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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
[log in to unmask]
On 2/21/13 1:49 PM, "Kurt Thorn" <[log in to unmask]> 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
>><[log in to unmask]>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: [log in to unmask]
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>>[mailto:[log in to unmask]]
>>> On Behalf Of Watkins, Simon C
>>> Sent: 20 February 2013 18:18
>>> To: [log in to unmask]
>>> 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" <[log in to unmask]> 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:[log in to unmask]]
>>>> On Behalf Of Dmitry Sokolov
>>>> Sent: Tuesday, February 19, 2013 8:53 PM
>>>> To: [log in to unmask]
>>>> 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***
>>>> [log in to unmask] <mailto:[log in to unmask]>
>>>>
>>>> 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:[log in to unmask]] On Behalf Of Periasamy,
>>>>> Ammasi
>>>>> (ap3t)
>>>>> Sent: Tuesday, February 19, 2013 10:17 PM
>>>>> To: [log in to unmask]
>>>>> 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:[log in to unmask] 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:[log in to unmask]] On Behalf Of Watkins, Simon
>>>>> C
>>>>> Sent: Tuesday, February 19, 2013 10:00 PM
>>>>> To: [log in to unmask]
>>>>> 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" <[log in to unmask]>
>>>>> 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:
>>>>>> [log in to unmask]<mailto:[log in to unmask]>
>>>>>> Phone: +61 (0)3 9035-7749
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> On 20/02/2013, at 7:01 AM, Adam White
>>>>>> <[log in to unmask]<mailto:[log in to unmask]>> 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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