Long term water immersion imaging

[Adam White]

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

Hello All,

Thanks a lot for your input regarding my question.  We are attempting to
implement a few of the different strategies suggested and will be happy to
update with more info once we give them a try.  For now, we were able to
do some experiments using Genteal gel as an immersion medium and actually
had a lot of success.  We managed about 16 hours of imaging without a
humidified chamber.  The only note I would add is that we left the glove
finger in place on the objective and I used a pretty liberal amount of
gel.  

Thanks again,
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
212-851-4613
[hidden email]


   


On 2/21/13 1:49 PM, "Kurt Thorn" <[hidden email]> wrote:

>*****
>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: [phone]
>>> 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: *[phone]***
>>>> [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:[phone];
>>>>> 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: [phone]
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> 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)
>>>> ---------------------------------------------------------------------
>>
>

[Adrian Smith-6]

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

Hi all,

We are keen to try out the Genteal that was mentioned recently.

In Australia there are two formulations sold and neither of the packages look the same as the pictures on Amazon.

There is:-

Genteal Lubricant Eye Drops - <http://www.chemistaustralia.com.au/products/genteal-lubricant-eye-drop-0-3-15ml.html>
and
Genteal Lubricating Eye Gel - <http://www.chemistaustralia.com.au/products/genteal-lubricant-eye-drop-0-3-15ml.html>

From the Novartis site it looks likes both have hypromellose whereas the Gel adds carbomer 980.
<http://www.novartis.com.au/PI_PDF/ged_geg.pdf>


Can the Genteal users please point us in the right direction :)

Thanks,

Adrian Smith,
Centenary Institute, Sydney, Australia


On 20/02/2013, at 2:00 PM, Watkins, Simon C wrote:

> *****
> 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: [phone]
>>
>>
>>
>>
>>
>>
>>
>> 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

[Dan Focht]

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

List

Does anybody have time to throw some Genteal in a fluorometer and see
how it behaves throughout the spectrum?
Just curious.

Dan


On 3/3/2013 8:24 PM, Adrian Smith wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> We are keen to try out the Genteal that was mentioned recently.
>
> In Australia there are two formulations sold and neither of the packages look the same as the pictures on Amazon.
>
> There is:-
>
> Genteal Lubricant Eye Drops - <http://www.chemistaustralia.com.au/products/genteal-lubricant-eye-drop-0-3-15ml.html>
> and
> Genteal Lubricating Eye Gel - <http://www.chemistaustralia.com.au/products/genteal-lubricant-eye-drop-0-3-15ml.html>
>
>  From the Novartis site it looks likes both have hypromellose whereas the Gel adds carbomer 980.
> <http://www.novartis.com.au/PI_PDF/ged_geg.pdf>
>
>
> Can the Genteal users please point us in the right direction :)
>
> Thanks,
>
> Adrian Smith,
> Centenary Institute, Sydney, Australia
>
>
> On 20/02/2013, at 2:00 PM, Watkins, Simon C wrote:
>
>> *****
>> 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: [phone]
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> 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

--
Dan Focht Bioptechs Inc. V: [phone] www.bioptechs.com

[Stephen Cody-2]

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

G'day Adam,

I've posted a link to a pdf which shows my setup. I have used gravity for
the drip feed, and peristaltic pumps for the scavenge system. Also I setup
a manual syringe for manual additions for short term (eg. 2 hour)
experiments .

More recently I use the "house" vacuum supply and a water trap for the
scavenge. My system uses poly vinyl tubing as the "moat" and holder for the
tubes, I think this is easier and a bit safer than brass rings that the
commercial systems have adapted from my original idea. A national hardware
chain in Australia sell very short lengths of tubing (70mm in length) in
various diameters, to act as joiners for tubing sold by the meter. This is
a great way to get a collection of different diameter tubing. Another trick
taught to me by my friend (the late Robin Cole) is that if the tubing is
just a little to loose, you can make it smaller by using a hose clamp
(obviously its not on an objective). Tighten the metal hose clamp. heat the
tubing and hose clamp with a heat gun. Remove heat and allow to cool. Once
cool you can remove the clamp and the tubing will now be a smaller diameter.

https://www.dropbox.com/s/lyzxfc7mvl1m1qf/Cody_Lens_re-watering_system_post.pdf

I scanned this pdf for viruses before posting.

Cheers
Steve

On 2 March 2013 02:27, 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
> *****
>
> Hello All,
>
> Thanks a lot for your input regarding my question.  We are attempting to
> implement a few of the different strategies suggested and will be happy to
> update with more info once we give them a try.  For now, we were able to
> do some experiments using Genteal gel as an immersion medium and actually
> had a lot of success.  We managed about 16 hours of imaging without a
> humidified chamber.  The only note I would add is that we left the glove
> finger in place on the objective and I used a pretty liberal amount of
> gel.
>
> Thanks again,
> 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
> 212-851-4613
> [hidden email]
>
>
>
>
>
> On 2/21/13 1:49 PM, "Kurt Thorn" <[hidden email]> wrote:
>
> >*****
> >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: [phone]
> >>> 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: *[phone]***
> >>>> [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:[phone];
> >>>>> 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: [phone]
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>> 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)
> >>>> ---------------------------------------------------------------------
> >>
> >
>