Multiple-position long-term time-lapse with oil immersion lens

[ramachan]

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Dear All,

One of our users is doing long-term time-lapse imaging in multi-well plates with reflection based autofocus. The sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked perfectly well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the oil immersion could have interfered  with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I'm wondering now, if the hypothesis of the bubble is correct, how likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second.

What is your experience with the use of oil immersion in such experiments?

Thanks! Best wishes,
Radek

Radek MACHAN, Ph.D. (Senior Research Fellow)
SCELSE Advanced Biofilm Imaging Facility<http://www.scelse.sg/Page/imaging-facility> Manager
Nanyang Technological University
#B2, 60 Nanyang Drive, SBS-01N-27
Singapore 637551


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[TEFK]

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Dear Radek,

We have quite some experience with multi-well plates and immersion oil objective lenses. An air bubble could have caused the auto focus to have failed, but that excess or too little oil can cause the auto-focus system to fail as the oil runs off the lens or gets spread too thin shortly after starting acquisition. Most microscope vendors include the auto-focus status in the raw file metadata for each timepoint and position. You could check back and see if the auto-focus system failed by looking at the status code to trace if the focus or another malfunction occurred.

Here some tips that might prevent issue in the future:
- We use a folded piece of lenspaper to distribute a thin oil layer on the glass and remove the lenspaper on a part of the glass you will not image (air bubbles are introduced when lifting the paper off the glass!)
- Move around all xy positions a few times to evenly smear the immersion oil and then start setting up auto focus
- use 96w or 348w plates if possible and space samples close together, also this allows more conditions to be imaged simultaneously.
- slow the xy-stage movement down if hardware supports this and if it doesn't impact the experimental setup too much

Best  wishes,

Timo

Timo E.F. Kuijt, PhD
Oncode Institute | Kops lab
Hubrecht Institute | Room E2.22
Uppsalalaan 8 | 3584 CT Utrecht
The Netherlands
Tel: [phone]
P.O. Box 85164 | 3508 AD | Utrecht
The Netherlands                          

On 21/06/2020, 08:57, "Confocal Microscopy List on behalf of Radek Machan (Dr)" <[hidden email] on behalf of [hidden email]> wrote:

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    Dear All,
   
    One of our users is doing long-term time-lapse imaging in multi-well plates with reflection based autofocus. The sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked perfectly well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the oil immersion could have interfered  with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I'm wondering now, if the hypothesis of the bubble is correct, how likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second.
   
    What is your experience with the use of oil immersion in such experiments?
   
    Thanks! Best wishes,
    Radek
   
    Radek MACHAN, Ph.D. (Senior Research Fellow)
    SCELSE Advanced Biofilm Imaging Facility<http://www.scelse.sg/Page/imaging-facility> Manager
    Nanyang Technological University
    #B2, 60 Nanyang Drive, SBS-01N-27
    Singapore 637551
   
   
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[Sylvie Le Guyader]

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Hi Radek

If the sample is in water, I would use a water objective and have an oil with RI 1.33 as immersion medium. You will get better image quality since your sample is very likely to be thicker than 10um. On top of that the oil with low RI is less viscous so less likely to have bubbles.
I would apply the oil on the objective with a rod, not with a bottle that needs to be flipped and squeezed.
You will need to check that the hw autofocus works with the water objective.
Depending on how many weeks you are imaging, another reason for the loss of focus might be that the oil was spread too thin on the plate.

Med vänlig hälsning / Best regards

Sylvie

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Live Cell Imaging Facility Manager
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From: Confocal Microscopy List <[hidden email]> on behalf of Radek Machan (Dr) <[hidden email]>
Sent: Sunday, June 21, 2020 8:56:58 AM
To: [hidden email] <[hidden email]>
Subject: Multiple-position long-term time-lapse with oil immersion lens

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Dear All,

One of our users is doing long-term time-lapse imaging in multi-well plates with reflection based autofocus. The sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked perfectly well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the oil immersion could have interfered  with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I'm wondering now, if the hypothesis of the bubble is correct, how likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second.

What is your experience with the use of oil immersion in such experiments?

Thanks! Best wishes,
Radek

Radek MACHAN, Ph.D. (Senior Research Fellow)
SCELSE Advanced Biofilm Imaging Facility<https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.scelse.sg%2FPage%2Fimaging-facility&amp;data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Cfcdcbdbf32bf4863cd5808d815b05ec6%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637283194539194146&amp;sdata=4MCjV7DDUCjRkk2YJng7%2Bnt5gHwmmOpfBFeT8fy2h6c%3D&amp;reserved=0> Manager
Nanyang Technological University
#B2, 60 Nanyang Drive, SBS-01N-27
Singapore 637551


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[Montero Llopis, Paula De La Milagrosa]

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Hi Radek,

I think your hypothesis is very likely. Additionally what could have happened is that during the stage movement the oil was dragged and did not cover the top of the lens when the acquisition happened. Or the focal plane across the plate plane was not the same.

In general, multipoint timelapse with immersion oil is very challenging because of the issues you are bringing up. Plates aren’t completely flat either and slight changes in Z will either make the experiment fail or damage the lens.

Things you could do is to reduce the speed of the the stage, “paint” immersion oil on the glass (this will make a huge mess and can potentially damage the stage if oil gets inside), or determine a map of Z positions throughout the plate so that the nosepiece can adjust for the different focal plane and hopefully damage the lens less.

Thank you!

Paula

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________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Timo Kuijt <[hidden email]>
Sent: Sunday, June 21, 2020 8:59:33 AM
To: [hidden email] <[hidden email]>
Subject: Re: Multiple-position long-term time-lapse with oil immersion lens

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Dear Radek,

We have quite some experience with multi-well plates and immersion oil objective lenses. An air bubble could have caused the auto focus to have failed, but that excess or too little oil can cause the auto-focus system to fail as the oil runs off the lens or gets spread too thin shortly after starting acquisition. Most microscope vendors include the auto-focus status in the raw file metadata for each timepoint and position. You could check back and see if the auto-focus system failed by looking at the status code to trace if the focus or another malfunction occurred.

Here some tips that might prevent issue in the future:
- We use a folded piece of lenspaper to distribute a thin oil layer on the glass and remove the lenspaper on a part of the glass you will not image (air bubbles are introduced when lifting the paper off the glass!)
- Move around all xy positions a few times to evenly smear the immersion oil and then start setting up auto focus
- use 96w or 348w plates if possible and space samples close together, also this allows more conditions to be imaged simultaneously.
- slow the xy-stage movement down if hardware supports this and if it doesn't impact the experimental setup too much

Best  wishes,

Timo

Timo E.F. Kuijt, PhD
Oncode Institute | Kops lab
Hubrecht Institute | Room E2.22
Uppsalalaan 8 | 3584 CT Utrecht
The Netherlands
Tel: [phone]
P.O. Box 85164 | 3508 AD | Utrecht
The Netherlands

On 21/06/2020, 08:57, "Confocal Microscopy List on behalf of Radek Machan (Dr)" <[hidden email] on behalf of [hidden email]> wrote:

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    Dear All,

    One of our users is doing long-term time-lapse imaging in multi-well plates with reflection based autofocus. The sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked perfectly well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the oil immersion could have interfered  with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I'm wondering now, if the hypothesis of the bubble is correct, how likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second.

    What is your experience with the use of oil immersion in such experiments?

    Thanks! Best wishes,
    Radek

    Radek MACHAN, Ph.D. (Senior Research Fellow)
    SCELSE Advanced Biofilm Imaging Facility<http://www.scelse.sg/Page/imaging-facility> Manager
    Nanyang Technological University
    #B2, 60 Nanyang Drive, SBS-01N-27
    Singapore 637551


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[Tim Feinstein]

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Hi Radek,

I do that experiment quite a lot and yes, bubbles have been the bane of my existence.  Before I start an experiment I always do a quick bubble test before I do anything else - just move the stage around and look carefully at the meniscus.  You can spot bubbles because they move like bearings, at about half the speed the stage is moving.  If you spot a bubble, take the imaging chamber off and clean everything thoroughly, then oil it and check again.  You can sometimes remove a bubble with the edge of a lens tissue but more often that just makes things worse.  

Keep in mind that it doesn't have to be a bubble - sometimes these systems just lose their lock and never get it back.  For example, if you don't quite have enough oil then you can easily break the focus lock partway through an experiment.  Also not that if your system has the option to choose between continuous and point-by-point mode, continuous mode is a lot more likely to scotch the entire experiment if it momentarily loses a lock.  For this reason I generally use point-by-point mode for long term multipoint time lapse experiments, and continuous mode for higher-speed and short term acquisitions.  

Another non bubble-related problem has to do with how far the stage moves between points.  Most systems default their stage to move very fast between multipoints to save time, but moving fast over a long distance can break autofocus lock.  Think about this when you set up your multipoints - try to minimize how far the stage moves from point to point, and keep an eye on how far it has to move between your last point and the first point.  If necessary you should create 'dummy' positions between distant points so that the stage can pause and re-calibrate.  Nikon (no commercial interest) has a great feature that automatically re-orders the multipoints to minimize stage travel; with other scope makers you just have to keep all this in mind while you're setting up the experiment.   Slowing down the stage movement during multipoint helps a lot, especially with the continuous focus setting.  

If you are using an infrared beam autofocus (Nikon, Leica, etc) then I don't recommend 1.33 RI water-matched oil.  Those autofocus implementations need a refractive index mismatch at the coverslip, so they often struggle to get & hold a lock with water or water-mimicking oil immersion.  

I agree with others that it's a good idea to thoroughly spread the oil around before starting the experiment.  Also, use an objective heating collar and give the whole system an hour or two to thermally stabilize before you leave it unattended.  

All the best,


T

Timothy Feinstein, Ph.D.
Research Scientist
Department of Developmental Biology
University of Pittsburgh

 



On 6/21/20, 9:11 AM, "Confocal Microscopy List on behalf of Timo Kuijt" <[hidden email] on behalf of [hidden email]> wrote:

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    Dear Radek,
   
    We have quite some experience with multi-well plates and immersion oil objective lenses. An air bubble could have caused the auto focus to have failed, but that excess or too little oil can cause the auto-focus system to fail as the oil runs off the lens or gets spread too thin shortly after starting acquisition. Most microscope vendors include the auto-focus status in the raw file metadata for each timepoint and position. You could check back and see if the auto-focus system failed by looking at the status code to trace if the focus or another malfunction occurred.
   
    Here some tips that might prevent issue in the future:
    - We use a folded piece of lenspaper to distribute a thin oil layer on the glass and remove the lenspaper on a part of the glass you will not image (air bubbles are introduced when lifting the paper off the glass!)
    - Move around all xy positions a few times to evenly smear the immersion oil and then start setting up auto focus
    - use 96w or 348w plates if possible and space samples close together, also this allows more conditions to be imaged simultaneously.
    - slow the xy-stage movement down if hardware supports this and if it doesn't impact the experimental setup too much
   
    Best  wishes,
   
    Timo
   
    Timo E.F. Kuijt, PhD
    Oncode Institute | Kops lab
    Hubrecht Institute | Room E2.22
    Uppsalalaan 8 | 3584 CT Utrecht
    The Netherlands
    Tel: [phone]
    P.O. Box 85164 | 3508 AD | Utrecht
    The Netherlands                          
   
    On 21/06/2020, 08:57, "Confocal Microscopy List on behalf of Radek Machan (Dr)" <[hidden email] on behalf of [hidden email]> wrote:
   
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        Dear All,
       
        One of our users is doing long-term time-lapse imaging in multi-well plates with reflection based autofocus. The sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked perfectly well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the oil immersion could have interfered  with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I'm wondering now, if the hypothesis of the bubble is correct, how likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second.
       
        What is your experience with the use of oil immersion in such experiments?
       
        Thanks! Best wishes,
        Radek
       
        Radek MACHAN, Ph.D. (Senior Research Fellow)
        SCELSE Advanced Biofilm Imaging Facility<https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.scelse.sg%2FPage%2Fimaging-facility&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7C35fe2300425b4c36128908d815e4ac54%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C637283419180080427&amp;sdata=cTYvhaAyjO2j%2BMHVDFJmdPjFiVOW3OoO7hDF6riM5GM%3D&amp;reserved=0> Manager
        Nanyang Technological University
        #B2, 60 Nanyang Drive, SBS-01N-27
        Singapore 637551
       
       
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[Gaurav Joshi]

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Hi Radek,

In an exact set up as yours, I had encountered similar problems. To
overcome the issues I did the following
- Before putting the multiwell chamber on to the stage, I made sure that
the microscope was well equilibrated with the incubation chamber at 37C
(please see the note toward the end)
- The bottom part of the glass coverslip was coated with film of oil by
rolling the cylinder part of the glass tip applicator. In my experience,
using the glass tip applicator reduced the bubbles
- Oil was put on the objective and the plate was set on the stage
- A contact was made between the objective and the plate and the cells were
brought in focus
- The plate was allowed to equilibrate at 37C for 30 minutes
- All the region of interests on the multiwell plate were selected in the
software and the perfect focus (reflection based focus value) set for each
point. Because of slight slant, as I moved from well to well the focus
changed. Not waiting for 30 minutes to equilibrate also resulted in
incorrect reflect based focus values for the entirety of the experiment
- The plate was made to travel through this path manually a couple of times
to make sure that oil is evenly spread out and there is no possibility of a
bubble forming later due to an insufficient oil

Note: Initially, my experiments failed because the thermal drift with only
the stage top incubator was overwhelming for a reflection based system to
manage after a few hours. Using both the stage top incubator and an
incubator that encloses an entire microscope, I have done time-lapse for
upto 20 hours.

Happy to elaborate or clarify any of these points.

Best,
Gaurav.

*Gaurav Joshi, PhD*  |  Imaging Scientist
Health Sciences Research Building (HSRB)
EG72, 1760 Haygood Drive, Atlanta, GA 30322

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On Sun, Jun 21, 2020 at 2:57 AM Radek Machan (Dr) <[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.
> *****
>
> Dear All,
>
> One of our users is doing long-term time-lapse imaging in multi-well
> plates with reflection based autofocus. The sample is quite flat, so an oil
> immersion lens seemed the best choice in terms of light collection
> efficiency and image quality. Our first run worked perfectly well. However,
> during the second experiment the focusing failed quite early during the
> time-lapse and the sample remained out of focus for most of the experiment.
> I suspect that an air bubble in the oil immersion could have interfered
> with the focusing and because of high viscosity of the oil, a bubble can
> persist there for quite some time. I'm wondering now, if the hypothesis of
> the bubble is correct, how likely is such an incident to happen - whether
> we were particularly lucky in the first experiment or particularly unlucky
> in the second.
>
> What is your experience with the use of oil immersion in such experiments?
>
> Thanks! Best wishes,
> Radek
>
> Radek MACHAN, Ph.D. (Senior Research Fellow)
> SCELSE Advanced Biofilm Imaging Facility<
> http://www.scelse.sg/Page/imaging-facility> Manager
> Nanyang Technological University
> #B2, 60 Nanyang Drive, SBS-01N-27
> Singapore 637551
>
>
> ________________________________
>
> CONFIDENTIALITY: This email is intended solely for the person(s) named and
> may be confidential and/or privileged. If you are not the intended
> recipient, please delete it, notify us and do not copy, use, or disclose
> its contents.
> Towards a sustainable earth: Print only when necessary. Thank you.
>

[ramachan]

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Dear All,

Thank you for the suggestions. We'll implement them in our next experiment.

Best wishes,
Radek


Radek MACHAN, Ph.D. (Senior Research Fellow)
SCELSE Advanced Biofilm Imaging Facility<http://www.scelse.sg/Page/imaging-facility> Manager
Nanyang Technological University
#B2, 60 Nanyang Drive, SBS-01N-27
Singapore 637551


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From: Radek Machan (Dr)
Sent: Sunday, June 21, 2020 14:56
To: [hidden email] <[hidden email]>
Subject: Multiple-position long-term time-lapse with oil immersion lens

Dear All,

One of our users is doing long-term time-lapse imaging in multi-well plates with reflection based autofocus. The sample is quite flat, so an oil immersion lens seemed the best choice in terms of light collection efficiency and image quality. Our first run worked perfectly well. However, during the second experiment the focusing failed quite early during the time-lapse and the sample remained out of focus for most of the experiment. I suspect that an air bubble in the oil immersion could have interfered  with the focusing and because of high viscosity of the oil, a bubble can persist there for quite some time. I'm wondering now, if the hypothesis of the bubble is correct, how likely is such an incident to happen - whether we were particularly lucky in the first experiment or particularly unlucky in the second.

What is your experience with the use of oil immersion in such experiments?

Thanks! Best wishes,
Radek

Radek MACHAN, Ph.D. (Senior Research Fellow)
SCELSE Advanced Biofilm Imaging Facility<http://www.scelse.sg/Page/imaging-facility> Manager
Nanyang Technological University
#B2, 60 Nanyang Drive, SBS-01N-27
Singapore 637551


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