System stabilty and reproducibility

[Konstantín Levitskiy]

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Dear colleagues,

We have a quiet new confocal system with STORM on the same equipment Nikon
A1R+. We observed some drifting of the definite point in time lapse
scanning. These drifts happened on 3 XYZ directions. Technical Services
could reduce the XY drifts. PFS (perfect focus system) goes well for
maintaining the focus in Z, but when you need to make a Z-stack acquisition
you can’t use PFS, since in this case is when the drift on Z occurs
remarkably. After some adjustments of the vibration isolation table,
discharging the cables and tubes from touching the table and putting OKO-Lab
all microscope chamber to 29ºC, the resting Z-drift measured was about
0.5mkm/10 min, (however it seems to me that it was for 5 min). And they say
that is normal working system. The equipment is still in 5-years warranty
period.

 <http://imgur.com/a/t9BV7> http://imgur.com/a/t9BV7

Has anybody experienced similar problems and what drift could be acceptable
if it can`t be totally removed?

The experiments performed on the system: acquisition of 150 mkm of mouse
embryo, 2048x2048, with step of 2.5 mkm for one colour and same Z-width,
same resolution, step 0.95 mkm (Nyquist condition) for other channels. With
our Z-drift we can’t combine separately acquired series. And sometimes we
find complete black in the middle of  the images (focus out positive) or
some planes seem to be repeated (focus in negative).

Best regards,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email:  <mailto:[hidden email]> [hidden email]

Web:  <http://www.ibis-sevilla.es/> www.ibis-sevilla.es

 

[Sylvie Le Guyader]

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Hi Konstantin

Very slow z stacks are really prone to showing z drift due to temperature changes which will occur anyway even if your incubator is set to 29 deg. There is always a cyclical temperature variation in the incubator.

One thing that we noticed is that even though our systems are set to 37 deg all the time, turning on the microscope anyway induces some z drift which takes hours to set. For that reason we leave on all the time not just the incubator but also the microscopes (only the microscopes, not the rest of the equipment like stage, lasers, PC...). Each user turns the microscope off then immediately on again at the start of his/her imaging session just to make sure it is reset to default values. This has helped a lot in getting more reproducible point spread functions.

Also presumably your sample is fixed and might be stored in the fridge. For slow and long (and painful) z stacks, it is very important to put the sample in the incubator, on the stage, for a super long time before starting to image. This is especially true when imaging with an oil objective as the heat transfers through the oil. If you take a sample from the fridge and put it on the microscope at 29 deg and start imaging with an oil objective, you will see that there is a z drift for several hours.

Another concern if you get movements in xy is how your sample is made. This is a thick sample. With a Nyquist xy sampling at 0.95 um I suppose you are using a 20x air objective. If your sample is on the slide, not on the coverslip where it should be, and you have been rather generous with the mounting medium, then you might be touching the coverslip with the objective when imaging the top of the section. This can happen even with a working distance of 1mm. I would put the sample on the coverslip.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Konstantín Levitskiy
Sent: den 4 april 2017 10:28
To: [hidden email]
Subject: System stabilty and reproducibility

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com andinclude the link in your posting.
*****

Dear colleagues,

We have a quiet new confocal system with STORM on the same equipment Nikon
A1R+. We observed some drifting of the definite point in time lapse
scanning. These drifts happened on 3 XYZ directions. Technical Services could reduce the XY drifts. PFS (perfect focus system) goes well for maintaining the focus in Z, but when you need to make a Z-stack acquisition you can’t use PFS, since in this case is when the drift on Z occurs remarkably. After some adjustments of the vibration isolation table, discharging the cables and tubes from touching the table and putting OKO-Lab all microscope chamber to 29ºC, the resting Z-drift measured was about
0.5mkm/10 min, (however it seems to me that it was for 5 min). And they say that is normal working system. The equipment is still in 5-years warranty period.

<http://imgur.com/a/t9BV7>;http://imgur.com/a/t9BV7

Has anybody experienced similar problems and what drift could be acceptable if it can`t be totally removed?

The experiments performed on the system: acquisition of 150 mkm of mouse embryo, 2048x2048, with step of 2.5 mkm for one colour and same Z-width, same resolution, step 0.95 mkm (Nyquist condition) for other channels. With our Z-drift we can’t combine separately acquired series. And sometimes we find complete black in the middle of  the images (focus out positive) or some planes seem to be repeated (focus in negative).

Best regards,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email:  <mailto:[hidden email]>[hidden email]

Web:  <http://www.ibis-sevilla.es/>; www.ibis-sevilla.es

[Jeremy Adler-5]

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Hi Sylvie,

We spotted some wonderful Z axis distortions.  
It was noteworthy that an enclosure, particularly when made air tight, minimized drift in all axes.
As a sign of the times, most of our users opted for ease of access and simply removed the enclosure - choice turned out to be a design fault.

J. Adler and S.N. Pagakis (2003)
Reducing Image Distortions Due To Temperature Related Microscope Stage Drift
J. Microscopy, 210, 131-137.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: den 6 april 2017 08:43
To: [hidden email]
Subject: Re: System stabilty and reproducibility

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Konstantin

Very slow z stacks are really prone to showing z drift due to temperature changes which will occur anyway even if your incubator is set to 29 deg. There is always a cyclical temperature variation in the incubator.

One thing that we noticed is that even though our systems are set to 37 deg all the time, turning on the microscope anyway induces some z drift which takes hours to set. For that reason we leave on all the time not just the incubator but also the microscopes (only the microscopes, not the rest of the equipment like stage, lasers, PC...). Each user turns the microscope off then immediately on again at the start of his/her imaging session just to make sure it is reset to default values. This has helped a lot in getting more reproducible point spread functions.

Also presumably your sample is fixed and might be stored in the fridge. For slow and long (and painful) z stacks, it is very important to put the sample in the incubator, on the stage, for a super long time before starting to image. This is especially true when imaging with an oil objective as the heat transfers through the oil. If you take a sample from the fridge and put it on the microscope at 29 deg and start imaging with an oil objective, you will see that there is a z drift for several hours.

Another concern if you get movements in xy is how your sample is made. This is a thick sample. With a Nyquist xy sampling at 0.95 um I suppose you are using a 20x air objective. If your sample is on the slide, not on the coverslip where it should be, and you have been rather generous with the mounting medium, then you might be touching the coverslip with the objective when imaging the top of the section. This can happen even with a working distance of 1mm. I would put the sample on the coverslip.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Konstantín Levitskiy
Sent: den 4 april 2017 10:28
To: [hidden email]
Subject: System stabilty and reproducibility

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com andinclude the link in your posting.
*****

Dear colleagues,

We have a quiet new confocal system with STORM on the same equipment Nikon
A1R+. We observed some drifting of the definite point in time lapse
scanning. These drifts happened on 3 XYZ directions. Technical Services could reduce the XY drifts. PFS (perfect focus system) goes well for maintaining the focus in Z, but when you need to make a Z-stack acquisition you can’t use PFS, since in this case is when the drift on Z occurs remarkably. After some adjustments of the vibration isolation table, discharging the cables and tubes from touching the table and putting OKO-Lab all microscope chamber to 29ºC, the resting Z-drift measured was about
0.5mkm/10 min, (however it seems to me that it was for 5 min). And they say that is normal working system. The equipment is still in 5-years warranty period.

<http://imgur.com/a/t9BV7>;http://imgur.com/a/t9BV7

Has anybody experienced similar problems and what drift could be acceptable if it can`t be totally removed?

The experiments performed on the system: acquisition of 150 mkm of mouse embryo, 2048x2048, with step of 2.5 mkm for one colour and same Z-width, same resolution, step 0.95 mkm (Nyquist condition) for other channels. With our Z-drift we can’t combine separately acquired series. And sometimes we find complete black in the middle of  the images (focus out positive) or some planes seem to be repeated (focus in negative).

Best regards,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email:  <mailto:[hidden email]>[hidden email]

Web:  <http://www.ibis-sevilla.es/>; www.ibis-sevilla.es