Oil objective maximum area/distance for long-term timelapse imaging

[Kenneth Chen]

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Hi all,

We're in the process of designing microfluidic devices for multi-day
time-lapse imaging. In the past we've had some trouble with image quality
for very large devices deteriorating due to the loss/thinness of the
immersion oil layer between sample and objective when very large areas are
scanned. We have only used traditional oil objectives (no water or
glycerol) Does anyone have any strategies to mitigate this or rules of
thumb about "safe" maximum sampling areas or distances to travel?

Thanks,
Ken Chen

[Sylvie Le Guyader]

Hi Ken



My experience: imaging 2 cells 1.7mm apart (thickness of the well wall in the MW plates we were using then) every 10 min for 12h with a 60x oil objective and a hardware autofocus at 37 deg resulted in a high risk (about 25% of the cases?) of losing focus. There go years of sweat and tears…



We were lucky to get to test a super device manufactured by EMBLEM, the commercial side of EMBL. You send your objective. They custom make a cap and send you a kit containing cap, tubes, pump and instructions. It is a bit clumsy because you need to remove all other objectives and you need to be careful not to put oil all over the place but it works a charm. I have imaged 10 positions in each well over 15 wells every 10 min for 15 h with this device without losing focus. :) You could contact them<http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers> to ask if they commercialize it.



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] New number!

LCI website





-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kenneth Chen
Sent: den 2 mars 2016 13:22
To: [hidden email]
Subject: Oil objective maximum area/distance for long-term timelapse imaging



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*****



Hi all,



We're in the process of designing microfluidic devices for multi-day time-lapse imaging. In the past we've had some trouble with image quality for very large devices deteriorating due to the loss/thinness of the immersion oil layer between sample and objective when very large areas are scanned. We have only used traditional oil objectives (no water or

glycerol) Does anyone have any strategies to mitigate this or rules of thumb about "safe" maximum sampling areas or distances to travel?



Thanks,

Ken Chen

[Gary Laevsky]

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We regularly image the 4 center wells (about 50 total points) of a Labtek 8
well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.

First we put the plate on, one drop of oil on the 60X or 100X objective,
and drive to all four wells.

Then we take the plate off, and add one more drop of oil, and we're good to
go.

We also slow the stage speed way down so PFS can follow the glass.

Hope this helps.

Gary

On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader <[hidden email]>
wrote:

> Hi Ken
>
>
>
> My experience: imaging 2 cells 1.7mm apart (thickness of the well wall in
> the MW plates we were using then) every 10 min for 12h with a 60x oil
> objective and a hardware autofocus at 37 deg resulted in a high risk (about
> 25% of the cases?) of losing focus. There go years of sweat and tears…
>
>
>
> We were lucky to get to test a super device manufactured by EMBLEM, the
> commercial side of EMBL. You send your objective. They custom make a cap
> and send you a kit containing cap, tubes, pump and instructions. It is a
> bit clumsy because you need to remove all other objectives and you need to
> be careful not to put oil all over the place but it works a charm. I have
> imaged 10 positions in each well over 15 wells every 10 min for 15 h with
> this device without losing focus. :) You could contact them<
> http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> to ask if they commercialize it.
>
>
>
> 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] New number!
>
> LCI website
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Kenneth Chen
> Sent: den 2 mars 2016 13:22
> To: [hidden email]
> Subject: Oil objective maximum area/distance for long-term timelapse
> imaging
>
>
>
> *****
>
> 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 all,
>
>
>
> We're in the process of designing microfluidic devices for multi-day
> time-lapse imaging. In the past we've had some trouble with image quality
> for very large devices deteriorating due to the loss/thinness of the
> immersion oil layer between sample and objective when very large areas are
> scanned. We have only used traditional oil objectives (no water or
>
> glycerol) Does anyone have any strategies to mitigate this or rules of
> thumb about "safe" maximum sampling areas or distances to travel?
>
>
>
> Thanks,
>
> Ken Chen
>

--
Best,

Gary Laevsky, Ph.D.
Confocal Imaging Facility Manager
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310

[Emmanuel Levy]

*****
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.
*****

It may also help to use oil with high viscosity index so it remains spread
on the glass.
All the best,
Emmanuel


On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We regularly image the 4 center wells (about 50 total points) of a Labtek 8
> well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
>
> First we put the plate on, one drop of oil on the 60X or 100X objective,
> and drive to all four wells.
>
> Then we take the plate off, and add one more drop of oil, and we're good to
> go.
>
> We also slow the stage speed way down so PFS can follow the glass.
>
> Hope this helps.
>
> Gary
>
> On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader <[hidden email]
> >
> wrote:
>
> > Hi Ken
> >
> >
> >
> > My experience: imaging 2 cells 1.7mm apart (thickness of the well wall in
> > the MW plates we were using then) every 10 min for 12h with a 60x oil
> > objective and a hardware autofocus at 37 deg resulted in a high risk
> (about
> > 25% of the cases?) of losing focus. There go years of sweat and tears…
> >
> >
> >
> > We were lucky to get to test a super device manufactured by EMBLEM, the
> > commercial side of EMBL. You send your objective. They custom make a cap
> > and send you a kit containing cap, tubes, pump and instructions. It is a
> > bit clumsy because you need to remove all other objectives and you need
> to
> > be careful not to put oil all over the place but it works a charm. I have
> > imaged 10 positions in each well over 15 wells every 10 min for 15 h with
> > this device without losing focus. :) You could contact them<
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> > to ask if they commercialize it.
> >
> >
> >
> > 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] New number!
> >
> > LCI website
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Kenneth Chen
> > Sent: den 2 mars 2016 13:22
> > To: [hidden email]
> > Subject: Oil objective maximum area/distance for long-term timelapse
> > imaging
> >
> >
> >
> > *****
> >
> > 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 all,
> >
> >
> >
> > We're in the process of designing microfluidic devices for multi-day
> > time-lapse imaging. In the past we've had some trouble with image quality
> > for very large devices deteriorating due to the loss/thinness of the
> > immersion oil layer between sample and objective when very large areas
> are
> > scanned. We have only used traditional oil objectives (no water or
> >
> > glycerol) Does anyone have any strategies to mitigate this or rules of
> > thumb about "safe" maximum sampling areas or distances to travel?
> >
> >
> >
> > Thanks,
> >
> > Ken Chen
> >
>
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Confocal Imaging Facility Manager
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>

[Smith, Benjamin E.]

*****
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.
*****

I can also testify to the benefit of greatly reducing the stage velocity during long-term live imaging.  We have done imaging of up to 48-hours, and having the stage crawl along allows the immersion fluid to stay wetted to the objective, and also eliminates any sample motion.  

One other consideration we've done is to setup the run early in the morning so you can check up on it throughout the day.  That way, you can also add immersion fluid as needed.  Normally, you will find that towards the beginning of the run you may need to add a little more to the objective, but after about 3-4 hours everything stabilizes, and the system is good to go.  

I also make sure that there is a sufficient pause between scans so that I have enough time to adjust things and still start the next scan at exactly the next time interval.

Good luck,
   Ben Smith

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Emmanuel Levy <[hidden email]>
Sent: Wednesday, March 2, 2016 8:46 AM
To: [hidden email]
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging

*****
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.
*****

It may also help to use oil with high viscosity index so it remains spread
on the glass.
All the best,
Emmanuel


On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We regularly image the 4 center wells (about 50 total points) of a Labtek 8
> well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
>
> First we put the plate on, one drop of oil on the 60X or 100X objective,
> and drive to all four wells.
>
> Then we take the plate off, and add one more drop of oil, and we're good to
> go.
>
> We also slow the stage speed way down so PFS can follow the glass.
>
> Hope this helps.
>
> Gary
>
> On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader <[hidden email]
> >
> wrote:
>
> > Hi Ken
> >
> >
> >
> > My experience: imaging 2 cells 1.7mm apart (thickness of the well wall in
> > the MW plates we were using then) every 10 min for 12h with a 60x oil
> > objective and a hardware autofocus at 37 deg resulted in a high risk
> (about
> > 25% of the cases?) of losing focus. There go years of sweat and tears…
> >
> >
> >
> > We were lucky to get to test a super device manufactured by EMBLEM, the
> > commercial side of EMBL. You send your objective. They custom make a cap
> > and send you a kit containing cap, tubes, pump and instructions. It is a
> > bit clumsy because you need to remove all other objectives and you need
> to
> > be careful not to put oil all over the place but it works a charm. I have
> > imaged 10 positions in each well over 15 wells every 10 min for 15 h with
> > this device without losing focus. :) You could contact them<
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> > to ask if they commercialize it.
> >
> >
> >
> > 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] New number!
> >
> > LCI website
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Kenneth Chen
> > Sent: den 2 mars 2016 13:22
> > To: [hidden email]
> > Subject: Oil objective maximum area/distance for long-term timelapse
> > imaging
> >
> >
> >
> > *****
> >
> > 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 all,
> >
> >
> >
> > We're in the process of designing microfluidic devices for multi-day
> > time-lapse imaging. In the past we've had some trouble with image quality
> > for very large devices deteriorating due to the loss/thinness of the
> > immersion oil layer between sample and objective when very large areas
> are
> > scanned. We have only used traditional oil objectives (no water or
> >
> > glycerol) Does anyone have any strategies to mitigate this or rules of
> > thumb about "safe" maximum sampling areas or distances to travel?
> >
> >
> >
> > Thanks,
> >
> > Ken Chen
> >
>
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Confocal Imaging Facility Manager
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>

[Müller,Tobias]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hello,

I've noticed that if the software allows you to set a settle time after the stage movement, this can aid in having a uniform oil layer between objective and glass before acquisition.

Best,
Tobias

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, Benjamin E.
Sent: Wednesday, March 02, 2016 3:56 PM
To: [hidden email]
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging

*****
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.
*****

I can also testify to the benefit of greatly reducing the stage velocity during long-term live imaging.  We have done imaging of up to 48-hours, and having the stage crawl along allows the immersion fluid to stay wetted to the objective, and also eliminates any sample motion.  

One other consideration we've done is to setup the run early in the morning so you can check up on it throughout the day.  That way, you can also add immersion fluid as needed.  Normally, you will find that towards the beginning of the run you may need to add a little more to the objective, but after about 3-4 hours everything stabilizes, and the system is good to go.  

I also make sure that there is a sufficient pause between scans so that I have enough time to adjust things and still start the next scan at exactly the next time interval.

Good luck,
   Ben Smith

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Emmanuel Levy <[hidden email]>
Sent: Wednesday, March 2, 2016 8:46 AM
To: [hidden email]
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging

*****
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.
*****

It may also help to use oil with high viscosity index so it remains spread on the glass.
All the best,
Emmanuel


On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We regularly image the 4 center wells (about 50 total points) of a
> Labtek 8 well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
>
> First we put the plate on, one drop of oil on the 60X or 100X
> objective, and drive to all four wells.
>
> Then we take the plate off, and add one more drop of oil, and we're
> good to go.
>
> We also slow the stage speed way down so PFS can follow the glass.
>
> Hope this helps.
>
> Gary
>
> On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> <[hidden email]
> >
> wrote:
>
> > Hi Ken
> >
> >
> >
> > My experience: imaging 2 cells 1.7mm apart (thickness of the well
> > wall in the MW plates we were using then) every 10 min for 12h with
> > a 60x oil objective and a hardware autofocus at 37 deg resulted in a
> > high risk
> (about
> > 25% of the cases?) of losing focus. There go years of sweat and
> > tears.
> >
> >
> >
> > We were lucky to get to test a super device manufactured by EMBLEM,
> > the commercial side of EMBL. You send your objective. They custom
> > make a cap and send you a kit containing cap, tubes, pump and
> > instructions. It is a bit clumsy because you need to remove all
> > other objectives and you need
> to
> > be careful not to put oil all over the place but it works a charm. I
> > have imaged 10 positions in each well over 15 wells every 10 min for
> > 15 h with this device without losing focus. :) You could contact
> > them<
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offe
> > rs>
> > to ask if they commercialize it.
> >
> >
> >
> > 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] New number!
> >
> > LCI website
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]]
> > On Behalf Of Kenneth Chen
> > Sent: den 2 mars 2016 13:22
> > To: [hidden email]
> > Subject: Oil objective maximum area/distance for long-term timelapse
> > imaging
> >
> >
> >
> > *****
> >
> > 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 all,
> >
> >
> >
> > We're in the process of designing microfluidic devices for multi-day
> > time-lapse imaging. In the past we've had some trouble with image
> > quality for very large devices deteriorating due to the
> > loss/thinness of the immersion oil layer between sample and
> > objective when very large areas
> are
> > scanned. We have only used traditional oil objectives (no water or
> >
> > glycerol) Does anyone have any strategies to mitigate this or rules
> > of thumb about "safe" maximum sampling areas or distances to travel?
> >
> >
> >
> > Thanks,
> >
> > Ken Chen
> >
>
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Confocal Imaging Facility Manager
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>

[Sylvie Le Guyader]

*****
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.
*****

I forgot to write that what the magic oil device does is that it refills the immersion oil on the objectuf lense. :-)

I agree that reducing the stage speed helps but we never got to a fully reliable system. Might take magic hands... Sigh!

Ann. What do you mean by 'it did not keep'? Did it bleach? The GattaQuant website specifically says that it virtually doesn't bleach! :-(

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

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


---- Smith, Benjamin E. wrote ----

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

I can also testify to the benefit of greatly reducing the stage velocity during long-term live imaging.  We have done imaging of up to 48-hours, and having the stage crawl along allows the immersion fluid to stay wetted to the objective, and also eliminates any sample motion.

One other consideration we've done is to setup the run early in the morning so you can check up on it throughout the day.  That way, you can also add immersion fluid as needed.  Normally, you will find that towards the beginning of the run you may need to add a little more to the objective, but after about 3-4 hours everything stabilizes, and the system is good to go.

I also make sure that there is a sufficient pause between scans so that I have enough time to adjust things and still start the next scan at exactly the next time interval.

Good luck,
   Ben Smith

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Emmanuel Levy <[hidden email]>
Sent: Wednesday, March 2, 2016 8:46 AM
To: [hidden email]
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging

*****
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.
*****

It may also help to use oil with high viscosity index so it remains spread
on the glass.
All the best,
Emmanuel


On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We regularly image the 4 center wells (about 50 total points) of a Labtek 8
> well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
>
> First we put the plate on, one drop of oil on the 60X or 100X objective,
> and drive to all four wells.
>
> Then we take the plate off, and add one more drop of oil, and we're good to
> go.
>
> We also slow the stage speed way down so PFS can follow the glass.
>
> Hope this helps.
>
> Gary
>
> On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader <[hidden email]
> >
> wrote:
>
> > Hi Ken
> >
> >
> >
> > My experience: imaging 2 cells 1.7mm apart (thickness of the well wall in
> > the MW plates we were using then) every 10 min for 12h with a 60x oil
> > objective and a hardware autofocus at 37 deg resulted in a high risk
> (about
> > 25% of the cases?) of losing focus. There go years of sweat and tears…
> >
> >
> >
> > We were lucky to get to test a super device manufactured by EMBLEM, the
> > commercial side of EMBL. You send your objective. They custom make a cap
> > and send you a kit containing cap, tubes, pump and instructions. It is a
> > bit clumsy because you need to remove all other objectives and you need
> to
> > be careful not to put oil all over the place but it works a charm. I have
> > imaged 10 positions in each well over 15 wells every 10 min for 15 h with
> > this device without losing focus. :) You could contact them<
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> > to ask if they commercialize it.
> >
> >
> >
> > 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] New number!
> >
> > LCI website
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Kenneth Chen
> > Sent: den 2 mars 2016 13:22
> > To: [hidden email]
> > Subject: Oil objective maximum area/distance for long-term timelapse
> > imaging
> >
> >
> >
> > *****
> >
> > 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 all,
> >
> >
> >
> > We're in the process of designing microfluidic devices for multi-day
> > time-lapse imaging. In the past we've had some trouble with image quality
> > for very large devices deteriorating due to the loss/thinness of the
> > immersion oil layer between sample and objective when very large areas
> are
> > scanned. We have only used traditional oil objectives (no water or
> >
> > glycerol) Does anyone have any strategies to mitigate this or rules of
> > thumb about "safe" maximum sampling areas or distances to travel?
> >
> >
> >
> > Thanks,
> >
> > Ken Chen
> >
>
>
>
> --
> Best,
>
> Gary Laevsky, Ph.D.
> Confocal Imaging Facility Manager
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310
>

[Jason Miller]

*****
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.
*****

We tried to solve this problem by using water instead of oil, acknowledging
that we lost the increased NA. We were using an inverted setup, and similar
to Sylvie, we machined a cap that goes around the objective with a drip
setup - so water drips into this cap, which encases the objective, and
keeps water in the cap at all times. You have to empirically test what drip
rate you need to counter evaporation. We found that the viscous water
substitutes (e.g. ultrasound gel, gonisol, etc.) did not have enough
capillary action to stay sealed between the objective and the bottom of the
plate. But water did. This also allows for very fast imaging - you don't
have to slow down the speed with which the stage travels between wells or
within wells at all.

Regards-
Jason

-------

Jason Miller, MD, PhD

University of Michigan Kellogg Eye Center


Home address:

117 Worden Ave

Ann Arbor, MI 48103

Cell: [phone]<tel:%28415%29%20225-2134>

E-mail: *[hidden email]<mailto:[hidden email]> <[hidden email]<mailto:[hidden email]>>*


"Forgiveness does not change the past, but it does enlarge the future." -
Paul Boese

On Wed, Mar 2, 2016 at 12:05 PM, Sylvie Le Guyader <[hidden email]<mailto:[hidden email]>>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your posting.
> *****
>
> I forgot to write that what the magic oil device does is that it refills
> the immersion oil on the objectuf lense. :-)
>
> I agree that reducing the stage speed helps but we never got to a fully
> reliable system. Might take magic hands... Sigh!
>
> Ann. What do you mean by 'it did not keep'? Did it bleach? The GattaQuant
> website specifically says that it virtually doesn't bleach! :-(
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader, PhD
> Live Cell Imaging Unit Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7,
> Novum, G lift, floor 6
> 14157 Huddinge
> Sweden
> mobile: [phone]<tel:%2B46%20%280%29%2073%20733%205008>
> office: [phone]<tel:%2B46%20%280%29%208%205248%201107>
> LCI website
>
>
> ---- Smith, Benjamin E. wrote ----
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your posting.
> *****
>
> I can also testify to the benefit of greatly reducing the stage velocity
> during long-term live imaging.  We have done imaging of up to 48-hours,
> and having the stage crawl along allows the immersion fluid to stay wetted
> to the objective, and also eliminates any sample motion.
>
> One other consideration we've done is to setup the run early in the
> morning so you can check up on it throughout the day.  That way, you can
> also add immersion fluid as needed.  Normally, you will find that towards
> the beginning of the run you may need to add a little more to the
> objective, but after about 3-4 hours everything stabilizes, and the system
> is good to go.
>
> I also make sure that there is a sufficient pause between scans so that I
> have enough time to adjust things and still start the next scan at exactly
> the next time interval.
>
> Good luck,
>    Ben Smith
>
> ________________________________________
> From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> on
> behalf of Emmanuel Levy <[hidden email]<mailto:[hidden email]>>
> Sent: Wednesday, March 2, 2016 8:46 AM
> To: [hidden email]<mailto:[hidden email]>
> Subject: Re: Oil objective maximum area/distance for long-term timelapse
> imaging
>
> *****
> 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<http://www.imgur.com/> and include the link in your posting.
> *****
>
> It may also help to use oil with high viscosity index so it remains spread
> on the glass.
> All the best,
> Emmanuel
>
>
> On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]<mailto:[hidden email]>> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your
> posting.
> > *****
> >
> > We regularly image the 4 center wells (about 50 total points) of a
> Labtek 8
> > well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
> >
> > First we put the plate on, one drop of oil on the 60X or 100X objective,
> > and drive to all four wells.
> >
> > Then we take the plate off, and add one more drop of oil, and we're good
> to
> > go.
> >
> > We also slow the stage speed way down so PFS can follow the glass.
> >
> > Hope this helps.
> >
> > Gary
> >
> > On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> <[hidden email]<mailto:[hidden email]>
> > >
> > wrote:
> >
> > > Hi Ken
> > >
> > >
> > >
> > > My experience: imaging 2 cells 1.7mm apart (thickness of the well wall
> in
> > > the MW plates we were using then) every 10 min for 12h with a 60x oil
> > > objective and a hardware autofocus at 37 deg resulted in a high risk
> > (about
> > > 25% of the cases?) of losing focus. There go years of sweat and tears...
> > >
> > >
> > >
> > > We were lucky to get to test a super device manufactured by EMBLEM,
> the
> > > commercial side of EMBL. You send your objective. They custom make a
> cap
> > > and send you a kit containing cap, tubes, pump and instructions. It is
> a
> > > bit clumsy because you need to remove all other objectives and you
> need
> > to
> > > be careful not to put oil all over the place but it works a charm. I
> have
> > > imaged 10 positions in each well over 15 wells every 10 min for 15 h
> with
> > > this device without losing focus. :) You could contact them<
> > >
> http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> > > to ask if they commercialize it.
> > >
> > >
> > >
> > > 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]<tel:%2B46%20%280%29%2073%20733%205008>
> > >
> > > office: [phone] New number!
> > >
> > > LCI website
> > >
> > >
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List
> [mailto:[hidden email]<mailto:[hidden email]>]
> > > On Behalf Of Kenneth Chen
> > > Sent: den 2 mars 2016 13:22
> > > To: [hidden email]<mailto:[hidden email]>
> > > Subject: Oil objective maximum area/distance for long-term timelapse
> > > imaging
> > >
> > >
> > >
> > > *****
> > >
> > > 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<http://www.imgur.com/> and include the link in your
> > posting.
> > >
> > > *****
> > >
> > >
> > >
> > > Hi all,
> > >
> > >
> > >
> > > We're in the process of designing microfluidic devices for multi-day
> > > time-lapse imaging. In the past we've had some trouble with image
> quality
> > > for very large devices deteriorating due to the loss/thinness of the
> > > immersion oil layer between sample and objective when very large areas
> > are
> > > scanned. We have only used traditional oil objectives (no water or
> > >
> > > glycerol) Does anyone have any strategies to mitigate this or rules of
> > > thumb about "safe" maximum sampling areas or distances to travel?
> > >
> > >
> > >
> > > Thanks,
> > >
> > > Ken Chen
> > >
> >
> >
> >
> > --
> > Best,
> >
> > Gary Laevsky, Ph.D.
> > Confocal Imaging Facility Manager
> > Dept. of Molecular Biology
> > Washington Rd.
> > Princeton University
> > Princeton, New Jersey, 08544-1014
> > (O) 609 258 5432<tel:609%20258%205432>
> > (C) 508 507 1310<tel:508%20507%201310>
> >
>

**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

[Emmanuel Levy]

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

Dear Jason,

I've got a question about using water immersion. In my experience the
hardware autofocus locks on the interface having the largest difference in
refractive index. When using water immersion, that means both the top and
bottom interface of the glass yield equal such difference. Haven't you had
problems with that? May I ask which hardware autofocus you've been using?

Thank you,

Emmanuel



On 2 March 2016 at 19:19, Miller, Jason <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We tried to solve this problem by using water instead of oil, acknowledging
> that we lost the increased NA. We were using an inverted setup, and similar
> to Sylvie, we machined a cap that goes around the objective with a drip
> setup - so water drips into this cap, which encases the objective, and
> keeps water in the cap at all times. You have to empirically test what drip
> rate you need to counter evaporation. We found that the viscous water
> substitutes (e.g. ultrasound gel, gonisol, etc.) did not have enough
> capillary action to stay sealed between the objective and the bottom of the
> plate. But water did. This also allows for very fast imaging - you don't
> have to slow down the speed with which the stage travels between wells or
> within wells at all.
>
> Regards-
> Jason
>
> -------
>
> Jason Miller, MD, PhD
>
> University of Michigan Kellogg Eye Center
>
>
> Home address:
>
> 117 Worden Ave
>
> Ann Arbor, MI 48103
>
> Cell: [phone]<tel:%28415%29%20225-2134>
>
> E-mail: *[hidden email]<mailto:
> [hidden email]> <[hidden email]<mailto:
> [hidden email]>>*
>
>
> "Forgiveness does not change the past, but it does enlarge the future." -
> Paul Boese
>
> On Wed, Mar 2, 2016 at 12:05 PM, Sylvie Le Guyader <
> [hidden email]<mailto:[hidden email]>>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com<http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > I forgot to write that what the magic oil device does is that it refills
> > the immersion oil on the objectuf lense. :-)
> >
> > I agree that reducing the stage speed helps but we never got to a fully
> > reliable system. Might take magic hands... Sigh!
> >
> > Ann. What do you mean by 'it did not keep'? Did it bleach? The GattaQuant
> > website specifically says that it virtually doesn't bleach! :-(
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader, PhD
> > Live Cell Imaging Unit Manager
> > Karolinska Institutet- Bionut Dpt
> > Hälsovägen 7,
> > Novum, G lift, floor 6
> > 14157 Huddinge
> > Sweden
> > mobile: [phone]<tel:%2B46%20%280%29%2073%20733%205008>
> > office: [phone]<tel:%2B46%20%280%29%208%205248%201107>
> > LCI website
> >
> >
> > ---- Smith, Benjamin E. wrote ----
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com<http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > I can also testify to the benefit of greatly reducing the stage velocity
> > during long-term live imaging.  We have done imaging of up to 48-hours,
> > and having the stage crawl along allows the immersion fluid to stay
> wetted
> > to the objective, and also eliminates any sample motion.
> >
> > One other consideration we've done is to setup the run early in the
> > morning so you can check up on it throughout the day.  That way, you can
> > also add immersion fluid as needed.  Normally, you will find that towards
> > the beginning of the run you may need to add a little more to the
> > objective, but after about 3-4 hours everything stabilizes, and the
> system
> > is good to go.
> >
> > I also make sure that there is a sufficient pause between scans so that I
> > have enough time to adjust things and still start the next scan at
> exactly
> > the next time interval.
> >
> > Good luck,
> >    Ben Smith
> >
> > ________________________________________
> > From: Confocal Microscopy List <[hidden email]<mailto:
> [hidden email]>> on
> > behalf of Emmanuel Levy <[hidden email]<mailto:
> [hidden email]>>
> > Sent: Wednesday, March 2, 2016 8:46 AM
> > To: [hidden email]<mailto:
> [hidden email]>
> > Subject: Re: Oil objective maximum area/distance for long-term timelapse
> > imaging
> >
> > *****
> > 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<http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > It may also help to use oil with high viscosity index so it remains
> spread
> > on the glass.
> > All the best,
> > Emmanuel
> >
> >
> > On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]<mailto:
> [hidden email]>> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com<http://www.imgur.com/> and
> include the link in your
> > posting.
> > > *****
> > >
> > > We regularly image the 4 center wells (about 50 total points) of a
> > Labtek 8
> > > well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
> > >
> > > First we put the plate on, one drop of oil on the 60X or 100X
> objective,
> > > and drive to all four wells.
> > >
> > > Then we take the plate off, and add one more drop of oil, and we're
> good
> > to
> > > go.
> > >
> > > We also slow the stage speed way down so PFS can follow the glass.
> > >
> > > Hope this helps.
> > >
> > > Gary
> > >
> > > On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> > <[hidden email]<mailto:[hidden email]>
> > > >
> > > wrote:
> > >
> > > > Hi Ken
> > > >
> > > >
> > > >
> > > > My experience: imaging 2 cells 1.7mm apart (thickness of the well
> wall
> > in
> > > > the MW plates we were using then) every 10 min for 12h with a 60x oil
> > > > objective and a hardware autofocus at 37 deg resulted in a high risk
> > > (about
> > > > 25% of the cases?) of losing focus. There go years of sweat and
> tears...
> > > >
> > > >
> > > >
> > > > We were lucky to get to test a super device manufactured by EMBLEM,
> > the
> > > > commercial side of EMBL. You send your objective. They custom make a
> > cap
> > > > and send you a kit containing cap, tubes, pump and instructions. It
> is
> > a
> > > > bit clumsy because you need to remove all other objectives and you
> > need
> > > to
> > > > be careful not to put oil all over the place but it works a charm. I
> > have
> > > > imaged 10 positions in each well over 15 wells every 10 min for 15 h
> > with
> > > > this device without losing focus. :) You could contact them<
> > > >
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> > > > to ask if they commercialize it.
> > > >
> > > >
> > > >
> > > > 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]<tel:%2B46%20%280%29%2073%20733%205008>
> > > >
> > > > office: [phone] New number!
> > > >
> > > > LCI website
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > -----Original Message-----
> > > > From: Confocal Microscopy List
> > [mailto:[hidden email]<mailto:
> [hidden email]>]
> > > > On Behalf Of Kenneth Chen
> > > > Sent: den 2 mars 2016 13:22
> > > > To: [hidden email]<mailto:
> [hidden email]>
> > > > Subject: Oil objective maximum area/distance for long-term timelapse
> > > > imaging
> > > >
> > > >
> > > >
> > > > *****
> > > >
> > > > 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<http://www.imgur.com/> and
> include the link in your
> > > posting.
> > > >
> > > > *****
> > > >
> > > >
> > > >
> > > > Hi all,
> > > >
> > > >
> > > >
> > > > We're in the process of designing microfluidic devices for multi-day
> > > > time-lapse imaging. In the past we've had some trouble with image
> > quality
> > > > for very large devices deteriorating due to the loss/thinness of the
> > > > immersion oil layer between sample and objective when very large
> areas
> > > are
> > > > scanned. We have only used traditional oil objectives (no water or
> > > >
> > > > glycerol) Does anyone have any strategies to mitigate this or rules
> of
> > > > thumb about "safe" maximum sampling areas or distances to travel?
> > > >
> > > >
> > > >
> > > > Thanks,
> > > >
> > > > Ken Chen
> > > >
> > >
> > >
> > >
> > > --
> > > Best,
> > >
> > > Gary Laevsky, Ph.D.
> > > Confocal Imaging Facility Manager
> > > Dept. of Molecular Biology
> > > Washington Rd.
> > > Princeton University
> > > Princeton, New Jersey, 08544-1014
> > > (O) 609 258 5432<tel:609%20258%205432>
> > > (C) 508 507 1310<tel:508%20507%201310>
> > >
> >
> **********************************************************
> Electronic Mail is not secure, may not be read every day, and should not
> be used for urgent or sensitive issues
>

[Jason Miller]

*****
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 Emmanuel-

I thought I was the only one who had noticed this problem! I've not seen it discussed on the listserve, and when I contacted Nikon, they hadn't heard of the problem. But it soon became clear to me that the issue you bring up is exactly what's going on. Here's what I've noticed about the phenomenon and some things I've done to avoid the problem. We use the Nikon PFS system, which as you know, relies on an IR beam reflecting off a refractive index mismatched surface. We see the problem manifest as a "jitter" as PFS rapidly jumps between planes (the objective water-glass interface, and then the glass surface-cell interface).


a)     If you have confluent cells, especially if they are densely packed, that decreases the problem since the RI of the cells is different than pure water.

b)    The higher the NA objective, the worse the problem. Although to be honest, I haven't distinguished whether it's higher objective magnification or higher NA specifically.

c)     The thinner the glass, the worse the problem. If you are using an objective with a correction collar, that means you can try for glass that is thicker and adjust by adjusting the correction collar.

Our main solution for automated imaging involved writing a micromanager script. What we found out was that if perfect focus starts to jitter at a spot (jumping between the top and bottom of the glass), we let PFS go for a few seconds and take the average Z position of where it is during the jitter (get Z position every x ms, and average that position over the few seconds we let PFS go). Then we turn PFS off, have the stage move to this Z position (the average Z depth), and turn PFS back on. 95% of the time, that's all we need. Occasionally, that doesn't work. If it doesn't, we try one more time. And then if that doesn't work, we assume we are instead out of PFS range, and we have the script automatically move X um up, then X um down from the original Z focus spot and reattempt PFS focus there. The X um is determined empirically, but once you determine it, it's remarkably reproducible. We've been able to do runs over entire plates for 40+ hours, imaging every 20 minutes to 1 hour, using this type of setup.

-Jason


On 2 March 2016 at 19:19, Miller, Jason <[hidden email]<mailto:[hidden email]>> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your posting.
> *****
>
> We tried to solve this problem by using water instead of oil,
> acknowledging
> that we lost the increased NA. We were using an inverted setup, and
> similar
> to Sylvie, we machined a cap that goes around the objective with a drip
> setup - so water drips into this cap, which encases the objective, and
> keeps water in the cap at all times. You have to empirically test what
> drip
> rate you need to counter evaporation. We found that the viscous water
> substitutes (e.g. ultrasound gel, gonisol, etc.) did not have enough
> capillary action to stay sealed between the objective and the bottom of
> the
> plate. But water did. This also allows for very fast imaging - you don't
> have to slow down the speed with which the stage travels between wells or
> within wells at all.
>
> Regards-
> Jason
>
> -------
>
> Jason Miller, MD, PhD
>
> University of Michigan Kellogg Eye Center
>
>
> Home address:
>
> 117 Worden Ave
>
> Ann Arbor, MI 48103
>
> Cell: [phone]<tel:%28415%29%20225-2134><tel:%28415%29%20225-2134>
>
> E-mail: *[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>> <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>>*
>
>
> "Forgiveness does not change the past, but it does enlarge the future." -
> Paul Boese
>
> On Wed, Mar 2, 2016 at 12:05 PM, Sylvie Le Guyader <
> [hidden email]<mailto:[hidden email]><mailto:[hidden email]<mailto:[hidden email]>>>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com<http://www.imgur.com/><http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > I forgot to write that what the magic oil device does is that it refills
> > the immersion oil on the objectuf lense. :-)
> >
> > I agree that reducing the stage speed helps but we never got to a fully
> > reliable system. Might take magic hands... Sigh!
> >
> > Ann. What do you mean by 'it did not keep'? Did it bleach? The
> > GattaQuant
> > website specifically says that it virtually doesn't bleach! :-(
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader, PhD
> > Live Cell Imaging Unit Manager
> > Karolinska Institutet- Bionut Dpt
> > Hälsovägen 7,
> > Novum, G lift, floor 6
> > 14157 Huddinge
> > Sweden
> > mobile: [phone]<tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%2073%20733%205008>
> > office: [phone]<tel:%2B46%20%280%29%208%205248%201107>
> > LCI website
> >
> >
> > ---- Smith, Benjamin E. wrote ----
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com<http://www.imgur.com/><http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > I can also testify to the benefit of greatly reducing the stage velocity
> > during long-term live imaging.  We have done imaging of up to 48-hours,
> > and having the stage crawl along allows the immersion fluid to stay
> wetted
> > to the objective, and also eliminates any sample motion.
> >
> > One other consideration we've done is to setup the run early in the
> > morning so you can check up on it throughout the day.  That way, you can
> > also add immersion fluid as needed.  Normally, you will find that
> > towards
> > the beginning of the run you may need to add a little more to the
> > objective, but after about 3-4 hours everything stabilizes, and the
> system
> > is good to go.
> >
> > I also make sure that there is a sufficient pause between scans so that
> > I
> > have enough time to adjust things and still start the next scan at
> exactly
> > the next time interval.
> >
> > Good luck,
> >    Ben Smith
> >
> > ________________________________________
> > From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>> on
> > behalf of Emmanuel Levy <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>>
> > Sent: Wednesday, March 2, 2016 8:46 AM
> > To: [hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>
> > Subject: Re: Oil objective maximum area/distance for long-term timelapse
> > imaging
> >
> > *****
> > 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<http://www.imgur.com/><http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > It may also help to use oil with high viscosity index so it remains
> spread
> > on the glass.
> > All the best,
> > Emmanuel
> >
> >
> > On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com<http://www.imgur.com/><http://www.imgur.com/> and
> include the link in your
> > posting.
> > > *****
> > >
> > > We regularly image the 4 center wells (about 50 total points) of a
> > Labtek 8
> > > well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
> > >
> > > First we put the plate on, one drop of oil on the 60X or 100X
> objective,
> > > and drive to all four wells.
> > >
> > > Then we take the plate off, and add one more drop of oil, and we're
> good
> > to
> > > go.
> > >
> > > We also slow the stage speed way down so PFS can follow the glass.
> > >
> > > Hope this helps.
> > >
> > > Gary
> > >
> > > On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> > <[hidden email]<mailto:[hidden email]><mailto:[hidden email]<mailto:[hidden email]>>
> > > >
> > > wrote:
> > >
> > > > Hi Ken
> > > >
> > > >
> > > >
> > > > My experience: imaging 2 cells 1.7mm apart (thickness of the well
> wall
> > in
> > > > the MW plates we were using then) every 10 min for 12h with a 60x
> > > > oil
> > > > objective and a hardware autofocus at 37 deg resulted in a high risk
> > > (about
> > > > 25% of the cases?) of losing focus. There go years of sweat and
> tears...
> > > >
> > > >
> > > >
> > > > We were lucky to get to test a super device manufactured by EMBLEM,
> > the
> > > > commercial side of EMBL. You send your objective. They custom make a
> > cap
> > > > and send you a kit containing cap, tubes, pump and instructions. It
> is
> > a
> > > > bit clumsy because you need to remove all other objectives and you
> > need
> > > to
> > > > be careful not to put oil all over the place but it works a charm. I
> > have
> > > > imaged 10 positions in each well over 15 wells every 10 min for 15 h
> > with
> > > > this device without losing focus. :) You could contact them<
> > > >
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers>
> > > > to ask if they commercialize it.
> > > >
> > > >
> > > >
> > > > 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]<tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%2073%20733%205008>
> > > >
> > > > office: [phone] New number!
> > > >
> > > > LCI website
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > -----Original Message-----
> > > > From: Confocal Microscopy List
> > [mailto:[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>]
> > > > On Behalf Of Kenneth Chen
> > > > Sent: den 2 mars 2016 13:22
> > > > To: [hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>
> > > > Subject: Oil objective maximum area/distance for long-term timelapse
> > > > imaging
> > > >
> > > >
> > > >
> > > > *****
> > > >
> > > > 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<http://www.imgur.com/><http://www.imgur.com/> and
> include the link in your
> > > posting.
> > > >
> > > > *****
> > > >
> > > >
> > > >
> > > > Hi all,
> > > >
> > > >
> > > >
> > > > We're in the process of designing microfluidic devices for multi-day
> > > > time-lapse imaging. In the past we've had some trouble with image
> > quality
> > > > for very large devices deteriorating due to the loss/thinness of the
> > > > immersion oil layer between sample and objective when very large
> areas
> > > are
> > > > scanned. We have only used traditional oil objectives (no water or
> > > >
> > > > glycerol) Does anyone have any strategies to mitigate this or rules
> of
> > > > thumb about "safe" maximum sampling areas or distances to travel?
> > > >
> > > >
> > > >
> > > > Thanks,
> > > >
> > > > Ken Chen
> > > >
> > >
> > >
> > >
> > > --
> > > Best,
> > >
> > > Gary Laevsky, Ph.D.
> > > Confocal Imaging Facility Manager
> > > Dept. of Molecular Biology
> > > Washington Rd.
> > > Princeton University
> > > Princeton, New Jersey, 08544-1014
> > > (O) 609 258 5432<tel:609%20258%205432><tel:609%20258%205432>
> > > (C) 508 507 1310<tel:508%20507%201310><tel:508%20507%201310>
> > >
> >
> **********************************************************
> Electronic Mail is not secure, may not be read every day, and should not
> be used for urgent or sensitive issues
>

**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

[Emmanuel Levy]

*****
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 Jason,

Thanks for your detailed answer. This problem means we have avoided using
water immersion objectives but it's good to know that it can be
circumvented using a macro as you described.

We generally cover the full plate with oil and image it. In terms of
time-lapse we have not worked over entire plates, only across a couple of
wells for ~24h and it's been working well. Like everyone described, it is
important to slow down stage movements. Also, as I mentioned in a previous
post, the viscosity of the oil matters. The one we use now is from
Cargille, HF 700 cSt.

All the best,

Emmanuel



On 2 March 2016 at 23:32, Miller, Jason <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Emmanuel-
>
> I thought I was the only one who had noticed this problem! I've not seen
> it discussed on the listserve, and when I contacted Nikon, they hadn't
> heard of the problem. But it soon became clear to me that the issue you
> bring up is exactly what's going on. Here's what I've noticed about the
> phenomenon and some things I've done to avoid the problem. We use the Nikon
> PFS system, which as you know, relies on an IR beam reflecting off a
> refractive index mismatched surface. We see the problem manifest as a
> "jitter" as PFS rapidly jumps between planes (the objective water-glass
> interface, and then the glass surface-cell interface).
>
>
> a)     If you have confluent cells, especially if they are densely packed,
> that decreases the problem since the RI of the cells is different than pure
> water.
>
> b)    The higher the NA objective, the worse the problem. Although to be
> honest, I haven't distinguished whether it's higher objective magnification
> or higher NA specifically.
>
> c)     The thinner the glass, the worse the problem. If you are using an
> objective with a correction collar, that means you can try for glass that
> is thicker and adjust by adjusting the correction collar.
>
> Our main solution for automated imaging involved writing a micromanager
> script. What we found out was that if perfect focus starts to jitter at a
> spot (jumping between the top and bottom of the glass), we let PFS go for a
> few seconds and take the average Z position of where it is during the
> jitter (get Z position every x ms, and average that position over the few
> seconds we let PFS go). Then we turn PFS off, have the stage move to this Z
> position (the average Z depth), and turn PFS back on. 95% of the time,
> that's all we need. Occasionally, that doesn't work. If it doesn't, we try
> one more time. And then if that doesn't work, we assume we are instead out
> of PFS range, and we have the script automatically move X um up, then X um
> down from the original Z focus spot and reattempt PFS focus there. The X um
> is determined empirically, but once you determine it, it's remarkably
> reproducible. We've been able to do runs over entire plates for 40+ hours,
> imaging every 20 minutes to 1 hour, using this type of setup.
>
> -Jason
>
>
> On 2 March 2016 at 19:19, Miller, Jason <[hidden email]<mailto:
> [hidden email]>> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com<http://www.imgur.com/> and include
> the link in your posting.
> > *****
> >
> > We tried to solve this problem by using water instead of oil,
> > acknowledging
> > that we lost the increased NA. We were using an inverted setup, and
> > similar
> > to Sylvie, we machined a cap that goes around the objective with a drip
> > setup - so water drips into this cap, which encases the objective, and
> > keeps water in the cap at all times. You have to empirically test what
> > drip
> > rate you need to counter evaporation. We found that the viscous water
> > substitutes (e.g. ultrasound gel, gonisol, etc.) did not have enough
> > capillary action to stay sealed between the objective and the bottom of
> > the
> > plate. But water did. This also allows for very fast imaging - you don't
> > have to slow down the speed with which the stage travels between wells or
> > within wells at all.
> >
> > Regards-
> > Jason
> >
> > -------
> >
> > Jason Miller, MD, PhD
> >
> > University of Michigan Kellogg Eye Center
> >
> >
> > Home address:
> >
> > 117 Worden Ave
> >
> > Ann Arbor, MI 48103
> >
> > Cell: [phone]<tel:%28415%29%20225-2134><tel:%28415%29%20225-2134>
> >
> > E-mail: *[hidden email]<mailto:
> [hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]>>
> <[hidden email]<mailto:[hidden email]
> ><mailto:
> > [hidden email]<mailto:[hidden email]
> >>>*
> >
> >
> > "Forgiveness does not change the past, but it does enlarge the future." -
> > Paul Boese
> >
> > On Wed, Mar 2, 2016 at 12:05 PM, Sylvie Le Guyader <
> > [hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>>
> > wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com<http://www.imgur.com/><
> http://www.imgur.com/> and include
> > the link in your posting.
> > > *****
> > >
> > > I forgot to write that what the magic oil device does is that it
> refills
> > > the immersion oil on the objectuf lense. :-)
> > >
> > > I agree that reducing the stage speed helps but we never got to a fully
> > > reliable system. Might take magic hands... Sigh!
> > >
> > > Ann. What do you mean by 'it did not keep'? Did it bleach? The
> > > GattaQuant
> > > website specifically says that it virtually doesn't bleach! :-(
> > >
> > > Med vänlig hälsning / Best regards
> > >
> > > Sylvie
> > >
> > > @@@@@@@@@@@@@@@@@@@@@@@@
> > >
> > > Sylvie Le Guyader, PhD
> > > Live Cell Imaging Unit Manager
> > > Karolinska Institutet- Bionut Dpt
> > > Hälsovägen 7,
> > > Novum, G lift, floor 6
> > > 14157 Huddinge
> > > Sweden
> > > mobile: [phone]
> <tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%2073%20733%205008>
> > > office: [phone]<tel:%2B46%20%280%29%208%205248%201107>
> > > LCI website
> > >
> > >
> > > ---- Smith, Benjamin E. wrote ----
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com<http://www.imgur.com/><
> http://www.imgur.com/> and include
> > the link in your posting.
> > > *****
> > >
> > > I can also testify to the benefit of greatly reducing the stage
> velocity
> > > during long-term live imaging.  We have done imaging of up to 48-hours,
> > > and having the stage crawl along allows the immersion fluid to stay
> > wetted
> > > to the objective, and also eliminates any sample motion.
> > >
> > > One other consideration we've done is to setup the run early in the
> > > morning so you can check up on it throughout the day.  That way, you
> can
> > > also add immersion fluid as needed.  Normally, you will find that
> > > towards
> > > the beginning of the run you may need to add a little more to the
> > > objective, but after about 3-4 hours everything stabilizes, and the
> > system
> > > is good to go.
> > >
> > > I also make sure that there is a sufficient pause between scans so that
> > > I
> > > have enough time to adjust things and still start the next scan at
> > exactly
> > > the next time interval.
> > >
> > > Good luck,
> > >    Ben Smith
> > >
> > > ________________________________________
> > > From: Confocal Microscopy List <[hidden email]
> <mailto:[hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]>>>
> on
> > > behalf of Emmanuel Levy <[hidden email]<mailto:
> [hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]>>>
> > > Sent: Wednesday, March 2, 2016 8:46 AM
> > > To: [hidden email]<mailto:
> [hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]
> >>
> > > Subject: Re: Oil objective maximum area/distance for long-term
> timelapse
> > > imaging
> > >
> > > *****
> > > 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<http://www.imgur.com/><
> http://www.imgur.com/> and include
> > the link in your posting.
> > > *****
> > >
> > > It may also help to use oil with high viscosity index so it remains
> > spread
> > > on the glass.
> > > All the best,
> > > Emmanuel
> > >
> > >
> > > On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]
> <mailto:[hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]>>> wrote:
> > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > > Post images on http://www.imgur.com<http://www.imgur.com/><
> http://www.imgur.com/> and
> > include the link in your
> > > posting.
> > > > *****
> > > >
> > > > We regularly image the 4 center wells (about 50 total points) of a
> > > Labtek 8
> > > > well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
> > > >
> > > > First we put the plate on, one drop of oil on the 60X or 100X
> > objective,
> > > > and drive to all four wells.
> > > >
> > > > Then we take the plate off, and add one more drop of oil, and we're
> > good
> > > to
> > > > go.
> > > >
> > > > We also slow the stage speed way down so PFS can follow the glass.
> > > >
> > > > Hope this helps.
> > > >
> > > > Gary
> > > >
> > > > On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> > > <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>
> > > > >
> > > > wrote:
> > > >
> > > > > Hi Ken
> > > > >
> > > > >
> > > > >
> > > > > My experience: imaging 2 cells 1.7mm apart (thickness of the well
> > wall
> > > in
> > > > > the MW plates we were using then) every 10 min for 12h with a 60x
> > > > > oil
> > > > > objective and a hardware autofocus at 37 deg resulted in a high
> risk
> > > > (about
> > > > > 25% of the cases?) of losing focus. There go years of sweat and
> > tears...
> > > > >
> > > > >
> > > > >
> > > > > We were lucky to get to test a super device manufactured by EMBLEM,
> > > the
> > > > > commercial side of EMBL. You send your objective. They custom make
> a
> > > cap
> > > > > and send you a kit containing cap, tubes, pump and instructions. It
> > is
> > > a
> > > > > bit clumsy because you need to remove all other objectives and you
> > > need
> > > > to
> > > > > be careful not to put oil all over the place but it works a charm.
> I
> > > have
> > > > > imaged 10 positions in each well over 15 wells every 10 min for 15
> h
> > > with
> > > > > this device without losing focus. :) You could contact them<
> > > > >
> > > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offers
> >
> > > > > to ask if they commercialize it.
> > > > >
> > > > >
> > > > >
> > > > > 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]
> <tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%2073%20733%205008>
> > > > >
> > > > > office: [phone] New number!
> > > > >
> > > > > LCI website
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > -----Original Message-----
> > > > > From: Confocal Microscopy List
> > > [mailto:[hidden email]<mailto:
> [hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]
> >>]
> > > > > On Behalf Of Kenneth Chen
> > > > > Sent: den 2 mars 2016 13:22
> > > > > To: [hidden email]<mailto:
> [hidden email]><mailto:
> > [hidden email]<mailto:[hidden email]
> >>
> > > > > Subject: Oil objective maximum area/distance for long-term
> timelapse
> > > > > imaging
> > > > >
> > > > >
> > > > >
> > > > > *****
> > > > >
> > > > > 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<http://www.imgur.com/><
> http://www.imgur.com/> and
> > include the link in your
> > > > posting.
> > > > >
> > > > > *****
> > > > >
> > > > >
> > > > >
> > > > > Hi all,
> > > > >
> > > > >
> > > > >
> > > > > We're in the process of designing microfluidic devices for
> multi-day
> > > > > time-lapse imaging. In the past we've had some trouble with image
> > > quality
> > > > > for very large devices deteriorating due to the loss/thinness of
> the
> > > > > immersion oil layer between sample and objective when very large
> > areas
> > > > are
> > > > > scanned. We have only used traditional oil objectives (no water or
> > > > >
> > > > > glycerol) Does anyone have any strategies to mitigate this or rules
> > of
> > > > > thumb about "safe" maximum sampling areas or distances to travel?
> > > > >
> > > > >
> > > > >
> > > > > Thanks,
> > > > >
> > > > > Ken Chen
> > > > >
> > > >
> > > >
> > > >
> > > > --
> > > > Best,
> > > >
> > > > Gary Laevsky, Ph.D.
> > > > Confocal Imaging Facility Manager
> > > > Dept. of Molecular Biology
> > > > Washington Rd.
> > > > Princeton University
> > > > Princeton, New Jersey, 08544-1014
> > > > (O) 609 258 5432<tel:609%20258%205432><tel:609%20258%205432>
> > > > (C) 508 507 1310<tel:508%20507%201310><tel:508%20507%201310>
> > > >
> > >
> > **********************************************************
> > Electronic Mail is not secure, may not be read every day, and should not
> > be used for urgent or sensitive issues
> >
> **********************************************************
> Electronic Mail is not secure, may not be read every day, and should not
> be used for urgent or sensitive issues
>

[Sylvie Le Guyader]

*****
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 Emmanuel and Jason

We have seen this problem before on our Nikon systems but this seem to have been a software problem so it was fixed by an update. We are currently running on NIS 4.40 and do not experience this anymore either with the 20x air or the 60x oil objectives. You might want to ask Nikon for a hot fix in case you are running on an older version.

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] New number!
LCI website


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Miller, Jason
Sent: den 2 mars 2016 22:32
To: [hidden email]
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging

*****
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*****

Hi Emmanuel-

I thought I was the only one who had noticed this problem! I've not seen it discussed on the listserve, and when I contacted Nikon, they hadn't heard of the problem. But it soon became clear to me that the issue you bring up is exactly what's going on. Here's what I've noticed about the phenomenon and some things I've done to avoid the problem. We use the Nikon PFS system, which as you know, relies on an IR beam reflecting off a refractive index mismatched surface. We see the problem manifest as a "jitter" as PFS rapidly jumps between planes (the objective water-glass interface, and then the glass surface-cell interface).


a)     If you have confluent cells, especially if they are densely packed, that decreases the problem since the RI of the cells is different than pure water.

b)    The higher the NA objective, the worse the problem. Although to be honest, I haven't distinguished whether it's higher objective magnification or higher NA specifically.

c)     The thinner the glass, the worse the problem. If you are using an objective with a correction collar, that means you can try for glass that is thicker and adjust by adjusting the correction collar.

Our main solution for automated imaging involved writing a micromanager script. What we found out was that if perfect focus starts to jitter at a spot (jumping between the top and bottom of the glass), we let PFS go for a few seconds and take the average Z position of where it is during the jitter (get Z position every x ms, and average that position over the few seconds we let PFS go). Then we turn PFS off, have the stage move to this Z position (the average Z depth), and turn PFS back on. 95% of the time, that's all we need. Occasionally, that doesn't work. If it doesn't, we try one more time. And then if that doesn't work, we assume we are instead out of PFS range, and we have the script automatically move X um up, then X um down from the original Z focus spot and reattempt PFS focus there. The X um is determined empirically, but once you determine it, it's remarkably reproducible. We've been able to do runs over entire plates for 40+ hours, imaging every 20 minutes to 1 hour, using this type of setup.

-Jason


On 2 March 2016 at 19:19, Miller, Jason <[hidden email]<mailto:[hidden email]>> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com<http://www.imgur.com/> and include the link in your posting.
> *****
>
> We tried to solve this problem by using water instead of oil,
> acknowledging that we lost the increased NA. We were using an inverted
> setup, and similar to Sylvie, we machined a cap that goes around the
> objective with a drip setup - so water drips into this cap, which
> encases the objective, and keeps water in the cap at all times. You
> have to empirically test what drip rate you need to counter
> evaporation. We found that the viscous water substitutes (e.g.
> ultrasound gel, gonisol, etc.) did not have enough capillary action to
> stay sealed between the objective and the bottom of the plate. But
> water did. This also allows for very fast imaging - you don't have to
> slow down the speed with which the stage travels between wells or
> within wells at all.
>
> Regards-
> Jason
>
> -------
>
> Jason Miller, MD, PhD
>
> University of Michigan Kellogg Eye Center
>
>
> Home address:
>
> 117 Worden Ave
>
> Ann Arbor, MI 48103
>
> Cell: (415)
> 225-2134<tel:%28415%29%20225-2134><tel:%28415%29%20225-2134>
>
> E-mail: *[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>> <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]
> >>>*
>
>
> "Forgiveness does not change the past, but it does enlarge the
> future." - Paul Boese
>
> On Wed, Mar 2, 2016 at 12:05 PM, Sylvie Le Guyader <
> [hidden email]<mailto:[hidden email]><mailto:Sylvie.
> [hidden email]<mailto:[hidden email]>>>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on
> > http://www.imgur.com<http://www.imgur.com/><http://www.imgur.com/>
> > and include
> the link in your posting.
> > *****
> >
> > I forgot to write that what the magic oil device does is that it
> > refills the immersion oil on the objectuf lense. :-)
> >
> > I agree that reducing the stage speed helps but we never got to a
> > fully reliable system. Might take magic hands... Sigh!
> >
> > Ann. What do you mean by 'it did not keep'? Did it bleach? The
> > GattaQuant website specifically says that it virtually doesn't
> > bleach! :-(
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader, PhD
> > Live Cell Imaging Unit Manager
> > Karolinska Institutet- Bionut Dpt
> > Hälsovägen 7,
> > Novum, G lift, floor 6
> > 14157 Huddinge
> > Sweden
> > mobile: [phone]
> > 5008<tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%2073
> > %20733%205008>
> > office: [phone]<tel:%2B46%20%280%29%208%205248%201107>
> > LCI website
> >
> >
> > ---- Smith, Benjamin E. wrote ----
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on
> > http://www.imgur.com<http://www.imgur.com/><http://www.imgur.com/>
> > and include
> the link in your posting.
> > *****
> >
> > I can also testify to the benefit of greatly reducing the stage
> > velocity during long-term live imaging.  We have done imaging of up
> > to 48-hours, and having the stage crawl along allows the immersion
> > fluid to stay
> wetted
> > to the objective, and also eliminates any sample motion.
> >
> > One other consideration we've done is to setup the run early in the
> > morning so you can check up on it throughout the day.  That way, you
> > can also add immersion fluid as needed.  Normally, you will find
> > that towards the beginning of the run you may need to add a little
> > more to the objective, but after about 3-4 hours everything
> > stabilizes, and the
> system
> > is good to go.
> >
> > I also make sure that there is a sufficient pause between scans so
> > that I have enough time to adjust things and still start the next
> > scan at
> exactly
> > the next time interval.
> >
> > Good luck,
> >    Ben Smith
> >
> > ________________________________________
> > From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]
> DU>>> on
> > behalf of Emmanuel Levy <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>>
> > Sent: Wednesday, March 2, 2016 8:46 AM
> > To: [hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]
> DU>>
> > Subject: Re: Oil objective maximum area/distance for long-term
> > timelapse imaging
> >
> > *****
> > 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<http://www.imgur.com/><http://www.imgur.com/>
> > and include
> the link in your posting.
> > *****
> >
> > It may also help to use oil with high viscosity index so it remains
> spread
> > on the glass.
> > All the best,
> > Emmanuel
> >
> >
> > On 2 March 2016 at 16:18, Gary Laevsky <[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]>>> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on
> > > http://www.imgur.com<http://www.imgur.com/><http://www.imgur.com/>
> > > and
> include the link in your
> > posting.
> > > *****
> > >
> > > We regularly image the 4 center wells (about 50 total points) of a
> > Labtek 8
> > > well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
> > >
> > > First we put the plate on, one drop of oil on the 60X or 100X
> objective,
> > > and drive to all four wells.
> > >
> > > Then we take the plate off, and add one more drop of oil, and
> > > we're
> good
> > to
> > > go.
> > >
> > > We also slow the stage speed way down so PFS can follow the glass.
> > >
> > > Hope this helps.
> > >
> > > Gary
> > >
> > > On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> > <[hidden email]<mailto:[hidden email]><mailto:Sylv
> > [hidden email]<mailto:[hidden email]>>
> > > >
> > > wrote:
> > >
> > > > Hi Ken
> > > >
> > > >
> > > >
> > > > My experience: imaging 2 cells 1.7mm apart (thickness of the
> > > > well
> wall
> > in
> > > > the MW plates we were using then) every 10 min for 12h with a
> > > > 60x oil objective and a hardware autofocus at 37 deg resulted in
> > > > a high risk
> > > (about
> > > > 25% of the cases?) of losing focus. There go years of sweat and
> tears...
> > > >
> > > >
> > > >
> > > > We were lucky to get to test a super device manufactured by
> > > > EMBLEM,
> > the
> > > > commercial side of EMBL. You send your objective. They custom
> > > > make a
> > cap
> > > > and send you a kit containing cap, tubes, pump and instructions.
> > > > It
> is
> > a
> > > > bit clumsy because you need to remove all other objectives and
> > > > you
> > need
> > > to
> > > > be careful not to put oil all over the place but it works a
> > > > charm. I
> > have
> > > > imaged 10 positions in each well over 15 wells every 10 min for
> > > > 15 h
> > with
> > > > this device without losing focus. :) You could contact them<
> > > >
> > http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offe
> > rs>
> > > > to ask if they commercialize it.
> > > >
> > > >
> > > >
> > > > 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]
> > > > 5008<tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%
> > > > 2073%20733%205008>
> > > >
> > > > office: [phone] New number!
> > > >
> > > > LCI website
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > -----Original Message-----
> > > > From: Confocal Microscopy List
> > [mailto:[hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]
> DU>>]
> > > > On Behalf Of Kenneth Chen
> > > > Sent: den 2 mars 2016 13:22
> > > > To: [hidden email]<mailto:[hidden email]><mailto:
> [hidden email]<mailto:[hidden email]
> DU>>
> > > > Subject: Oil objective maximum area/distance for long-term
> > > > timelapse imaging
> > > >
> > > >
> > > >
> > > > *****
> > > >
> > > > 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<http://www.imgur.com/><http://www.imgur.com
> > > > /> and
> include the link in your
> > > posting.
> > > >
> > > > *****
> > > >
> > > >
> > > >
> > > > Hi all,
> > > >
> > > >
> > > >
> > > > We're in the process of designing microfluidic devices for
> > > > multi-day time-lapse imaging. In the past we've had some trouble
> > > > with image
> > quality
> > > > for very large devices deteriorating due to the loss/thinness of
> > > > the immersion oil layer between sample and objective when very
> > > > large
> areas
> > > are
> > > > scanned. We have only used traditional oil objectives (no water
> > > > or
> > > >
> > > > glycerol) Does anyone have any strategies to mitigate this or
> > > > rules
> of
> > > > thumb about "safe" maximum sampling areas or distances to travel?
> > > >
> > > >
> > > >
> > > > Thanks,
> > > >
> > > > Ken Chen
> > > >
> > >
> > >
> > >
> > > --
> > > Best,
> > >
> > > Gary Laevsky, Ph.D.
> > > Confocal Imaging Facility Manager
> > > Dept. of Molecular Biology
> > > Washington Rd.
> > > Princeton University
> > > Princeton, New Jersey, 08544-1014
> > > (O) 609 258 5432<tel:609%20258%205432><tel:609%20258%205432>
> > > (C) 508 507 1310<tel:508%20507%201310><tel:508%20507%201310>
> > >
> >
> **********************************************************
> Electronic Mail is not secure, may not be read every day, and should
> not be used for urgent or sensitive issues
>

**********************************************************
Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues