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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 <[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 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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