Long term water immersion imaging

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Adam White Adam White
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Long term water immersion imaging

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I did a search of the archive but I think I have exhausted most of the previous
suggestions... We are attempting to do some overnight live imaging
experiments using 2PE microscopy.  We are using the Nikon Apo LWD 25x/1.1
water immersion objective on an inverted stand and our main problem is
keeping the immersion medium in place for longer than a couple of hours.  We
have tried using the Cargille oil with a 1.335 RI but it does not have enough
viscosity/surface tension to be useful.  We have tried using ultrasound gel but
this dries out over time and only gives us about 5 hours of images.  I have also
tried all manner of different sealants/gloves/o-rings to varying degrees of
success/reproducibility.  The best I have found is to use a stretched-out glove
finger (plus a sealant) and just fill up the resulting "reservoir" with water.  This
has given us enough volume to get about 10 hours worth of imaging but is hard
to keep consistent.  Obviously, my question is whether someone has developed
or knows of a better system for doing this?  Perhaps a perfusion system or a
more durable glove-finger type solution?  I have seen the Leica system but this
won't fit our objective...  Any input you have to offer would be most
appreciated.

Best,
Adam


Adam B. White, Ph.D.
Confocal & Specialized Microscopy Shared Resource
Herbert Irving Comprehensive Cancer Center
Columbia University
1130 Saint Nicholas Ave, 222A
New York, NY 10032
Craig Brideau Craig Brideau
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Re: Long term water immersion imaging

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Wow, been there done that.  We've gone through all the trials and
tribulations you've outlined, including the ultrasound gel (so messy!).
 Basically the simplest way we've come up with (and we're only imaging for
a few hours, tops) has been a perfusion system.  Basically a good store of
solution and an peristaltic pump handling both input and output flows will
keep your sample from drying out.  The flow from the pump can cause some
vibration-like artifacts, so your chamber really needs dams and deflectors
to keep the pressure waves from moving things around.  It can work with
some fiddling though.

Craig


On Tue, Feb 19, 2013 at 1:01 PM, Adam White <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I did a search of the archive but I think I have exhausted most of the
> previous
> suggestions... We are attempting to do some overnight live imaging
> experiments using 2PE microscopy.  We are using the Nikon Apo LWD 25x/1.1
> water immersion objective on an inverted stand and our main problem is
> keeping the immersion medium in place for longer than a couple of hours.
>  We
> have tried using the Cargille oil with a 1.335 RI but it does not have
> enough
> viscosity/surface tension to be useful.  We have tried using ultrasound
> gel but
> this dries out over time and only gives us about 5 hours of images.  I
> have also
> tried all manner of different sealants/gloves/o-rings to varying degrees of
> success/reproducibility.  The best I have found is to use a stretched-out
> glove
> finger (plus a sealant) and just fill up the resulting "reservoir" with
> water.  This
> has given us enough volume to get about 10 hours worth of imaging but is
> hard
> to keep consistent.  Obviously, my question is whether someone has
> developed
> or knows of a better system for doing this?  Perhaps a perfusion system or
> a
> more durable glove-finger type solution?  I have seen the Leica system but
> this
> won't fit our objective...  Any input you have to offer would be most
> appreciated.
>
> Best,
> Adam
>
>
> Adam B. White, Ph.D.
> Confocal & Specialized Microscopy Shared Resource
> Herbert Irving Comprehensive Cancer Center
> Columbia University
> 1130 Saint Nicholas Ave, 222A
> New York, NY 10032
>
Chris Tully Chris Tully
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Re: Long term water immersion imaging

In reply to this post by Adam White
*****
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Adam,

I will suggest that you contact Biotechs (http://www.bioptechs.com). I have
previous previous experience selling their equipment, but no current ties
either as a sales rep or as a customer.

They make a coverslip bottom culture dish which can be heated and have
buffer perfused through it on a computer controlled schedule.

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-475-9753 (c)

[image: View my profile on LinkedIn]<http://www.linkedin.com/in/christully/>


On Tue, Feb 19, 2013 at 3:01 PM, Adam White <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I did a search of the archive but I think I have exhausted most of the
> previous
> suggestions... We are attempting to do some overnight live imaging
> experiments using 2PE microscopy.  We are using the Nikon Apo LWD 25x/1.1
> water immersion objective on an inverted stand and our main problem is
> keeping the immersion medium in place for longer than a couple of hours.
>  We
> have tried using the Cargille oil with a 1.335 RI but it does not have
> enough
> viscosity/surface tension to be useful.  We have tried using ultrasound
> gel but
> this dries out over time and only gives us about 5 hours of images.  I
> have also
> tried all manner of different sealants/gloves/o-rings to varying degrees of
> success/reproducibility.  The best I have found is to use a stretched-out
> glove
> finger (plus a sealant) and just fill up the resulting "reservoir" with
> water.  This
> has given us enough volume to get about 10 hours worth of imaging but is
> hard
> to keep consistent.  Obviously, my question is whether someone has
> developed
> or knows of a better system for doing this?  Perhaps a perfusion system or
> a
> more durable glove-finger type solution?  I have seen the Leica system but
> this
> won't fit our objective...  Any input you have to offer would be most
> appreciated.
>
> Best,
> Adam
>
>
> Adam B. White, Ph.D.
> Confocal & Specialized Microscopy Shared Resource
> Herbert Irving Comprehensive Cancer Center
> Columbia University
> 1130 Saint Nicholas Ave, 222A
> New York, NY 10032
>
Douglas Richardson Douglas Richardson
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Re: Long term water immersion imaging

In reply to this post by Adam White
*****
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*****

We're having luck using Zeiss Immersol 2010 W as a substitute immersion
media for water objectives in long term confocal/2photon imaging (
https://www.micro-shop.zeiss.com/?l=en&p=us&f=a&i=400000002130&h=25&n=0&sd=444969-0000-000).
It's refractive index is matched to water, but it's less-prone to
evaporation.  In cooperation with your glove finger, this might do the
trick.

-Doug

On Tue, Feb 19, 2013 at 3:01 PM, Adam White <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I did a search of the archive but I think I have exhausted most of the
> previous
> suggestions... We are attempting to do some overnight live imaging
> experiments using 2PE microscopy.  We are using the Nikon Apo LWD 25x/1.1
> water immersion objective on an inverted stand and our main problem is
> keeping the immersion medium in place for longer than a couple of hours.
>  We
> have tried using the Cargille oil with a 1.335 RI but it does not have
> enough
> viscosity/surface tension to be useful.  We have tried using ultrasound
> gel but
> this dries out over time and only gives us about 5 hours of images.  I
> have also
> tried all manner of different sealants/gloves/o-rings to varying degrees of
> success/reproducibility.  The best I have found is to use a stretched-out
> glove
> finger (plus a sealant) and just fill up the resulting "reservoir" with
> water.  This
> has given us enough volume to get about 10 hours worth of imaging but is
> hard
> to keep consistent.  Obviously, my question is whether someone has
> developed
> or knows of a better system for doing this?  Perhaps a perfusion system or
> a
> more durable glove-finger type solution?  I have seen the Leica system but
> this
> won't fit our objective...  Any input you have to offer would be most
> appreciated.
>
> Best,
> Adam
>
>
> Adam B. White, Ph.D.
> Confocal & Specialized Microscopy Shared Resource
> Herbert Irving Comprehensive Cancer Center
> Columbia University
> 1130 Saint Nicholas Ave, 222A
> New York, NY 10032
>
Benjamin Hibbs Benjamin Hibbs
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Re: Long term water immersion imaging

In reply to this post by Adam White
*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Adam,

I'm sure some of the other members of the list have more experience than I. However, perhaps you could try making a continual water source like the leica system. I know Steve Cody has developed some nifty techniques to maintain the immersion water automatically, but even a manual top-up every few hours could help you in conjunction with your reservoir approach.

Best of luck,

Ben


Ben Hibbs
Platform Support Officer—Advanced Fluorescence Imaging
The Melbourne Materials Institute (MMI)
University of Melbourne, Victoria 3010, Australia
Email: [hidden email]<mailto:[hidden email]>
Phone: +61 (0)3 9035-7749







On 20/02/2013, at 7:01 AM, Adam White <[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
*****

I did a search of the archive but I think I have exhausted most of the previous
suggestions... We are attempting to do some overnight live imaging
experiments using 2PE microscopy.  We are using the Nikon Apo LWD 25x/1.1
water immersion objective on an inverted stand and our main problem is
keeping the immersion medium in place for longer than a couple of hours.  We
have tried using the Cargille oil with a 1.335 RI but it does not have enough
viscosity/surface tension to be useful.  We have tried using ultrasound gel but
this dries out over time and only gives us about 5 hours of images.  I have also
tried all manner of different sealants/gloves/o-rings to varying degrees of
success/reproducibility.  The best I have found is to use a stretched-out glove
finger (plus a sealant) and just fill up the resulting "reservoir" with water.  This
has given us enough volume to get about 10 hours worth of imaging but is hard
to keep consistent.  Obviously, my question is whether someone has developed
or knows of a better system for doing this?  Perhaps a perfusion system or a
more durable glove-finger type solution?  I have seen the Leica system but this
won't fit our objective...  Any input you have to offer would be most
appreciated.

Best,
Adam


Adam B. White, Ph.D.
Confocal & Specialized Microscopy Shared Resource
Herbert Irving Comprehensive Cancer Center
Columbia University
1130 Saint Nicholas Ave, 222A
New York, NY 10032
leoncio vergara leoncio vergara
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Re: Long term water immersion imaging

In reply to this post by Craig Brideau
*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you just need enough water flow to overcome evaporation, it doesn't sound that you need an endless supply of water even for many hrs... maybe a push-pull syringe pump arrangement at minimum flow rate could do the same as the peristaltic pump without the vibrations. I have never tried any of this of course.

Leoncio

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, February 19, 2013 3:02 PM
To: [hidden email]
Subject: Re: Long term water immersion imaging

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

Wow, been there done that.  We've gone through all the trials and tribulations you've outlined, including the ultrasound gel (so messy!).
 Basically the simplest way we've come up with (and we're only imaging for a few hours, tops) has been a perfusion system.  Basically a good store of solution and an peristaltic pump handling both input and output flows will keep your sample from drying out.  The flow from the pump can cause some vibration-like artifacts, so your chamber really needs dams and deflectors to keep the pressure waves from moving things around.  It can work with some fiddling though.

Craig


On Tue, Feb 19, 2013 at 1:01 PM, Adam White <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I did a search of the archive but I think I have exhausted most of the
> previous suggestions... We are attempting to do some overnight live
> imaging experiments using 2PE microscopy.  We are using the Nikon Apo
> LWD 25x/1.1 water immersion objective on an inverted stand and our
> main problem is keeping the immersion medium in place for longer than
> a couple of hours.
>  We
> have tried using the Cargille oil with a 1.335 RI but it does not have
> enough viscosity/surface tension to be useful.  We have tried using
> ultrasound gel but this dries out over time and only gives us about 5
> hours of images.  I have also tried all manner of different
> sealants/gloves/o-rings to varying degrees of success/reproducibility.  
> The best I have found is to use a stretched-out glove finger (plus a
> sealant) and just fill up the resulting "reservoir" with water.  This
> has given us enough volume to get about 10 hours worth of imaging but
> is hard to keep consistent.  Obviously, my question is whether someone
> has developed or knows of a better system for doing this?  Perhaps a
> perfusion system or a more durable glove-finger type solution?  I have
> seen the Leica system but this won't fit our objective...  Any input
> you have to offer would be most appreciated.
>
> Best,
> Adam
>
>
> Adam B. White, Ph.D.
> Confocal & Specialized Microscopy Shared Resource Herbert Irving
> Comprehensive Cancer Center Columbia University
> 1130 Saint Nicholas Ave, 222A
> New York, NY 10032
>
Watkins, Simon C Watkins, Simon C
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Re: Long term water immersion imaging

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

For us genteal gel
http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBIS7
Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in some
conditions.. Though this was within an humidified chamber

Simon Watkins Ph.D

Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
University of Pittsburgh
Bsts 225 3550 terrace st
Pittsburgh PA 15261
Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
412-352-2277






On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Adam,
>
>I'm sure some of the other members of the list have more experience than
>I. However, perhaps you could try making a continual water source like
>the leica system. I know Steve Cody has developed some nifty techniques
>to maintain the immersion water automatically, but even a manual top-up
>every few hours could help you in conjunction with your reservoir
>approach.
>
>Best of luck,
>
>Ben
>
>
>Ben Hibbs
>Platform Support Officer‹Advanced Fluorescence Imaging
>The Melbourne Materials Institute (MMI)
>University of Melbourne, Victoria 3010, Australia
>Email: [hidden email]<mailto:[hidden email]>
>Phone: +61 (0)3 9035-7749
>
>
>
>
>
>
>
>On 20/02/2013, at 7:01 AM, Adam White
><[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
>*****
>
>I did a search of the archive but I think I have exhausted most of the
>previous
>suggestions... We are attempting to do some overnight live imaging
>experiments using 2PE microscopy.  We are using the Nikon Apo LWD 25x/1.1
>water immersion objective on an inverted stand and our main problem is
>keeping the immersion medium in place for longer than a couple of hours.
>We
>have tried using the Cargille oil with a 1.335 RI but it does not have
>enough
>viscosity/surface tension to be useful.  We have tried using ultrasound
>gel but
>this dries out over time and only gives us about 5 hours of images.  I
>have also
>tried all manner of different sealants/gloves/o-rings to varying degrees
>of
>success/reproducibility.  The best I have found is to use a stretched-out
>glove
>finger (plus a sealant) and just fill up the resulting "reservoir" with
>water.  This
>has given us enough volume to get about 10 hours worth of imaging but is
>hard
>to keep consistent.  Obviously, my question is whether someone has
>developed
>or knows of a better system for doing this?  Perhaps a perfusion system
>or a
>more durable glove-finger type solution?  I have seen the Leica system
>but this
>won't fit our objective...  Any input you have to offer would be most
>appreciated.
>
>Best,
>Adam
>
>
>Adam B. White, Ph.D.
>Confocal & Specialized Microscopy Shared Resource
>Herbert Irving Comprehensive Cancer Center
>Columbia University
>1130 Saint Nicholas Ave, 222A
>New York, NY 10032
Periasamy, Ammasi (ap3t) Periasamy, Ammasi (ap3t)
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Re: Long term water immersion imaging

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

Dear All
For water immersion lens, yes if you use water, particularly for 2photon imaging, it will evaporate within few minutes. We went through this for 2p imaging. I thought there should be a medium of refractive index same as water. I discussed this issue with our Zeiss sales representative. She came to my office next day and provided valuable information. The zeiss sells an immersion oil and its refractive index is same as water. We have been using this for 2photon or Confocal time lapse imaging (24 hrs) since 2008. No problem and the image quality is great. I am not sure anyone raised this question before in the list server. I apologize for missing this kind of question/help. The price is super high compared to the regular immersion oil. The price is about $125-150. Here is the part#
000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml
Visit the Zeiss market place web site to order it. www.micro-shop.zeiss.com/us/us_en
Hope this helps.
Best wishes,
Ammasi


Dr. Ammasi Periasamy
Professor & Center Director
W.M. Keck Center for Cellular Imaging (KCCI)
(A University Imaging Center)
Biology, University of Virginia
Mail or FedEx: 485 McCormick Rd.
Charlottesville, VA 22904.
Office Location: Physical Life Sciences Building (B005)
90, Geldard Drive, Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:434-982-5210; Email:[hidden email]
http://www.kcci.virginia.edu/contact/peri.php
************************
12th Annual Workshop on FRET Microscopy, March 11-16, 2013
http://www.kcci.virginia.edu/workshop/workshop2013/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Watkins, Simon C
Sent: Tuesday, February 19, 2013 10:00 PM
To: [hidden email]
Subject: Re: Long term water immersion imaging

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

For us genteal gel
http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBIS7
Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in some conditions.. Though this was within an humidified chamber

Simon Watkins Ph.D

Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
University of Pittsburgh
Bsts 225 3550 terrace st
Pittsburgh PA 15261
Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
412-352-2277






On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Adam,
>
>I'm sure some of the other members of the list have more experience
>than I. However, perhaps you could try making a continual water source
>like the leica system. I know Steve Cody has developed some nifty
>techniques to maintain the immersion water automatically, but even a
>manual top-up every few hours could help you in conjunction with your
>reservoir approach.
>
>Best of luck,
>
>Ben
>
>
>Ben Hibbs
>Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>Materials Institute (MMI) University of Melbourne, Victoria 3010,
>Australia
>Email:
>[hidden email]<mailto:[hidden email]>
>Phone: +61 (0)3 9035-7749
>
>
>
>
>
>
>
>On 20/02/2013, at 7:01 AM, Adam White
><[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
>*****
>
>I did a search of the archive but I think I have exhausted most of the
>previous suggestions... We are attempting to do some overnight live
>imaging experiments using 2PE microscopy.  We are using the Nikon Apo
>LWD 25x/1.1 water immersion objective on an inverted stand and our main
>problem is keeping the immersion medium in place for longer than a
>couple of hours.
>We
>have tried using the Cargille oil with a 1.335 RI but it does not have
>enough viscosity/surface tension to be useful.  We have tried using
>ultrasound gel but this dries out over time and only gives us about 5
>hours of images.  I have also tried all manner of different
>sealants/gloves/o-rings to varying degrees of success/reproducibility.  
>The best I have found is to use a stretched-out glove finger (plus a
>sealant) and just fill up the resulting "reservoir" with water.  This
>has given us enough volume to get about 10 hours worth of imaging but
>is hard to keep consistent.  Obviously, my question is whether someone
>has developed or knows of a better system for doing this?  Perhaps a
>perfusion system or a more durable glove-finger type solution?  I have
>seen the Leica system but this won't fit our objective...  Any input
>you have to offer would be most appreciated.
>
>Best,
>Adam
>
>
>Adam B. White, Ph.D.
>Confocal & Specialized Microscopy Shared Resource Herbert Irving
>Comprehensive Cancer Center Columbia University
>1130 Saint Nicholas Ave, 222A
>New York, NY 10032
Dmitry Sokolov Dmitry Sokolov
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Re: Long term water immersion imaging

In reply to this post by leoncio vergara
*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

What about a closed but not sealed large shallow reservoir levelled with
Petri dish?

I tried to summarise the options at MIAWiki Knowledge Network:
http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20water%20immersion%20imaging

The other options and comments will be appreciated. They can be left at
the bottom of the page.

Cheers,
Dmitry

*Advanced Knowledge Management*
for *MICROSCOPY *and *Image Analysis *
------------------------------------------------------------------------
*Dmitry Sokolov*, Ph.D.
Mob: *+64 21 063 5382***
[hidden email] <mailto:[hidden email]>



20.02.2013 15:07, Vergara, Leoncio A. ?????:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you just need enough water flow to overcome evaporation, it doesn't sound that you need an endless supply of water even for many hrs... maybe a push-pull syringe pump arrangement at minimum flow rate could do the same as the peristaltic pump without the vibrations. I have never tried any of this of course.
>
> Leoncio
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Tuesday, February 19, 2013 3:02 PM
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Wow, been there done that.  We've gone through all the trials and tribulations you've outlined, including the ultrasound gel (so messy!).
>   Basically the simplest way we've come up with (and we're only imaging for a few hours, tops) has been a perfusion system.  Basically a good store of solution and an peristaltic pump handling both input and output flows will keep your sample from drying out.  The flow from the pump can cause some vibration-like artifacts, so your chamber really needs dams and deflectors to keep the pressure waves from moving things around.  It can work with some fiddling though.
>
> Craig
>
>
> On Tue, Feb 19, 2013 at 1:01 PM, Adam White <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I did a search of the archive but I think I have exhausted most of the
>> previous suggestions... We are attempting to do some overnight live
>> imaging experiments using 2PE microscopy.  We are using the Nikon Apo
>> LWD 25x/1.1 water immersion objective on an inverted stand and our
>> main problem is keeping the immersion medium in place for longer than
>> a couple of hours.
>>   We
>> have tried using the Cargille oil with a 1.335 RI but it does not have
>> enough viscosity/surface tension to be useful.  We have tried using
>> ultrasound gel but this dries out over time and only gives us about 5
>> hours of images.  I have also tried all manner of different
>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
>> The best I have found is to use a stretched-out glove finger (plus a
>> sealant) and just fill up the resulting "reservoir" with water.  This
>> has given us enough volume to get about 10 hours worth of imaging but
>> is hard to keep consistent.  Obviously, my question is whether someone
>> has developed or knows of a better system for doing this?  Perhaps a
>> perfusion system or a more durable glove-finger type solution?  I have
>> seen the Leica system but this won't fit our objective...  Any input
>> you have to offer would be most appreciated.
>>
>> Best,
>> Adam
>>
>>
>> Adam B. White, Ph.D.
>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
>> Comprehensive Cancer Center Columbia University
>> 1130 Saint Nicholas Ave, 222A
>> New York, NY 10032
>>
Periasamy, Ammasi (ap3t) Periasamy, Ammasi (ap3t)
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Re: Long term water immersion imaging

In reply to this post by Periasamy, Ammasi (ap3t)
*****
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*****

Sorry, I forgot to mention...
This water immersion oil can be used for any commercially available objective lens.
We are using this for Nikon, Leica and Olympus. It works.
Good luck.
Ammasi


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
Sent: Tuesday, February 19, 2013 10:17 PM
To: [hidden email]
Subject: Re: Long term water immersion imaging

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

Dear All
For water immersion lens, yes if you use water, particularly for 2photon imaging, it will evaporate within few minutes. We went through this for 2p imaging. I thought there should be a medium of refractive index same as water. I discussed this issue with our Zeiss sales representative. She came to my office next day and provided valuable information. The zeiss sells an immersion oil and its refractive index is same as water. We have been using this for 2photon or Confocal time lapse imaging (24 hrs) since 2008. No problem and the image quality is great. I am not sure anyone raised this question before in the list server. I apologize for missing this kind of question/help. The price is super high compared to the regular immersion oil. The price is about $125-150. Here is the part#
000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the Zeiss market place web site to order it. www.micro-shop.zeiss.com/us/us_en Hope this helps.
Best wishes,
Ammasi


Dr. Ammasi Periasamy
Professor & Center Director
W.M. Keck Center for Cellular Imaging (KCCI)
(A University Imaging Center)
Biology, University of Virginia
Mail or FedEx: 485 McCormick Rd.
Charlottesville, VA 22904.
Office Location: Physical Life Sciences Building (B005)
90, Geldard Drive, Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:434-982-5210; Email:[hidden email]
http://www.kcci.virginia.edu/contact/peri.php
************************
12th Annual Workshop on FRET Microscopy, March 11-16, 2013
http://www.kcci.virginia.edu/workshop/workshop2013/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Watkins, Simon C
Sent: Tuesday, February 19, 2013 10:00 PM
To: [hidden email]
Subject: Re: Long term water immersion imaging

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

For us genteal gel
http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBIS7
Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in some conditions.. Though this was within an humidified chamber

Simon Watkins Ph.D

Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
University of Pittsburgh
Bsts 225 3550 terrace st
Pittsburgh PA 15261
Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
412-352-2277






On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Adam,
>
>I'm sure some of the other members of the list have more experience
>than I. However, perhaps you could try making a continual water source
>like the leica system. I know Steve Cody has developed some nifty
>techniques to maintain the immersion water automatically, but even a
>manual top-up every few hours could help you in conjunction with your
>reservoir approach.
>
>Best of luck,
>
>Ben
>
>
>Ben Hibbs
>Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>Materials Institute (MMI) University of Melbourne, Victoria 3010,
>Australia
>Email:
>[hidden email]<mailto:[hidden email]>
>Phone: +61 (0)3 9035-7749
>
>
>
>
>
>
>
>On 20/02/2013, at 7:01 AM, Adam White
><[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
>*****
>
>I did a search of the archive but I think I have exhausted most of the
>previous suggestions... We are attempting to do some overnight live
>imaging experiments using 2PE microscopy.  We are using the Nikon Apo
>LWD 25x/1.1 water immersion objective on an inverted stand and our main
>problem is keeping the immersion medium in place for longer than a
>couple of hours.
>We
>have tried using the Cargille oil with a 1.335 RI but it does not have
>enough viscosity/surface tension to be useful.  We have tried using
>ultrasound gel but this dries out over time and only gives us about 5
>hours of images.  I have also tried all manner of different
>sealants/gloves/o-rings to varying degrees of success/reproducibility.  
>The best I have found is to use a stretched-out glove finger (plus a
>sealant) and just fill up the resulting "reservoir" with water.  This
>has given us enough volume to get about 10 hours worth of imaging but
>is hard to keep consistent.  Obviously, my question is whether someone
>has developed or knows of a better system for doing this?  Perhaps a
>perfusion system or a more durable glove-finger type solution?  I have
>seen the Leica system but this won't fit our objective...  Any input
>you have to offer would be most appreciated.
>
>Best,
>Adam
>
>
>Adam B. White, Ph.D.
>Confocal & Specialized Microscopy Shared Resource Herbert Irving
>Comprehensive Cancer Center Columbia University
>1130 Saint Nicholas Ave, 222A
>New York, NY 10032
Dmitry Sokolov Dmitry Sokolov
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Re: Long term water immersion imaging

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

Hi Ammasi,

sorry, I probably missed how the immersion oil was applied: on the top
of water or on the top of sample:
http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20water%20immersion%20imaging

Thank you beforehand,
Dmitry

*Advanced Knowledge Management*
for *MICROSCOPY *and *Image Analysis *
------------------------------------------------------------------------
*Dmitry Sokolov*, Ph.D.
Mob: *+64 21 063 5382***
[hidden email] <mailto:[hidden email]>

20.02.2013 16:19, Periasamy, Ammasi (ap3t) ?????:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sorry, I forgot to mention...
> This water immersion oil can be used for any commercially available objective lens.
> We are using this for Nikon, Leica and Olympus. It works.
> Good luck.
> Ammasi
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, February 19, 2013 10:17 PM
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All
> For water immersion lens, yes if you use water, particularly for 2photon imaging, it will evaporate within few minutes. We went through this for 2p imaging. I thought there should be a medium of refractive index same as water. I discussed this issue with our Zeiss sales representative. She came to my office next day and provided valuable information. The zeiss sells an immersion oil and its refractive index is same as water. We have been using this for 2photon or Confocal time lapse imaging (24 hrs) since 2008. No problem and the image quality is great. I am not sure anyone raised this question before in the list server. I apologize for missing this kind of question/help. The price is super high compared to the regular immersion oil. The price is about $125-150. Here is the part#
> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the Zeiss market place web site to order it. www.micro-shop.zeiss.com/us/us_en Hope this helps.
> Best wishes,
> Ammasi
>
>
> Dr. Ammasi Periasamy
> Professor & Center Director
> W.M. Keck Center for Cellular Imaging (KCCI)
> (A University Imaging Center)
> Biology, University of Virginia
> Mail or FedEx: 485 McCormick Rd.
> Charlottesville, VA 22904.
> Office Location: Physical Life Sciences Building (B005)
> 90, Geldard Drive, Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab)
> Fax:434-982-5210; Email:[hidden email]
> http://www.kcci.virginia.edu/contact/peri.php
> ************************
> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Watkins, Simon C
> Sent: Tuesday, February 19, 2013 10:00 PM
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For us genteal gel
> http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBIS7
> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in some conditions.. Though this was within an humidified chamber
>
> Simon Watkins Ph.D
>
> Professor and Vice Chair Cell Biology
> Professor Immunology
> Director Center for Biologic Imaging
> University of Pittsburgh
> Bsts 225 3550 terrace st
> Pittsburgh PA 15261
> Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
> 412-352-2277
>
>
>
>
>
>
> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Adam,
>>
>> I'm sure some of the other members of the list have more experience
>> than I. However, perhaps you could try making a continual water source
>> like the leica system. I know Steve Cody has developed some nifty
>> techniques to maintain the immersion water automatically, but even a
>> manual top-up every few hours could help you in conjunction with your
>> reservoir approach.
>>
>> Best of luck,
>>
>> Ben
>>
>>
>> Ben Hibbs
>> Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>> Materials Institute (MMI) University of Melbourne, Victoria 3010,
>> Australia
>> Email:
>> [hidden email]<mailto:[hidden email]>
>> Phone: +61 (0)3 9035-7749
>>
>>
>>
>>
>>
>>
>>
>> On 20/02/2013, at 7:01 AM, Adam White
>> <[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
>> *****
>>
>> I did a search of the archive but I think I have exhausted most of the
>> previous suggestions... We are attempting to do some overnight live
>> imaging experiments using 2PE microscopy.  We are using the Nikon Apo
>> LWD 25x/1.1 water immersion objective on an inverted stand and our main
>> problem is keeping the immersion medium in place for longer than a
>> couple of hours.
>> We
>> have tried using the Cargille oil with a 1.335 RI but it does not have
>> enough viscosity/surface tension to be useful.  We have tried using
>> ultrasound gel but this dries out over time and only gives us about 5
>> hours of images.  I have also tried all manner of different
>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
>> The best I have found is to use a stretched-out glove finger (plus a
>> sealant) and just fill up the resulting "reservoir" with water.  This
>> has given us enough volume to get about 10 hours worth of imaging but
>> is hard to keep consistent.  Obviously, my question is whether someone
>> has developed or knows of a better system for doing this?  Perhaps a
>> perfusion system or a more durable glove-finger type solution?  I have
>> seen the Leica system but this won't fit our objective...  Any input
>> you have to offer would be most appreciated.
>>
>> Best,
>> Adam
>>
>>
>> Adam B. White, Ph.D.
>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
>> Comprehensive Cancer Center Columbia University
>> 1130 Saint Nicholas Ave, 222A
>> New York, NY 10032
Martin Wessendorf-2 Martin Wessendorf-2
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Re: Long term water immersion imaging

In reply to this post by Craig Brideau
*****
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*****

Craig et al.--

Any chance that a siphon system could be used?  A siphon has the
advantage over a peristaltic pump of not introducing mechanical or
electrical noise.  THe design I'm thinking of is hard to describe so
I've uploaded a crude sketch onto my University web site; it's available
here: https://www.myu.umn.edu/metadot/index.pl?iid=610740

--The design is one I learned from John Williams, a slice
electrophysiologist at OHSU--he uses it to supply baths on his
electrophys rigs.  The siphon is made from a syringe barrel (e.g. 60 ml)
with a single-hole stopper placed in the end.  A piece of glass tubing
is placed through the stopper into the water.  **The placement of the
bottom end of the tubing defines the height of the pressure-head and
should be approximately at the same level as the top of the lens--this
is a key feature and should provide fairly fine control over the flow
going to the lens.  As John uses it in tissue baths, suction is used to
remove excess water.  Not sure that that would be necessary for
microscopy, since the flow rate would be close to zero.

Mind you, *I've* never tried this on a microscope but I wonder if it
might work.

Martin

On 2/19/2013 3:02 PM, Craig Brideau wrote:

> Wow, been there done that.  We've gone through all the trials and
> tribulations you've outlined, including the ultrasound gel (so messy!).
>   Basically the simplest way we've come up with (and we're only imaging for
> a few hours, tops) has been a perfusion system.  Basically a good store of
> solution and an peristaltic pump handling both input and output flows will
> keep your sample from drying out.  The flow from the pump can cause some
> vibration-like artifacts, so your chamber really needs dams and deflectors
> to keep the pressure waves from moving things around.  It can work with
> some fiddling though.
>
> Craig


--
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
University of Minnesota             Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
Minneapolis, MN  55455                    e-mail: [hidden email]
Pascal Weber Pascal Weber
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Re: Long term water immersion imaging

In reply to this post by Adam White
*****
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*****

Few years i did this. And for that i used a silicon ring (from Zeiss) to keep the
water between the front lens and the specimen. I made an acquisition more than
96hrs without to refill it.
Sylvie Le Guyader Sylvie Le Guyader
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Re: Long term water immersion imaging

In reply to this post by Periasamy, Ammasi (ap3t)
*****
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*****

Hi there

The main problem with the Cargille or Zeiss oil for water immersion objectives is that it is a lot more fluid than immersion oil for oil objectives. In our hands it works very well on upright systems or shortish term imaging on an inverted system but it ends up running along the objectives on an inverted system especially in a warm incubator.

As mentioned in the past Steve Cody has generously shared his protocol on how to build a water dispenser for inverted microscopes. He sent it offline to interested people (including us) so you will not find it in the archive but you should ask him for it (or to give me the permission to send it to you).

Good luck! :)

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Hälsovägen 7
14157 Huddinge
Sweden
office: +46 (0) 8 5248 1107
LCI room: +46 (0) 8 5248 1172
mobile: +46 (0) 73 733 5008


> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Periasamy,
> Ammasi (ap3t)
> Sent: 20 February 2013 04:17
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All
> For water immersion lens, yes if you use water, particularly for 2photon imaging, it
> will evaporate within few minutes. We went through this for 2p imaging. I thought
> there should be a medium of refractive index same as water. I discussed this issue
> with our Zeiss sales representative. She came to my office next day and provided
> valuable information. The zeiss sells an immersion oil and its refractive index is
> same as water. We have been using this for 2photon or Confocal time lapse
> imaging (24 hrs) since 2008. No problem and the image quality is great. I am not
> sure anyone raised this question before in the list server. I apologize for missing
> this kind of question/help. The price is super high compared to the regular
> immersion oil. The price is about $125-150. Here is the part#
> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the Zeiss
> market place web site to order it. www.micro-shop.zeiss.com/us/us_en Hope this
> helps.
> Best wishes,
> Ammasi
>
>
> Dr. Ammasi Periasamy
> Professor & Center Director
> W.M. Keck Center for Cellular Imaging (KCCI)
> (A University Imaging Center)
> Biology, University of Virginia
> Mail or FedEx: 485 McCormick Rd.
> Charlottesville, VA 22904.
> Office Location: Physical Life Sciences Building (B005)
> 90, Geldard Drive, Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab)
> Fax:434-982-5210; Email:[hidden email]
> http://www.kcci.virginia.edu/contact/peri.php
> ************************
> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Watkins,
> Simon C
> Sent: Tuesday, February 19, 2013 10:00 PM
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For us genteal gel
> http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBIS7
> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in some
> conditions.. Though this was within an humidified chamber
>
> Simon Watkins Ph.D
>
> Professor and Vice Chair Cell Biology
> Professor Immunology
> Director Center for Biologic Imaging
> University of Pittsburgh
> Bsts 225 3550 terrace st
> Pittsburgh PA 15261
> Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
> 412-352-2277
>
>
>
>
>
>
> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]>
> wrote:
>
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >*****
> >
> >Hi Adam,
> >
> >I'm sure some of the other members of the list have more experience
> >than I. However, perhaps you could try making a continual water source
> >like the leica system. I know Steve Cody has developed some nifty
> >techniques to maintain the immersion water automatically, but even a
> >manual top-up every few hours could help you in conjunction with your
> >reservoir approach.
> >
> >Best of luck,
> >
> >Ben
> >
> >
> >Ben Hibbs
> >Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
> >Materials Institute (MMI) University of Melbourne, Victoria 3010,
> >Australia
> >Email:
> >[hidden email]<mailto:[hidden email]>
> >Phone: +61 (0)3 9035-7749
> >
> >
> >
> >
> >
> >
> >
> >On 20/02/2013, at 7:01 AM, Adam White
> ><[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
> >*****
> >
> >I did a search of the archive but I think I have exhausted most of the
> >previous suggestions... We are attempting to do some overnight live
> >imaging experiments using 2PE microscopy.  We are using the Nikon Apo
> >LWD 25x/1.1 water immersion objective on an inverted stand and our main
> >problem is keeping the immersion medium in place for longer than a
> >couple of hours.
> >We
> >have tried using the Cargille oil with a 1.335 RI but it does not have
> >enough viscosity/surface tension to be useful.  We have tried using
> >ultrasound gel but this dries out over time and only gives us about 5
> >hours of images.  I have also tried all manner of different
> >sealants/gloves/o-rings to varying degrees of success/reproducibility.
> >The best I have found is to use a stretched-out glove finger (plus a
> >sealant) and just fill up the resulting "reservoir" with water.  This
> >has given us enough volume to get about 10 hours worth of imaging but
> >is hard to keep consistent.  Obviously, my question is whether someone
> >has developed or knows of a better system for doing this?  Perhaps a
> >perfusion system or a more durable glove-finger type solution?  I have
> >seen the Leica system but this won't fit our objective...  Any input
> >you have to offer would be most appreciated.
> >
> >Best,
> >Adam
> >
> >
> >Adam B. White, Ph.D.
> >Confocal & Specialized Microscopy Shared Resource Herbert Irving
> >Comprehensive Cancer Center Columbia University
> >1130 Saint Nicholas Ave, 222A
> >New York, NY 10032
Pascal Weber Pascal Weber
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Re: Long term water immersion imaging

In reply to this post by Adam White
*****
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*****

It is still very expensive to use a water dispenser.
If you use the silicone collar (from Zeiss) that cost only a few euro. In addition it
homogenizes the temperature.
The real problem is the media itself and if you move your sample. Eg boxes, slides
or other.

I can send picture if someone need it.
Armstrong, Brian Armstrong, Brian
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Re: Long term water immersion imaging

In reply to this post by Dmitry Sokolov
*****
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*****

Hello, I think the problem is that these LWD 2P lenses are huge and need a large volume of water for the long working distance. The Zeiss immersion media will not hold surface tension in that volume (already mentioned [although works great for lenses such as 63x/1.2W]). What we did was purchase a variety of rubber washers in a "plumbing kit", the cost was a few dollars. We glued a rubber washer to the slide (silicone, VALAP, cyanoacrylate will all work here) and filled it with water. We imaged with this configuration over several days. A few years back I believe there was a thread on this listserve about using condoms as water dams for this purpose. The size of the condom will depend on the size of the objective. You can cut the condom to suit your needs.
Cheers,  

Brian D Armstrong PhD

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dmitry Sokolov
Sent: Tuesday, February 19, 2013 8:53 PM
To: [hidden email]
Subject: Re: Long term water immersion imaging

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

Hi Ammasi,

sorry, I probably missed how the immersion oil was applied: on the top
of water or on the top of sample:
http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20water%20immersion%20imaging

Thank you beforehand,
Dmitry

*Advanced Knowledge Management*
for *MICROSCOPY *and *Image Analysis *
------------------------------------------------------------------------
*Dmitry Sokolov*, Ph.D.
Mob: *+64 21 063 5382***
[hidden email] <mailto:[hidden email]>

20.02.2013 16:19, Periasamy, Ammasi (ap3t) ?????:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Sorry, I forgot to mention...
> This water immersion oil can be used for any commercially available objective lens.
> We are using this for Nikon, Leica and Olympus. It works.
> Good luck.
> Ammasi
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
> Sent: Tuesday, February 19, 2013 10:17 PM
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All
> For water immersion lens, yes if you use water, particularly for 2photon imaging, it will evaporate within few minutes. We went through this for 2p imaging. I thought there should be a medium of refractive index same as water. I discussed this issue with our Zeiss sales representative. She came to my office next day and provided valuable information. The zeiss sells an immersion oil and its refractive index is same as water. We have been using this for 2photon or Confocal time lapse imaging (24 hrs) since 2008. No problem and the image quality is great. I am not sure anyone raised this question before in the list server. I apologize for missing this kind of question/help. The price is super high compared to the regular immersion oil. The price is about $125-150. Here is the part#
> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the Zeiss market place web site to order it. www.micro-shop.zeiss.com/us/us_en Hope this helps.
> Best wishes,
> Ammasi
>
>
> Dr. Ammasi Periasamy
> Professor & Center Director
> W.M. Keck Center for Cellular Imaging (KCCI)
> (A University Imaging Center)
> Biology, University of Virginia
> Mail or FedEx: 485 McCormick Rd.
> Charlottesville, VA 22904.
> Office Location: Physical Life Sciences Building (B005)
> 90, Geldard Drive, Charlottesville, VA 22904
> Voice: 434-243-7602 (Office); 982-4869 (lab)
> Fax:434-982-5210; Email:[hidden email]
> http://www.kcci.virginia.edu/contact/peri.php
> ************************
> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
> *************************
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Watkins, Simon C
> Sent: Tuesday, February 19, 2013 10:00 PM
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For us genteal gel
> http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBIS7
> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in some conditions.. Though this was within an humidified chamber
>
> Simon Watkins Ph.D
>
> Professor and Vice Chair Cell Biology
> Professor Immunology
> Director Center for Biologic Imaging
> University of Pittsburgh
> Bsts 225 3550 terrace st
> Pittsburgh PA 15261
> Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
> 412-352-2277
>
>
>
>
>
>
> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Adam,
>>
>> I'm sure some of the other members of the list have more experience
>> than I. However, perhaps you could try making a continual water source
>> like the leica system. I know Steve Cody has developed some nifty
>> techniques to maintain the immersion water automatically, but even a
>> manual top-up every few hours could help you in conjunction with your
>> reservoir approach.
>>
>> Best of luck,
>>
>> Ben
>>
>>
>> Ben Hibbs
>> Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>> Materials Institute (MMI) University of Melbourne, Victoria 3010,
>> Australia
>> Email:
>> [hidden email]<mailto:[hidden email]>
>> Phone: +61 (0)3 9035-7749
>>
>>
>>
>>
>>
>>
>>
>> On 20/02/2013, at 7:01 AM, Adam White
>> <[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
>> *****
>>
>> I did a search of the archive but I think I have exhausted most of the
>> previous suggestions... We are attempting to do some overnight live
>> imaging experiments using 2PE microscopy.  We are using the Nikon Apo
>> LWD 25x/1.1 water immersion objective on an inverted stand and our main
>> problem is keeping the immersion medium in place for longer than a
>> couple of hours.
>> We
>> have tried using the Cargille oil with a 1.335 RI but it does not have
>> enough viscosity/surface tension to be useful.  We have tried using
>> ultrasound gel but this dries out over time and only gives us about 5
>> hours of images.  I have also tried all manner of different
>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
>> The best I have found is to use a stretched-out glove finger (plus a
>> sealant) and just fill up the resulting "reservoir" with water.  This
>> has given us enough volume to get about 10 hours worth of imaging but
>> is hard to keep consistent.  Obviously, my question is whether someone
>> has developed or knows of a better system for doing this?  Perhaps a
>> perfusion system or a more durable glove-finger type solution?  I have
>> seen the Leica system but this won't fit our objective...  Any input
>> you have to offer would be most appreciated.
>>
>> Best,
>> Adam
>>
>>
>> Adam B. White, Ph.D.
>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
>> Comprehensive Cancer Center Columbia University
>> 1130 Saint Nicholas Ave, 222A
>> New York, NY 10032


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Watkins, Simon C Watkins, Simon C
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Re: Long term water immersion imaging

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

I guess my last comment on the use of Genteal was a little cursory.  This
is a water based gel that was developed for folks with chronic dry eyes.
It has the same RI as water and works extremely well for long term
experiments with the new objectives (at least Oly and Nikon). It does work
better when the sample is mounted in a humidified chamber and allows the
full working distance of the lens to be used which generally will not work
with the water RI oils which simply do not generate sufficient surface
tension.  The best thing about Genteal, is that its an over the counter
medication and available from Amazon (see my earlier post)  which means it
works, and is in-expensive.  Working time for the stuff is about 24 hours
with a humidified chamber.  This also assumes the stage is being moved
around to set positions over time, which is very difficult to do with oil
As a last comment, as Genteal is water based, cleaning etc is a doddle
which can be a problem with the water RI oils.


Simon Watkins Ph.D

Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
University of Pittsburgh
Bsts 225 3550 terrace st
Pittsburgh PA 15261
Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
412-352-2277






On 2/20/13 11:40 AM, "Armstrong, Brian" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello, I think the problem is that these LWD 2P lenses are huge and need
>a large volume of water for the long working distance. The Zeiss
>immersion media will not hold surface tension in that volume (already
>mentioned [although works great for lenses such as 63x/1.2W]). What we
>did was purchase a variety of rubber washers in a "plumbing kit", the
>cost was a few dollars. We glued a rubber washer to the slide (silicone,
>VALAP, cyanoacrylate will all work here) and filled it with water. We
>imaged with this configuration over several days. A few years back I
>believe there was a thread on this listserve about using condoms as water
>dams for this purpose. The size of the condom will depend on the size of
>the objective. You can cut the condom to suit your needs.
>Cheers,  
>
>Brian D Armstrong PhD
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Dmitry Sokolov
>Sent: Tuesday, February 19, 2013 8:53 PM
>To: [hidden email]
>Subject: Re: Long term water immersion imaging
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Ammasi,
>
>sorry, I probably missed how the immersion oil was applied: on the top
>of water or on the top of sample:
><a href="http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20water%2">http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20water%2
>0immersion%20imaging
>
>Thank you beforehand,
>Dmitry
>
>*Advanced Knowledge Management*
>for *MICROSCOPY *and *Image Analysis *
>------------------------------------------------------------------------
>*Dmitry Sokolov*, Ph.D.
>Mob: *+64 21 063 5382***
>[hidden email] <mailto:[hidden email]>
>
>20.02.2013 16:19, Periasamy, Ammasi (ap3t) ?????:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Sorry, I forgot to mention...
>> This water immersion oil can be used for any commercially available
>>objective lens.
>> We are using this for Nikon, Leica and Olympus. It works.
>> Good luck.
>> Ammasi
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Periasamy, Ammasi
>>(ap3t)
>> Sent: Tuesday, February 19, 2013 10:17 PM
>> To: [hidden email]
>> Subject: Re: Long term water immersion imaging
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All
>> For water immersion lens, yes if you use water, particularly for
>>2photon imaging, it will evaporate within few minutes. We went through
>>this for 2p imaging. I thought there should be a medium of refractive
>>index same as water. I discussed this issue with our Zeiss sales
>>representative. She came to my office next day and provided valuable
>>information. The zeiss sells an immersion oil and its refractive index
>>is same as water. We have been using this for 2photon or Confocal time
>>lapse imaging (24 hrs) since 2008. No problem and the image quality is
>>great. I am not sure anyone raised this question before in the list
>>server. I apologize for missing this kind of question/help. The price is
>>super high compared to the regular immersion oil. The price is about
>>$125-150. Here is the part#
>> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the
>>Zeiss market place web site to order it.
>>www.micro-shop.zeiss.com/us/us_en Hope this helps.
>> Best wishes,
>> Ammasi
>>
>>
>> Dr. Ammasi Periasamy
>> Professor & Center Director
>> W.M. Keck Center for Cellular Imaging (KCCI)
>> (A University Imaging Center)
>> Biology, University of Virginia
>> Mail or FedEx: 485 McCormick Rd.
>> Charlottesville, VA 22904.
>> Office Location: Physical Life Sciences Building (B005)
>> 90, Geldard Drive, Charlottesville, VA 22904
>> Voice: 434-243-7602 (Office); 982-4869 (lab)
>> Fax:434-982-5210; Email:[hidden email]
>> http://www.kcci.virginia.edu/contact/peri.php
>> ************************
>> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
>> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
>> *************************
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Watkins, Simon C
>> Sent: Tuesday, February 19, 2013 10:00 PM
>> To: [hidden email]
>> Subject: Re: Long term water immersion imaging
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> For us genteal gel
>>
>>http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001GBI
>>S7
>> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in
>>some conditions.. Though this was within an humidified chamber
>>
>> Simon Watkins Ph.D
>>
>> Professor and Vice Chair Cell Biology
>> Professor Immunology
>> Director Center for Biologic Imaging
>> University of Pittsburgh
>> Bsts 225 3550 terrace st
>> Pittsburgh PA 15261
>> Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
>> 412-352-2277
>>
>>
>>
>>
>>
>>
>> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]>
>>wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Adam,
>>>
>>> I'm sure some of the other members of the list have more experience
>>> than I. However, perhaps you could try making a continual water source
>>> like the leica system. I know Steve Cody has developed some nifty
>>> techniques to maintain the immersion water automatically, but even a
>>> manual top-up every few hours could help you in conjunction with your
>>> reservoir approach.
>>>
>>> Best of luck,
>>>
>>> Ben
>>>
>>>
>>> Ben Hibbs
>>> Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>>> Materials Institute (MMI) University of Melbourne, Victoria 3010,
>>> Australia
>>> Email:
>>> [hidden email]<mailto:[hidden email]>
>>> Phone: +61 (0)3 9035-7749
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> On 20/02/2013, at 7:01 AM, Adam White
>>> <[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
>>> *****
>>>
>>> I did a search of the archive but I think I have exhausted most of the
>>> previous suggestions... We are attempting to do some overnight live
>>> imaging experiments using 2PE microscopy.  We are using the Nikon Apo
>>> LWD 25x/1.1 water immersion objective on an inverted stand and our main
>>> problem is keeping the immersion medium in place for longer than a
>>> couple of hours.
>>> We
>>> have tried using the Cargille oil with a 1.335 RI but it does not have
>>> enough viscosity/surface tension to be useful.  We have tried using
>>> ultrasound gel but this dries out over time and only gives us about 5
>>> hours of images.  I have also tried all manner of different
>>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
>>> The best I have found is to use a stretched-out glove finger (plus a
>>> sealant) and just fill up the resulting "reservoir" with water.  This
>>> has given us enough volume to get about 10 hours worth of imaging but
>>> is hard to keep consistent.  Obviously, my question is whether someone
>>> has developed or knows of a better system for doing this?  Perhaps a
>>> perfusion system or a more durable glove-finger type solution?  I have
>>> seen the Leica system but this won't fit our objective...  Any input
>>> you have to offer would be most appreciated.
>>>
>>> Best,
>>> Adam
>>>
>>>
>>> Adam B. White, Ph.D.
>>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
>>> Comprehensive Cancer Center Columbia University
>>> 1130 Saint Nicholas Ave, 222A
>>> New York, NY 10032
>
>
>---------------------------------------------------------------------
>*SECURITY/CONFIDENTIALITY WARNING:
>This message and any attachments are intended solely for the individual
>or entity to which they are addressed. This communication may contain
>information that is privileged, confidential, or exempt from disclosure
>under applicable law (e.g., personal health information, research data,
>financial information). Because this e-mail has been sent without
>encryption, individuals other than the intended recipient may be able to
>view the information, forward it to others or tamper with the information
>without the knowledge or consent of the sender. If you are not the
>intended recipient, or the employee or person responsible for delivering
>the message to the intended recipient, any dissemination, distribution or
>copying of the communication is strictly prohibited. If you received the
>communication in error, please notify the sender immediately by replying
>to this message and deleting the message and any accompanying files from
>your system. If, due to the security risks, you do not wish to receive
>further communications via e-mail, please reply to this message and
>inform the sender that you do not wish to receive further e-mail from the
>sender. (fpc5p)
>---------------------------------------------------------------------
Thomas Horn Thomas Horn
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Re: Long term water immersion imaging

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

Hello Adam,
I have just received from Nikon an immersion fluid replacement system for their water objectives. It consists of  objective-dedicated brass caps connected by tubing to a syringe pump. However, I have not tried it and I do not know if they offer it for your 25 x. Maybe worth contacting Nikon on that....
Best regards,
Thomas.
 

Dr. Thomas Horn,
The Single Cell Unit, U1.46
Department of Biosystems Science and Engineering (D-BSSE)
Swiss Federal Institute of Technology Zurich (ETH)
Mattenstrasse 26
CH 4048 Basel
Switzerland
Phone: +41 61 387 3373
mail: [hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Watkins, Simon C
Sent: 20 February 2013 18:18
To: [hidden email]
Subject: Re: Long term water immersion imaging

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

I guess my last comment on the use of Genteal was a little cursory.  This is a water based gel that was developed for folks with chronic dry eyes.
It has the same RI as water and works extremely well for long term experiments with the new objectives (at least Oly and Nikon). It does work better when the sample is mounted in a humidified chamber and allows the full working distance of the lens to be used which generally will not work with the water RI oils which simply do not generate sufficient surface tension.  The best thing about Genteal, is that its an over the counter medication and available from Amazon (see my earlier post)  which means it works, and is in-expensive.  Working time for the stuff is about 24 hours with a humidified chamber.  This also assumes the stage is being moved around to set positions over time, which is very difficult to do with oil As a last comment, as Genteal is water based, cleaning etc is a doddle which can be a problem with the water RI oils.


Simon Watkins Ph.D

Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
University of Pittsburgh
Bsts 225 3550 terrace st
Pittsburgh PA 15261
Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
412-352-2277






On 2/20/13 11:40 AM, "Armstrong, Brian" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hello, I think the problem is that these LWD 2P lenses are huge and
>need a large volume of water for the long working distance. The Zeiss
>immersion media will not hold surface tension in that volume (already
>mentioned [although works great for lenses such as 63x/1.2W]). What we
>did was purchase a variety of rubber washers in a "plumbing kit", the
>cost was a few dollars. We glued a rubber washer to the slide
>(silicone, VALAP, cyanoacrylate will all work here) and filled it with
>water. We imaged with this configuration over several days. A few years
>back I believe there was a thread on this listserve about using condoms
>as water dams for this purpose. The size of the condom will depend on
>the size of the objective. You can cut the condom to suit your needs.
>Cheers,  
>
>Brian D Armstrong PhD
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]]
>On Behalf Of Dmitry Sokolov
>Sent: Tuesday, February 19, 2013 8:53 PM
>To: [hidden email]
>Subject: Re: Long term water immersion imaging
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi Ammasi,
>
>sorry, I probably missed how the immersion oil was applied: on the top
>of water or on the top of sample:
>http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20wate
>r%2
>0immersion%20imaging
>
>Thank you beforehand,
>Dmitry
>
>*Advanced Knowledge Management*
>for *MICROSCOPY *and *Image Analysis *
>-----------------------------------------------------------------------
>-
>*Dmitry Sokolov*, Ph.D.
>Mob: *+64 21 063 5382***
>[hidden email] <mailto:[hidden email]>
>
>20.02.2013 16:19, Periasamy, Ammasi (ap3t) ?????:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Sorry, I forgot to mention...
>> This water immersion oil can be used for any commercially available
>>objective lens.
>> We are using this for Nikon, Leica and Olympus. It works.
>> Good luck.
>> Ammasi
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Periasamy,
>>Ammasi
>>(ap3t)
>> Sent: Tuesday, February 19, 2013 10:17 PM
>> To: [hidden email]
>> Subject: Re: Long term water immersion imaging
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All
>> For water immersion lens, yes if you use water, particularly for
>>2photon imaging, it will evaporate within few minutes. We went through
>>this for 2p imaging. I thought there should be a medium of refractive
>>index same as water. I discussed this issue with our Zeiss sales
>>representative. She came to my office next day and provided valuable
>>information. The zeiss sells an immersion oil and its refractive index
>>is same as water. We have been using this for 2photon or Confocal time
>>lapse imaging (24 hrs) since 2008. No problem and the image quality is
>>great. I am not sure anyone raised this question before in the list
>>server. I apologize for missing this kind of question/help. The price
>>is super high compared to the regular immersion oil. The price is
>>about $125-150. Here is the part#
>> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the
>>Zeiss market place web site to order it.
>>www.micro-shop.zeiss.com/us/us_en Hope this helps.
>> Best wishes,
>> Ammasi
>>
>>
>> Dr. Ammasi Periasamy
>> Professor & Center Director
>> W.M. Keck Center for Cellular Imaging (KCCI) (A University Imaging
>> Center) Biology, University of Virginia Mail or FedEx: 485 McCormick
>> Rd.
>> Charlottesville, VA 22904.
>> Office Location: Physical Life Sciences Building (B005) 90, Geldard
>> Drive, Charlottesville, VA 22904
>> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210;
>> Email:[hidden email] http://www.kcci.virginia.edu/contact/peri.php
>> ************************
>> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
>> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
>> *************************
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Watkins, Simon
>>C
>> Sent: Tuesday, February 19, 2013 10:00 PM
>> To: [hidden email]
>> Subject: Re: Long term water immersion imaging
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> For us genteal gel
>>
>>http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001
>>GBI
>>S7
>> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in
>>some conditions.. Though this was within an humidified chamber
>>
>> Simon Watkins Ph.D
>>
>> Professor and Vice Chair Cell Biology Professor Immunology Director
>> Center for Biologic Imaging University of Pittsburgh Bsts 225 3550
>> terrace st Pittsburgh PA 15261 Www.cbi.pitt.edu
>> <http://Www.cbi.pitt.edu/>
>> 412-352-2277
>>
>>
>>
>>
>>
>>
>> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]>
>>wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Adam,
>>>
>>> I'm sure some of the other members of the list have more experience
>>> than I. However, perhaps you could try making a continual water
>>> source like the leica system. I know Steve Cody has developed some
>>> nifty techniques to maintain the immersion water automatically, but
>>> even a manual top-up every few hours could help you in conjunction
>>> with your reservoir approach.
>>>
>>> Best of luck,
>>>
>>> Ben
>>>
>>>
>>> Ben Hibbs
>>> Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>>> Materials Institute (MMI) University of Melbourne, Victoria 3010,
>>> Australia
>>> Email:
>>> [hidden email]<mailto:[hidden email]>
>>> Phone: +61 (0)3 9035-7749
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> On 20/02/2013, at 7:01 AM, Adam White
>>> <[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
>>> *****
>>>
>>> I did a search of the archive but I think I have exhausted most of
>>> the previous suggestions... We are attempting to do some overnight
>>> live imaging experiments using 2PE microscopy.  We are using the
>>> Nikon Apo LWD 25x/1.1 water immersion objective on an inverted stand
>>> and our main problem is keeping the immersion medium in place for
>>> longer than a couple of hours.
>>> We
>>> have tried using the Cargille oil with a 1.335 RI but it does not
>>> have enough viscosity/surface tension to be useful.  We have tried
>>> using ultrasound gel but this dries out over time and only gives us
>>> about 5 hours of images.  I have also tried all manner of different
>>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
>>> The best I have found is to use a stretched-out glove finger (plus a
>>> sealant) and just fill up the resulting "reservoir" with water.  
>>> This has given us enough volume to get about 10 hours worth of
>>> imaging but is hard to keep consistent.  Obviously, my question is
>>> whether someone has developed or knows of a better system for doing
>>> this?  Perhaps a perfusion system or a more durable glove-finger
>>> type solution?  I have seen the Leica system but this won't fit our
>>> objective...  Any input you have to offer would be most appreciated.
>>>
>>> Best,
>>> Adam
>>>
>>>
>>> Adam B. White, Ph.D.
>>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
>>> Comprehensive Cancer Center Columbia University
>>> 1130 Saint Nicholas Ave, 222A
>>> New York, NY 10032
>
>
>---------------------------------------------------------------------
>*SECURITY/CONFIDENTIALITY WARNING:
>This message and any attachments are intended solely for the individual
>or entity to which they are addressed. This communication may contain
>information that is privileged, confidential, or exempt from disclosure
>under applicable law (e.g., personal health information, research data,
>financial information). Because this e-mail has been sent without
>encryption, individuals other than the intended recipient may be able
>to view the information, forward it to others or tamper with the
>information without the knowledge or consent of the sender. If you are
>not the intended recipient, or the employee or person responsible for
>delivering the message to the intended recipient, any dissemination,
>distribution or copying of the communication is strictly prohibited. If
>you received the communication in error, please notify the sender
>immediately by replying to this message and deleting the message and
>any accompanying files from your system. If, due to the security risks,
>you do not wish to receive further communications via e-mail, please
>reply to this message and inform the sender that you do not wish to
>receive further e-mail from the sender. (fpc5p)
>---------------------------------------------------------------------
Craig Brideau Craig Brideau
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|

Re: Long term water immersion imaging

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

I have that lens; I'm going to bug my Nikon rep about the cap right away!

Craig



On Thu, Feb 21, 2013 at 1:54 AM, Horn Thomas <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Adam,
> I have just received from Nikon an immersion fluid replacement system for
> their water objectives. It consists of  objective-dedicated brass caps
> connected by tubing to a syringe pump. However, I have not tried it and I
> do not know if they offer it for your 25 x. Maybe worth contacting Nikon on
> that....
> Best regards,
> Thomas.
>
>
> Dr. Thomas Horn,
> The Single Cell Unit, U1.46
> Department of Biosystems Science and Engineering (D-BSSE)
> Swiss Federal Institute of Technology Zurich (ETH)
> Mattenstrasse 26
> CH 4048 Basel
> Switzerland
> Phone: +41 61 387 3373
> mail: [hidden email]
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Watkins, Simon C
> Sent: 20 February 2013 18:18
> To: [hidden email]
> Subject: Re: Long term water immersion imaging
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I guess my last comment on the use of Genteal was a little cursory.  This
> is a water based gel that was developed for folks with chronic dry eyes.
> It has the same RI as water and works extremely well for long term
> experiments with the new objectives (at least Oly and Nikon). It does work
> better when the sample is mounted in a humidified chamber and allows the
> full working distance of the lens to be used which generally will not work
> with the water RI oils which simply do not generate sufficient surface
> tension.  The best thing about Genteal, is that its an over the counter
> medication and available from Amazon (see my earlier post)  which means it
> works, and is in-expensive.  Working time for the stuff is about 24 hours
> with a humidified chamber.  This also assumes the stage is being moved
> around to set positions over time, which is very difficult to do with oil
> As a last comment, as Genteal is water based, cleaning etc is a doddle
> which can be a problem with the water RI oils.
>
>
> Simon Watkins Ph.D
>
> Professor and Vice Chair Cell Biology
> Professor Immunology
> Director Center for Biologic Imaging
> University of Pittsburgh
> Bsts 225 3550 terrace st
> Pittsburgh PA 15261
> Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
> 412-352-2277
>
>
>
>
>
>
> On 2/20/13 11:40 AM, "Armstrong, Brian" <[hidden email]> wrote:
>
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >*****
> >
> >Hello, I think the problem is that these LWD 2P lenses are huge and
> >need a large volume of water for the long working distance. The Zeiss
> >immersion media will not hold surface tension in that volume (already
> >mentioned [although works great for lenses such as 63x/1.2W]). What we
> >did was purchase a variety of rubber washers in a "plumbing kit", the
> >cost was a few dollars. We glued a rubber washer to the slide
> >(silicone, VALAP, cyanoacrylate will all work here) and filled it with
> >water. We imaged with this configuration over several days. A few years
> >back I believe there was a thread on this listserve about using condoms
> >as water dams for this purpose. The size of the condom will depend on
> >the size of the objective. You can cut the condom to suit your needs.
> >Cheers,
> >
> >Brian D Armstrong PhD
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> >[mailto:[hidden email]]
> >On Behalf Of Dmitry Sokolov
> >Sent: Tuesday, February 19, 2013 8:53 PM
> >To: [hidden email]
> >Subject: Re: Long term water immersion imaging
> >
> >*****
> >To join, leave or search the confocal microscopy listserv, go to:
> >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >*****
> >
> >Hi Ammasi,
> >
> >sorry, I probably missed how the immersion oil was applied: on the top
> >of water or on the top of sample:
> >http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20wate
> >r%2
> >0immersion%20imaging
> >
> >Thank you beforehand,
> >Dmitry
> >
> >*Advanced Knowledge Management*
> >for *MICROSCOPY *and *Image Analysis *
> >-----------------------------------------------------------------------
> >-
> >*Dmitry Sokolov*, Ph.D.
> >Mob: *+64 21 063 5382***
> >[hidden email] <mailto:[hidden email]>
> >
> >20.02.2013 16:19, Periasamy, Ammasi (ap3t) ?????:
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Sorry, I forgot to mention...
> >> This water immersion oil can be used for any commercially available
> >>objective lens.
> >> We are using this for Nikon, Leica and Olympus. It works.
> >> Good luck.
> >> Ammasi
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >>[mailto:[hidden email]] On Behalf Of Periasamy,
> >>Ammasi
> >>(ap3t)
> >> Sent: Tuesday, February 19, 2013 10:17 PM
> >> To: [hidden email]
> >> Subject: Re: Long term water immersion imaging
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Dear All
> >> For water immersion lens, yes if you use water, particularly for
> >>2photon imaging, it will evaporate within few minutes. We went through
> >>this for 2p imaging. I thought there should be a medium of refractive
> >>index same as water. I discussed this issue with our Zeiss sales
> >>representative. She came to my office next day and provided valuable
> >>information. The zeiss sells an immersion oil and its refractive index
> >>is same as water. We have been using this for 2photon or Confocal time
> >>lapse imaging (24 hrs) since 2008. No problem and the image quality is
> >>great. I am not sure anyone raised this question before in the list
> >>server. I apologize for missing this kind of question/help. The price
> >>is super high compared to the regular immersion oil. The price is
> >>about $125-150. Here is the part#
> >> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the
> >>Zeiss market place web site to order it.
> >>www.micro-shop.zeiss.com/us/us_en Hope this helps.
> >> Best wishes,
> >> Ammasi
> >>
> >>
> >> Dr. Ammasi Periasamy
> >> Professor & Center Director
> >> W.M. Keck Center for Cellular Imaging (KCCI) (A University Imaging
> >> Center) Biology, University of Virginia Mail or FedEx: 485 McCormick
> >> Rd.
> >> Charlottesville, VA 22904.
> >> Office Location: Physical Life Sciences Building (B005) 90, Geldard
> >> Drive, Charlottesville, VA 22904
> >> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210;
> >> Email:[hidden email] http://www.kcci.virginia.edu/contact/peri.php
> >> ************************
> >> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
> >> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
> >> *************************
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >>[mailto:[hidden email]] On Behalf Of Watkins, Simon
> >>C
> >> Sent: Tuesday, February 19, 2013 10:00 PM
> >> To: [hidden email]
> >> Subject: Re: Long term water immersion imaging
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> For us genteal gel
> >>
> >>http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001
> >>GBI
> >>S7
> >> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in
> >>some conditions.. Though this was within an humidified chamber
> >>
> >> Simon Watkins Ph.D
> >>
> >> Professor and Vice Chair Cell Biology Professor Immunology Director
> >> Center for Biologic Imaging University of Pittsburgh Bsts 225 3550
> >> terrace st Pittsburgh PA 15261 Www.cbi.pitt.edu
> >> <http://Www.cbi.pitt.edu/>
> >> 412-352-2277
> >>
> >>
> >>
> >>
> >>
> >>
> >> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]>
> >>wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Hi Adam,
> >>>
> >>> I'm sure some of the other members of the list have more experience
> >>> than I. However, perhaps you could try making a continual water
> >>> source like the leica system. I know Steve Cody has developed some
> >>> nifty techniques to maintain the immersion water automatically, but
> >>> even a manual top-up every few hours could help you in conjunction
> >>> with your reservoir approach.
> >>>
> >>> Best of luck,
> >>>
> >>> Ben
> >>>
> >>>
> >>> Ben Hibbs
> >>> Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
> >>> Materials Institute (MMI) University of Melbourne, Victoria 3010,
> >>> Australia
> >>> Email:
> >>> [hidden email]<mailto:[hidden email]>
> >>> Phone: +61 (0)3 9035-7749
> >>>
> >>>
> >>>
> >>>
> >>>
> >>>
> >>>
> >>> On 20/02/2013, at 7:01 AM, Adam White
> >>> <[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
> >>> *****
> >>>
> >>> I did a search of the archive but I think I have exhausted most of
> >>> the previous suggestions... We are attempting to do some overnight
> >>> live imaging experiments using 2PE microscopy.  We are using the
> >>> Nikon Apo LWD 25x/1.1 water immersion objective on an inverted stand
> >>> and our main problem is keeping the immersion medium in place for
> >>> longer than a couple of hours.
> >>> We
> >>> have tried using the Cargille oil with a 1.335 RI but it does not
> >>> have enough viscosity/surface tension to be useful.  We have tried
> >>> using ultrasound gel but this dries out over time and only gives us
> >>> about 5 hours of images.  I have also tried all manner of different
> >>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
> >>> The best I have found is to use a stretched-out glove finger (plus a
> >>> sealant) and just fill up the resulting "reservoir" with water.
> >>> This has given us enough volume to get about 10 hours worth of
> >>> imaging but is hard to keep consistent.  Obviously, my question is
> >>> whether someone has developed or knows of a better system for doing
> >>> this?  Perhaps a perfusion system or a more durable glove-finger
> >>> type solution?  I have seen the Leica system but this won't fit our
> >>> objective...  Any input you have to offer would be most appreciated.
> >>>
> >>> Best,
> >>> Adam
> >>>
> >>>
> >>> Adam B. White, Ph.D.
> >>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
> >>> Comprehensive Cancer Center Columbia University
> >>> 1130 Saint Nicholas Ave, 222A
> >>> New York, NY 10032
> >
> >
> >---------------------------------------------------------------------
> >*SECURITY/CONFIDENTIALITY WARNING:
> >This message and any attachments are intended solely for the individual
> >or entity to which they are addressed. This communication may contain
> >information that is privileged, confidential, or exempt from disclosure
> >under applicable law (e.g., personal health information, research data,
> >financial information). Because this e-mail has been sent without
> >encryption, individuals other than the intended recipient may be able
> >to view the information, forward it to others or tamper with the
> >information without the knowledge or consent of the sender. If you are
> >not the intended recipient, or the employee or person responsible for
> >delivering the message to the intended recipient, any dissemination,
> >distribution or copying of the communication is strictly prohibited. If
> >you received the communication in error, please notify the sender
> >immediately by replying to this message and deleting the message and
> >any accompanying files from your system. If, due to the security risks,
> >you do not wish to receive further communications via e-mail, please
> >reply to this message and inform the sender that you do not wish to
> >receive further e-mail from the sender. (fpc5p)
> >---------------------------------------------------------------------
>
Kurt Thorn Kurt Thorn
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Open this post in threaded view
|

Re: Long term water immersion imaging

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

Sutter Instruments can also make custom brass caps for Nikon objectives
with a bored hole for a 22 gauge needle.  We have one for our 40x / 1.15
WI lens.

Kurt

On 2/21/2013 10:04 AM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have that lens; I'm going to bug my Nikon rep about the cap right away!
>
> Craig
>
>
>
> On Thu, Feb 21, 2013 at 1:54 AM, Horn Thomas <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello Adam,
>> I have just received from Nikon an immersion fluid replacement system for
>> their water objectives. It consists of  objective-dedicated brass caps
>> connected by tubing to a syringe pump. However, I have not tried it and I
>> do not know if they offer it for your 25 x. Maybe worth contacting Nikon on
>> that....
>> Best regards,
>> Thomas.
>>
>>
>> Dr. Thomas Horn,
>> The Single Cell Unit, U1.46
>> Department of Biosystems Science and Engineering (D-BSSE)
>> Swiss Federal Institute of Technology Zurich (ETH)
>> Mattenstrasse 26
>> CH 4048 Basel
>> Switzerland
>> Phone: +41 61 387 3373
>> mail: [hidden email]
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Watkins, Simon C
>> Sent: 20 February 2013 18:18
>> To: [hidden email]
>> Subject: Re: Long term water immersion imaging
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I guess my last comment on the use of Genteal was a little cursory.  This
>> is a water based gel that was developed for folks with chronic dry eyes.
>> It has the same RI as water and works extremely well for long term
>> experiments with the new objectives (at least Oly and Nikon). It does work
>> better when the sample is mounted in a humidified chamber and allows the
>> full working distance of the lens to be used which generally will not work
>> with the water RI oils which simply do not generate sufficient surface
>> tension.  The best thing about Genteal, is that its an over the counter
>> medication and available from Amazon (see my earlier post)  which means it
>> works, and is in-expensive.  Working time for the stuff is about 24 hours
>> with a humidified chamber.  This also assumes the stage is being moved
>> around to set positions over time, which is very difficult to do with oil
>> As a last comment, as Genteal is water based, cleaning etc is a doddle
>> which can be a problem with the water RI oils.
>>
>>
>> Simon Watkins Ph.D
>>
>> Professor and Vice Chair Cell Biology
>> Professor Immunology
>> Director Center for Biologic Imaging
>> University of Pittsburgh
>> Bsts 225 3550 terrace st
>> Pittsburgh PA 15261
>> Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
>> 412-352-2277
>>
>>
>>
>>
>>
>>
>> On 2/20/13 11:40 AM, "Armstrong, Brian" <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hello, I think the problem is that these LWD 2P lenses are huge and
>>> need a large volume of water for the long working distance. The Zeiss
>>> immersion media will not hold surface tension in that volume (already
>>> mentioned [although works great for lenses such as 63x/1.2W]). What we
>>> did was purchase a variety of rubber washers in a "plumbing kit", the
>>> cost was a few dollars. We glued a rubber washer to the slide
>>> (silicone, VALAP, cyanoacrylate will all work here) and filled it with
>>> water. We imaged with this configuration over several days. A few years
>>> back I believe there was a thread on this listserve about using condoms
>>> as water dams for this purpose. The size of the condom will depend on
>>> the size of the objective. You can cut the condom to suit your needs.
>>> Cheers,
>>>
>>> Brian D Armstrong PhD
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]]
>>> On Behalf Of Dmitry Sokolov
>>> Sent: Tuesday, February 19, 2013 8:53 PM
>>> To: [hidden email]
>>> Subject: Re: Long term water immersion imaging
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Ammasi,
>>>
>>> sorry, I probably missed how the immersion oil was applied: on the top
>>> of water or on the top of sample:
>>> http://confocal-manawatu.pbworks.com/w/page/63806798/Long%20term%20wate
>>> r%2
>>> 0immersion%20imaging
>>>
>>> Thank you beforehand,
>>> Dmitry
>>>
>>> *Advanced Knowledge Management*
>>> for *MICROSCOPY *and *Image Analysis *
>>> -----------------------------------------------------------------------
>>> -
>>> *Dmitry Sokolov*, Ph.D.
>>> Mob: *+64 21 063 5382***
>>> [hidden email] <mailto:[hidden email]>
>>>
>>> 20.02.2013 16:19, Periasamy, Ammasi (ap3t) ?????:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Sorry, I forgot to mention...
>>>> This water immersion oil can be used for any commercially available
>>>> objective lens.
>>>> We are using this for Nikon, Leica and Olympus. It works.
>>>> Good luck.
>>>> Ammasi
>>>>
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]] On Behalf Of Periasamy,
>>>> Ammasi
>>>> (ap3t)
>>>> Sent: Tuesday, February 19, 2013 10:17 PM
>>>> To: [hidden email]
>>>> Subject: Re: Long term water immersion imaging
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Dear All
>>>> For water immersion lens, yes if you use water, particularly for
>>>> 2photon imaging, it will evaporate within few minutes. We went through
>>>> this for 2p imaging. I thought there should be a medium of refractive
>>>> index same as water. I discussed this issue with our Zeiss sales
>>>> representative. She came to my office next day and provided valuable
>>>> information. The zeiss sells an immersion oil and its refractive index
>>>> is same as water. We have been using this for 2photon or Confocal time
>>>> lapse imaging (24 hrs) since 2008. No problem and the image quality is
>>>> great. I am not sure anyone raised this question before in the list
>>>> server. I apologize for missing this kind of question/help. The price
>>>> is super high compared to the regular immersion oil. The price is
>>>> about $125-150. Here is the part#
>>>> 000000-1252-136 Immersion medium "Immersol" W, oiler 20 ml Visit the
>>>> Zeiss market place web site to order it.
>>>> www.micro-shop.zeiss.com/us/us_en Hope this helps.
>>>> Best wishes,
>>>> Ammasi
>>>>
>>>>
>>>> Dr. Ammasi Periasamy
>>>> Professor & Center Director
>>>> W.M. Keck Center for Cellular Imaging (KCCI) (A University Imaging
>>>> Center) Biology, University of Virginia Mail or FedEx: 485 McCormick
>>>> Rd.
>>>> Charlottesville, VA 22904.
>>>> Office Location: Physical Life Sciences Building (B005) 90, Geldard
>>>> Drive, Charlottesville, VA 22904
>>>> Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210;
>>>> Email:[hidden email] http://www.kcci.virginia.edu/contact/peri.php
>>>> ************************
>>>> 12th Annual Workshop on FRET Microscopy, March 11-16, 2013
>>>> http://www.kcci.virginia.edu/workshop/workshop2013/index.php
>>>> *************************
>>>>
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]] On Behalf Of Watkins, Simon
>>>> C
>>>> Sent: Tuesday, February 19, 2013 10:00 PM
>>>> To: [hidden email]
>>>> Subject: Re: Long term water immersion imaging
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> For us genteal gel
>>>>
>>>> http://www.amazon.com/GenTeal-Lubricant-Moderate-Severe-Relief/dp/B001
>>>> GBI
>>>> S7
>>>> Y/ref=pd_bxgy_hpc_img_z works very well..we have used it overnight in
>>>> some conditions.. Though this was within an humidified chamber
>>>>
>>>> Simon Watkins Ph.D
>>>>
>>>> Professor and Vice Chair Cell Biology Professor Immunology Director
>>>> Center for Biologic Imaging University of Pittsburgh Bsts 225 3550
>>>> terrace st Pittsburgh PA 15261 Www.cbi.pitt.edu
>>>> <http://Www.cbi.pitt.edu/>
>>>> 412-352-2277
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>> On 2/19/13 6:04 PM, "Benjamin Hibbs" <[hidden email]>
>>>> wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Hi Adam,
>>>>>
>>>>> I'm sure some of the other members of the list have more experience
>>>>> than I. However, perhaps you could try making a continual water
>>>>> source like the leica system. I know Steve Cody has developed some
>>>>> nifty techniques to maintain the immersion water automatically, but
>>>>> even a manual top-up every few hours could help you in conjunction
>>>>> with your reservoir approach.
>>>>>
>>>>> Best of luck,
>>>>>
>>>>> Ben
>>>>>
>>>>>
>>>>> Ben Hibbs
>>>>> Platform Support Officer<Advanced Fluorescence Imaging The Melbourne
>>>>> Materials Institute (MMI) University of Melbourne, Victoria 3010,
>>>>> Australia
>>>>> Email:
>>>>> [hidden email]<mailto:[hidden email]>
>>>>> Phone: +61 (0)3 9035-7749
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> On 20/02/2013, at 7:01 AM, Adam White
>>>>> <[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
>>>>> *****
>>>>>
>>>>> I did a search of the archive but I think I have exhausted most of
>>>>> the previous suggestions... We are attempting to do some overnight
>>>>> live imaging experiments using 2PE microscopy.  We are using the
>>>>> Nikon Apo LWD 25x/1.1 water immersion objective on an inverted stand
>>>>> and our main problem is keeping the immersion medium in place for
>>>>> longer than a couple of hours.
>>>>> We
>>>>> have tried using the Cargille oil with a 1.335 RI but it does not
>>>>> have enough viscosity/surface tension to be useful.  We have tried
>>>>> using ultrasound gel but this dries out over time and only gives us
>>>>> about 5 hours of images.  I have also tried all manner of different
>>>>> sealants/gloves/o-rings to varying degrees of success/reproducibility.
>>>>> The best I have found is to use a stretched-out glove finger (plus a
>>>>> sealant) and just fill up the resulting "reservoir" with water.
>>>>> This has given us enough volume to get about 10 hours worth of
>>>>> imaging but is hard to keep consistent.  Obviously, my question is
>>>>> whether someone has developed or knows of a better system for doing
>>>>> this?  Perhaps a perfusion system or a more durable glove-finger
>>>>> type solution?  I have seen the Leica system but this won't fit our
>>>>> objective...  Any input you have to offer would be most appreciated.
>>>>>
>>>>> Best,
>>>>> Adam
>>>>>
>>>>>
>>>>> Adam B. White, Ph.D.
>>>>> Confocal & Specialized Microscopy Shared Resource Herbert Irving
>>>>> Comprehensive Cancer Center Columbia University
>>>>> 1130 Saint Nicholas Ave, 222A
>>>>> New York, NY 10032
>>>
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