubation system
>DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
>dish cover to maintain a temp of 37C.
>
>We have had persistent issues with drifts in X, Y and Z.  Initially the
>problems were sever, with shifts of up to a few microns in all 3 directions.
> We have made progressive adjustments to our protocol with some big
>improvements, but the problem is still significant.
>
>First, we found that by surrounding the open area below the stage (where you
>can access the objectives) with seran wrap, this provided a good way of
>protecting the system from air currents and thermal influence from the
>environment.  We also characterized the incubation system and found it works
>better to run it without feedback at a constant voltage (the feedback
>response was too slow because the incubation system is too much of a heat
>sink).  We typically turn on the incubator and let it equilibrate for at
>least 15min (with the dish inside as well, whenever possible).
>
>XYZ drift still remains, however.  It seems to come and go.  For one
>experiment it will be almost unnoticeable, but for another it will make the
>data practically unusable.  Sometimes the drifts are just in one direction.
> Other times the stack shifts in Z up and down several times in one time
>sequence.  It seems to be very irregular and so probably due to random
>fluctuations in the environment.
>
>XY shifts are not too bad, so long as the area of interest remains in view.
> There are several ways to adjust for the shift post-acquisition --- you
>have more wiggle room since there are all those pixels in every direction.
>Z shifts are the real issue that plagues us.  Any advice or ideas would be
>warmly welcomed.
>
>Daniel Murphy
>Albert Einstein College of Medicine
>Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham
>Parkway South Bronx, NY 10461 Ph 718-430-4027/8985

[Daniel Murphy]

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

Craig,

What exactly is image registration?  

At one point we thought of just oversampling in Z and then adjusting the
image digitally post-acquisition, depending on the Z-shifts.  This is
impractical for our application because we are looking at very small,
low-signal vesicles moving through the cell.  So oversampling would cause
issues with bleaching and we would lose too much time resolution.

If image registration is something else, then can you explain?

Thanks,

Dan

On Mon, 31 Jan 2011 10:23:06 -0700, Craig Brideau <[hidden email]>
wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>From a software side you can also just image volumes and then use image
>registration to undo the drifts in the data.  This may not be optimal for
>your application though.
>
>Craig
>
>
>On Mon, Jan 31, 2011 at 10:22 AM, Craig Brideau <[hidden email]>wrote:
>
>> Box in your scope; that is, build an enclosure around it.  The box will
>> block any air currents or other disturbances.  If temperature drift in the
>> room is more than 1 degree C you can also add temperature control to the box
>> to thermally isolate the microscope from the room.  Finally, what sort of
>> table is the microscope sitting on?
>>
>> Craig
>>
>>
>> On Mon, Jan 31, 2011 at 9:49 AM, Daniel Murphy <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hello,
>>>
>>> Some advice and a plea for help on XYZ drifts!
>>>
>>> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert
>>> 200M
>>> inverted scope.  Typically these go for 20min durations at 1frame/30sec.
>>>  We
>>> capture z-stacks that are typically 10-15 slices thick on a 40X Water with
>>> 2.5X optical zoom.  We also use a Warner instruments micro-incubation
>>> system
>>> DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
>>> dish cover to maintain a temp of 37C.
>>>
>>> We have had persistent issues with drifts in X, Y and Z.  Initially the
>>> problems were sever, with shifts of up to a few microns in all 3
>>> directions.
>>>  We have made progressive adjustments to our protocol with some big
>>> improvements, but the problem is still significant.
>>>
>>> First, we found that by surrounding the open area below the stage (where
>>> you
>>> can access the objectives) with seran wrap, this provided a good way of
>>> protecting the system from air currents and thermal influence from the
>>> environment.  We also characterized the incubation system and found it
>>> works
>>> better to run it without feedback at a constant voltage (the feedback
>>> response was too slow because the incubation system is too much of a heat
>>> sink).  We typically turn on the incubator and let it equilibrate for at
>>> least 15min (with the dish inside as well, whenever possible).
>>>
>>> XYZ drift still remains, however.  It seems to come and go.  For one
>>> experiment it will be almost unnoticeable, but for another it will make
>>> the
>>> data practically unusable.  Sometimes the drifts are just in one
>>> direction.
>>>  Other times the stack shifts in Z up and down several times in one time
>>> sequence.  It seems to be very irregular and so probably due to random
>>> fluctuations in the environment.
>>>
>>> XY shifts are not too bad, so long as the area of interest remains in
>>> view.
>>>  There are several ways to adjust for the shift post-acquisition --- you
>>> have more wiggle room since there are all those pixels in every direction.
>>> Z shifts are the real issue that plagues us.  Any advice or ideas would be
>>> warmly welcomed.
>>>
>>> Daniel Murphy
>>> Albert Einstein College of Medicine
>>> Optical Imaging Manager, Cell and Molecular Neuroimaging Core
>>> 1410 Pelham Parkway South
>>> Bronx, NY 10461
>>> Ph 718-430-4027/8985
>>>
>>
>>

[Armstrong, Brian]

Hi Dan, we have the same instrument and objective and have the same problem (Zeiss LSM510 inverted and 40x/1.2W). To get around this problem we purchased a new scope with the entire scope enclosed in the incubator, just as described by Kieth Morris, and I agree completely with his advice. The large XL incubator almost completely removes the focus drift problem, if the incubator is turned on around 2 hours prior to image acquisition. I was surprised and impressed by the difference the large incubator makes. As already described; the cost of the incubator system is really in the components and not the box so we purchased the Pecon acrylic box which is really quite nice to have as it is see-through.
 
Back to your immediate problem, as you already point out, if you turn on the small Pecon incubator at least two hours prior to imaging some of the focus drift problem is alleviated. We have tried the objective heater and the foam objective warmer, they are little help in my opinion. Something that did seem to help was the use of Zeiss Immersol W immersion oil for Water objectives.
Also, if you have the Zeiss multi-time macro (or MDA in AxioVision) you can pause your acquisition and refocus without stopping the experiment.

Hope this is helpful.
Cheers,

Brian D Armstrong PhD
Assistant Research Professor
Light Microscopy Core
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-Imaging/Pages/default.aspx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Daniel Murphy
Sent: Wednesday, February 02, 2011 9:02 AM
To: [hidden email]
Subject: Re: XYZ drifts - giving advice and looking for advice

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

All:

Thanks to all for the helpful and thorough advice.  From your comments, we
definitely have some ideas to work through to nail down the problem-causing
element(s).  From your comments and after speaking with a tech from Zeiss,
apparently this is an issue that previous users have also had with long
time-lapse imaging of incubated samples on the Axiovert 200M microscope.  He
had done previous studies and found that with a similar setup, except with
40x dry objective (instead of 40x water), there is a Z drift of about 13um
in the first 6 hours of turning the system on, at which point the Z drift is
more or less gone.  So for really stable time-lapse work, the system needs
to be on for at least 6hrs!  This is without an incubator enclosing the
entire scope like the Zeiss XL --- perhaps this would reduce the
equilibration time (any thoughts/experience?).

What solutions are there to the problem of the water evaporating off the
objective?  We use a water substitute with the same n but it is a little
thicker, and so less evaporation.

Thanks!

Dan

On Tue, 1 Feb 2011 13:51:12 -0000, Keith Morris <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Daniel,
>
>Danielle is quite right - to reduce Z focus drift significantly a Zeiss Live
>cell large XL3 incubator is probably required on your Axiovert 200 to close
>in the entire stage, nosepiece and objectives, and even then the stage has
>be on for at least 2 hours before the time-lapse and wait 10 minutes before
>starting the timelapse after opening the incubator doors [also a bit of
>blu-tack can help to hold the Petri-dish in one place, along with any
>'mounting screw' adjustments on the heatable Labtek insert P - although you
>would probably just put your Warner unit inside the XL3 incubator]. When you
>switch on the XL3 incubator and the temperature of the objectives/stage
>rises from 22oC to 37oC you get a focus drift of 30um or more [microscopes
>are still built largely with metal rather than composites and so suffer from
>thermal expansion effects]. We used to use the bijou Zeiss on-stage PeCon
>incubator-S units similar to yours, and the Z drift was very bad for
>time-lapse [fine for viewing live cells and capturing images as you could
>refocus all the time, but not so good for time lapse, where we had to take a
>z-stack at each time-point to track the focus point]. Once the large
>incubators were fitted things improved considerably [particularly if the
>microscope is on an anti-vibration air-table].
>
>Unfortunately a Zeiss XL3 incubator system sets you back �15k+ with the
>associated PeCon electronics, precise %CO2 control and heaters. A cheaper
>option would be a Zeiss objective heater that fits on the objective under
>the stage to minimize thermal expansion effects there [but that�s still
>thousands of pounds including TempController, and probably nowhere near as
>effective as the XL3 incubator, I've never used one]. Even cheaper still
>[and even less effective but better than nothing] is to use the foam
>objective insulators Zeiss manufacture specifically for the purpose to put
>around each objective. Worse still, with an oil objective and Mattek type
>culture dish the heat is conducted away from the culture vessel into the
>cold objective. Building you own XL3 type incubator enclosure might be a bit
>cheaper if you have a workshop, but you also need the air heater control
>sorted out and our in-house incubators were still �5k each for the Perspex
>enclosure alone [as our workshop charged an hourly rate]. You also might
>need some sort of humidity control for longer timelapses [we get our water
>vapour in via the 5% CO2 feed] or some use the PeCon FoilCovers to reduce
>media evaporation with culture dishes.
>
>See the XL3 incubator
>http://www.well.ox.ac.uk/live-cell-imaging
>Zeiss/PeCon incubator options
>http://www.pecon.biz/?page_id=55
>Foil cover
>http://www.pecon.biz/?page_id=245
>
>Antivibration feedback also helps if its just the rubber door-stop feet on
>the microscope base and no air-table - but these [Z motorized] focus issues
>are mainly thermal [air conditioning and lack of a regulated heated
>enclosure surround the entire objective/stage area] - the CRUK in London go
>as far as enclosing the entire microscope within an incubator enclosure. For
>cheap antivibration control reinforce the bench and add a large heavy slab
>of granite [or sealed in concrete if poor] under the microscope and rest the
>slab on anti-vibrational pads. It worked for us [we had a workshop to do the
>mods]. Vibration can cause XY movement of the manual stage and sample
>wobbling during capture. Once these slabs were in place you could tap the
>worktop without the specimen dancing on the screen.
>
>Also try and get the room air-conditioning adjusted - the standard office
>rated lab air-con is simply balanced to 22oC [knowledge of the Victorian
>thermostat now being lost]. You can upgrade the aircon wall temperature
>sensor electronics to make it more stable/accurate [cost us �1,000],
>assuming you have one, and possibly raise the room balance temperature to
>24oC. Simple cardboard deflectors, to make sure the cool air from the aircon
>ducting doesnt blow on the microscope, can also be highly effective.
>
>Good luck with the issue, Regards
>
>Keith
>
>No commercial interest
>
>---------------------------------------------------------------------------
>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:� [phone]
>Email:� [hidden email]
>Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Danielle Crippen
>Sent: 31 January 2011 17:51
>To: [hidden email]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>We've had similar problems and have found that we need to:
>
>A. Box in the system.  We've made several enclosures here...homemade ones
>are much less expensive than commercial sources (we have both) and work just
>as well...sometimes better.  Write to me off list and we can talk more about
>them if you like.  
>
>B. Isolate any fans in the box, so their vibration doesn't interfere with
>stage movement.
>
>C. Control the temperature and air current in the room. Even with a boxed-in
>microscope, temperature fluctuations in the surrounding environment have
>definitely caused Z drift in our experience.  It has also paid off for us to
>control where the air is blowing in the room (ie. not directly down on the
>microscope)...we just used some cardboard to re-direct the air current.
>
>Best of luck!!  This is a frustrating issue for sure!
>
>_______________________________
>Danielle Crippen
>Morphology and Imaging Core Manager
>Buck Institute for Research on Aging
>8001 Redwood Blvd
>Novato, CA 94945
>415-209-2046
>TheBuck.org
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Ramshesh, Venkat K
>Sent: Monday, January 31, 2011 9:41 AM
>To: [hidden email]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
>Hi Dan,
>
>  We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were
>caused by the stage controller joystick. We had to replace the controller.
>I am not sure if this applies to you but just a thought.
>Further as Tim has already pointed out the evaporation of water meniscus
>also causes drift problems.
>
>Best,
>Venkat
>
>Venkat Ramshesh, PhD
>Bioengineer/Facility Manager
>Cell and Molecular Imaging Core
>Hollings Cancer Center and Center for Cell Death, Injury and Regeneration
>Medical University of South Carolina
>QE302
>280 Calhoun Street, MSC 140
>Charleston, SC 29425
>
>Ph: 843-792-3530
>Fax: [phone]
>E-mail: [hidden email]
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On
>Behalf Of Daniel Murphy
>Sent: Monday, January 31, 2011 11:50 AM
>To: [hidden email]
>Subject: XYZ drifts - giving advice and looking for advice
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello,
>
>Some advice and a plea for help on XYZ drifts!
>
>We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
>inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
>capture z-stacks that are typically 10-15 slices thick on a 40X Water with
>2.5X optical zoom.  We also use a Warner instruments micro-incubation system
>DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
>dish cover to maintain a temp of 37C.
>
>We have had persistent issues with drifts in X, Y and Z.  Initially the
>problems were sever, with shifts of up to a few microns in all 3 directions.
> We have made progressive adjustments to our protocol with some big
>improvements, but the problem is still significant.
>
>First, we found that by surrounding the open area below the stage (where you
>can access the objectives) with seran wrap, this provided a good way of
>protecting the system from air currents and thermal influence from the
>environment.  We also characterized the incubation system and found it works
>better to run it without feedback at a constant voltage (the feedback
>response was too slow because the incubation system is too much of a heat
>sink).  We typically turn on the incubator and let it equilibrate for at
>least 15min (with the dish inside as well, whenever possible).
>
>XYZ drift still remains, however.  It seems to come and go.  For one
>experiment it will be almost unnoticeable, but for another it will make the
>data practically unusable.  Sometimes the drifts are just in one direction.
> Other times the stack shifts in Z up and down several times in one time
>sequence.  It seems to be very irregular and so probably due to random
>fluctuations in the environment.
>
>XY shifts are not too bad, so long as the area of interest remains in view.
> There are several ways to adjust for the shift post-acquisition --- you
>have more wiggle room since there are all those pixels in every direction.
>Z shifts are the real issue that plagues us.  Any advice or ideas would be
>warmly welcomed.
>
>Daniel Murphy
>Albert Einstein College of Medicine
>Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham
>Parkway South Bronx, NY 10461 Ph 718-430-4027/8985

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[Glen MacDonald-2]

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

There is most likely some variability between systems.    Our Axiovert 200M was installed with an ASI 2000 motorized stage.  Overnight timelapses with a brightfield specimen (fixed, stable) displayed several microns of drift in x,y and z using a 40X/.75 air objective.  Building an enclosure reduced this drift dramatically, and heating to 37C eliminated it entirely after  3 hours of pre-heating.

Regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]








On Feb 2, 2011, at 9:02 AM, Daniel Murphy wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> All:
>
> Thanks to all for the helpful and thorough advice.  From your comments, we
> definitely have some ideas to work through to nail down the problem-causing
> element(s).  From your comments and after speaking with a tech from Zeiss,
> apparently this is an issue that previous users have also had with long
> time-lapse imaging of incubated samples on the Axiovert 200M microscope.  He
> had done previous studies and found that with a similar setup, except with
> 40x dry objective (instead of 40x water), there is a Z drift of about 13um
> in the first 6 hours of turning the system on, at which point the Z drift is
> more or less gone.  So for really stable time-lapse work, the system needs
> to be on for at least 6hrs!  This is without an incubator enclosing the
> entire scope like the Zeiss XL --- perhaps this would reduce the
> equilibration time (any thoughts/experience?).
>
> What solutions are there to the problem of the water evaporating off the
> objective?  We use a water substitute with the same n but it is a little
> thicker, and so less evaporation.
>
> Thanks!
>
> Dan
>
> On Tue, 1 Feb 2011 13:51:12 -0000, Keith Morris <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Daniel,
>>
>> Danielle is quite right - to reduce Z focus drift significantly a Zeiss Live
>> cell large XL3 incubator is probably required on your Axiovert 200 to close
>> in the entire stage, nosepiece and objectives, and even then the stage has
>> be on for at least 2 hours before the time-lapse and wait 10 minutes before
>> starting the timelapse after opening the incubator doors [also a bit of
>> blu-tack can help to hold the Petri-dish in one place, along with any
>> 'mounting screw' adjustments on the heatable Labtek insert P - although you
>> would probably just put your Warner unit inside the XL3 incubator]. When you
>> switch on the XL3 incubator and the temperature of the objectives/stage
>> rises from 22oC to 37oC you get a focus drift of 30um or more [microscopes
>> are still built largely with metal rather than composites and so suffer from
>> thermal expansion effects]. We used to use the bijou Zeiss on-stage PeCon
>> incubator-S units similar to yours, and the Z drift was very bad for
>> time-lapse [fine for viewing live cells and capturing images as you could
>> refocus all the time, but not so good for time lapse, where we had to take a
>> z-stack at each time-point to track the focus point]. Once the large
>> incubators were fitted things improved considerably [particularly if the
>> microscope is on an anti-vibration air-table].
>>
>> Unfortunately a Zeiss XL3 incubator system sets you back �15k+ with the
>> associated PeCon electronics, precise %CO2 control and heaters. A cheaper
>> option would be a Zeiss objective heater that fits on the objective under
>> the stage to minimize thermal expansion effects there [but that�s still
>> thousands of pounds including TempController, and probably nowhere near as
>> effective as the XL3 incubator, I've never used one]. Even cheaper still
>> [and even less effective but better than nothing] is to use the foam
>> objective insulators Zeiss manufacture specifically for the purpose to put
>> around each objective. Worse still, with an oil objective and Mattek type
>> culture dish the heat is conducted away from the culture vessel into the
>> cold objective. Building you own XL3 type incubator enclosure might be a bit
>> cheaper if you have a workshop, but you also need the air heater control
>> sorted out and our in-house incubators were still �5k each for the Perspex
>> enclosure alone [as our workshop charged an hourly rate]. You also might
>> need some sort of humidity control for longer timelapses [we get our water
>> vapour in via the 5% CO2 feed] or some use the PeCon FoilCovers to reduce
>> media evaporation with culture dishes.
>>
>> See the XL3 incubator
>> http://www.well.ox.ac.uk/live-cell-imaging
>> Zeiss/PeCon incubator options
>> http://www.pecon.biz/?page_id=55
>> Foil cover
>> http://www.pecon.biz/?page_id=245
>>
>> Antivibration feedback also helps if its just the rubber door-stop feet on
>> the microscope base and no air-table - but these [Z motorized] focus issues
>> are mainly thermal [air conditioning and lack of a regulated heated
>> enclosure surround the entire objective/stage area] - the CRUK in London go
>> as far as enclosing the entire microscope within an incubator enclosure. For
>> cheap antivibration control reinforce the bench and add a large heavy slab
>> of granite [or sealed in concrete if poor] under the microscope and rest the
>> slab on anti-vibrational pads. It worked for us [we had a workshop to do the
>> mods]. Vibration can cause XY movement of the manual stage and sample
>> wobbling during capture. Once these slabs were in place you could tap the
>> worktop without the specimen dancing on the screen.
>>
>> Also try and get the room air-conditioning adjusted - the standard office
>> rated lab air-con is simply balanced to 22oC [knowledge of the Victorian
>> thermostat now being lost]. You can upgrade the aircon wall temperature
>> sensor electronics to make it more stable/accurate [cost us �1,000],
>> assuming you have one, and possibly raise the room balance temperature to
>> 24oC. Simple cardboard deflectors, to make sure the cool air from the aircon
>> ducting doesnt blow on the microscope, can also be highly effective.
>>
>> Good luck with the issue, Regards
>>
>> Keith
>>
>> No commercial interest
>>
>> ---------------------------------------------------------------------------
>> 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:� [phone]
>> Email:� [hidden email]
>> Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On
>> Behalf Of Danielle Crippen
>> Sent: 31 January 2011 17:51
>> To: [hidden email]
>> Subject: Re: XYZ drifts - giving advice and looking for advice
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We've had similar problems and have found that we need to:
>>
>> A. Box in the system.  We've made several enclosures here...homemade ones
>> are much less expensive than commercial sources (we have both) and work just
>> as well...sometimes better.  Write to me off list and we can talk more about
>> them if you like.  
>>
>> B. Isolate any fans in the box, so their vibration doesn't interfere with
>> stage movement.
>>
>> C. Control the temperature and air current in the room. Even with a boxed-in
>> microscope, temperature fluctuations in the surrounding environment have
>> definitely caused Z drift in our experience.  It has also paid off for us to
>> control where the air is blowing in the room (ie. not directly down on the
>> microscope)...we just used some cardboard to re-direct the air current.
>>
>> Best of luck!!  This is a frustrating issue for sure!
>>
>> _______________________________
>> Danielle Crippen
>> Morphology and Imaging Core Manager
>> Buck Institute for Research on Aging
>> 8001 Redwood Blvd
>> Novato, CA 94945
>> 415-209-2046
>> TheBuck.org
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On
>> Behalf Of Ramshesh, Venkat K
>> Sent: Monday, January 31, 2011 9:41 AM
>> To: [hidden email]
>> Subject: Re: XYZ drifts - giving advice and looking for advice
>>
>> Hi Dan,
>>
>> We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were
>> caused by the stage controller joystick. We had to replace the controller.
>> I am not sure if this applies to you but just a thought.
>> Further as Tim has already pointed out the evaporation of water meniscus
>> also causes drift problems.
>>
>> Best,
>> Venkat
>>
>> Venkat Ramshesh, PhD
>> Bioengineer/Facility Manager
>> Cell and Molecular Imaging Core
>> Hollings Cancer Center and Center for Cell Death, Injury and Regeneration
>> Medical University of South Carolina
>> QE302
>> 280 Calhoun Street, MSC 140
>> Charleston, SC 29425
>>
>> Ph: 843-792-3530
>> Fax: [phone]
>> E-mail: [hidden email]
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On
>> Behalf Of Daniel Murphy
>> Sent: Monday, January 31, 2011 11:50 AM
>> To: [hidden email]
>> Subject: XYZ drifts - giving advice and looking for advice
>>
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>>
>> Hello,
>>
>> Some advice and a plea for help on XYZ drifts!
>>
>> We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
>> inverted scope.  Typically these go for 20min durations at 1frame/30sec.  We
>> capture z-stacks that are typically 10-15 slices thick on a 40X Water with
>> 2.5X optical zoom.  We also use a Warner instruments micro-incubation system
>> DH40i (http://www.warneronline.com/product_info.cfm?id=1165) with heated
>> dish cover to maintain a temp of 37C.
>>
>> We have had persistent issues with drifts in X, Y and Z.  Initially the
>> problems were sever, with shifts of up to a few microns in all 3 directions.
>> We have made progressive adjustments to our protocol with some big
>> improvements, but the problem is still significant.
>>
>> First, we found that by surrounding the open area below the stage (where you
>> can access the objectives) with seran wrap, this provided a good way of
>> protecting the system from air currents and thermal influence from the
>> environment.  We also characterized the incubation system and found it works
>> better to run it without feedback at a constant voltage (the feedback
>> response was too slow because the incubation system is too much of a heat
>> sink).  We typically turn on the incubator and let it equilibrate for at
>> least 15min (with the dish inside as well, whenever possible).
>>
>> XYZ drift still remains, however.  It seems to come and go.  For one
>> experiment it will be almost unnoticeable, but for another it will make the
>> data practically unusable.  Sometimes the drifts are just in one direction.
>> Other times the stack shifts in Z up and down several times in one time
>> sequence.  It seems to be very irregular and so probably due to random
>> fluctuations in the environment.
>>
>> XY shifts are not too bad, so long as the area of interest remains in view.
>> There are several ways to adjust for the shift post-acquisition --- you
>> have more wiggle room since there are all those pixels in every direction.
>> Z shifts are the real issue that plagues us.  Any advice or ideas would be
>> warmly welcomed.
>>
>> Daniel Murphy
>> Albert Einstein College of Medicine
>> Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham
>> Parkway South Bronx, NY 10461 Ph 718-430-4027/8985

[Dan Stevens-2]

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Hi Daniel,

There has been some good feedback here, but I wanted to add two bits that I
did not see posted.

1 - I have seen a good number of people equilibrate their microscope and then
drop the sample in and begin imaging.  The sample needs to equilibrate.  You
can do a nice job of isolating whether the drift is associated with sample (most
often in my experience) or stand by using a fixed sample loosely fitted in the
carrier.  I have used PSF beads, so that the original plane of focus is easily
defined and the degree of drift can be found at the end of the time course.

2 - In performing heat shock experiments, I have noted xy drift.  These
experiments were done with perfusion, with the stage incubator kept at a
constant, over time periods where I expect there was effectively no heat
transfer beyond that carried by the bulk flow.  The use of a hardware based
autofocus device (zeiss definite focus), allowed us to measure the degree and
rate of z-focus drift, but a bit to my surprise we also saw significant xy drift.  
To my best recollection the heat shock was 5 degrees rise over 2 minutes, the
resulting z drift (measured by degree of system compensation to maintain the
plane) was 4um, and the xy drift was ~10um.  The xy drift reversed when we
dropped the temp back.

Thought that might be of some interest to you, given you saw drift in xy as
well as z.  

regards,

Dan Stevens, PhD
Application Specialist
Carl Zeiss Canada


*Strong commercial interest