Search the CONFOCAL archive at
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Dear Rob and List,
Sorry I've been on vacation since Christmas and so have been "Off the
Air".
As I previously discussed with Rob in an email, details of the paper
are:
Cody, S.H. & Williams, D.A. A novel organ bath design enables the use of
saline-immersible lenses on inverted microscopes. Journal of Microscopy
185(1): 94-97, (1997)
I think this technique would work for Bob's situation, although I'm sure
Guy's suggestion with the dental wax dam on an upright scope will also
work.
The trick is while the "latex tube" is sealed securely to the dipping
lens with an O-ring. It is not sealed directly to the specimen to be
imaged, but to a collar around a hole (lens access hole) in the base of
an open topped organ bath. The specimen itself is then either suspended
from manipulators as in Cody et al (1977). Or if used with a
"substratum" as in Bob's case (this could even be a slide or coverslip),
the substratum is placed at the bottom of the organ bath, with the
specimen side facing downward, suspended over the lens access hole. In
my bath the lens access hole was approx 30mm in diameter, so a coverslip
of dimensions 22 x 40mm could easily be placed over the hole. With
focusing up and down, excess water could easily pass either side of the
coverslip.
Why would you do all this? Well in my case it was simple, I had muscle
attached to force transducers and it was a simple way to image as well
as measure contraction forces. Obviously if you have tissue mounted on
manipulators, it may be a useful technique. It can also be useful if you
need to use a dipping lens, and only have access to a confocal on an
inverted microscope, not an upright system. If you do have access to an
upright microscope then Guy's suggestion is probably the simpler
solution. Hopefully my next purchase will be an upright scope so that I
can swap my compact Nikon-C1 scanhead easily between upright and
inverted microscopes for this very purpose.
I'll try and organise some pictures of the setup and post on my webpage.
I'll send the links when they are ready.
Cheers
Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute For Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville Victoria 3050
Australia
Tel: 61 3 9341 3155 Fax: 61 3 9341 3104
email: [log in to unmask]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal
-----Original Message-----
From: Robert J. Palmer Jr. [mailto:[log in to unmask]]
Sent: Thursday, 20 January 2005 5:38 AM
To: [log in to unmask]
Subject: Re: "special" technique
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Thanks for the reply! I'm a little confused as to how exactly this
works regardless of the "product" involved. In our particular case,
we have a very irregular opaque substratum on which our biology takes
place. We can image this nicely using a dipping lens on an upright,
but some colleagues of ours wish to do the same thing and have only
an invert. We can get the substratum attached to a slide with the
butter side down (protruding through the lens cutout in the
microscope stage, but we then must get a water film, actually about 2
mm of water, between the substratum and the lens. Furthermore, this
water meniscus must accommodate the motion of the stage during
confocal sectioning. I simply have a mental block when I try to
imagine how your glove finger would be attached to the lens to form a
pool of water in which the lens would operate. Doesn't the water
overflow when the stage is moved during sectioning? Doesn't the
glove touch the stage at some point? Do you have any pictures of this
setup?
Thanks for you advice!
By the way, another researcher here saw my post and also wants to
know what I learn. Maybe we can give you a "personal communication"
in our next publications :) :)
Rob Palmer
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Not sure where I first heard about the idea you're describing, Bob, but
it's
>probably been floating around for a while.
>
>A cheaper and less embarassing product more readily found product in a
lab
>environment ( no need for a trip to the corner drug store), I've found
that
>cutting one finger off a latex lab glove and slipping it over the lens
will
>serve much the same purpose in protecting objective lens from liquids.
>
>Trial and error quickly establishes what size of glove and correct
finger or
>thumb fits a given objective.
>
>Only caveat is the latex seemed to break down fairly rapidly, so
inspection
>for small holes or wear prior to use coupled with regular replacement
would be
>advisable.
>
>J. Scott Gens, PhD
>Post-doctoral Fellow
>Indiana University School of Medicine
>Division of Nephrology
>
>Quoting "Robert J. Palmer Jr." <[log in to unmask]>:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi all - I remember a post from a confocaler "down under" who had
>> developed an approach to using a water-immersible (dipping lens, no
>> coverslip) on an inverted platform. The approach involved a
>> prophylactic....
>> Can anyone put me on to this approach in the literature or put me in
>> contact with the person who advocated it? I would also be
interested
>> in other approaches to achieve this end.
>> Thanks!
>> Rob Palmer
>> --
>> Robert J. Palmer Jr., Ph.D.
>> Natl Inst Dental Craniofacial Res - Natl Insts Health
>> Oral Infection and Immunity Branch
>> Bldg 30, Room 310
>> 30 Convent Drive
>> Bethesda MD 20892
>> ph 301-594-0025
>> fax 301-402-0396
>>
>>
>>
--
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396
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