Posted by
Boswell, Carl A - (cboswell) on
URL: http://confocal-microscopy-list.275.s1.nabble.com/water-refill-device-for-WI-objective-tp5566734p5584935.html
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I'd like to see your ppt file, Steve, if for no other reason than to admire
your ingenuity and learn from the master.
C
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
----- Original Message -----
From: "Stephen Cody" <
[hidden email]>
To: <
[hidden email]>
Sent: Tuesday, September 28, 2010 8:00 PM
Subject: Re: water refill device for WI objective
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G'day Sylvie,
I've been building my own "lens re-watering" systems for water
immersion objectives for about ten years. Back then I let most, if not
all, the microscope companies know about it. They are very easy to
build.
My version uses a plastic pipette to add water via gravity feed. Any
excess flow is sucked away via a peristaltic pump.
All you need is some thin PE tubing (eg. Polyethylene Tubing I.D. =
0.58mm O.D. 0.96mm)
Actual diameters not important, anything roughly those dimensions will do.
You very slowly warm the tubing by holding it beside (NOT above), and
about 5cm away from the flame of a Bunsen burner. Rotating slowly
between thumbs and forefingers. Don't heat too quickly, slower the
better.
Keep the tubing under mild tension as you heat.
As soon as it starts to stretch, remove from heat, BEFORE it breaks.
That's the tricky part that needs practise.
Don't throw away the failures, they are useful too, more later.
Cut the PE tubing at it's thinnest point with a scalpel. You now have
two plastic pipettes, that will not damage your lens.
The failures end up curved, you can use these to suck away excess
water (you'll have no shortage of these!)
You now want to find a short length of some very large diameter Poly
Vinyl tubing that will fit snugly around the top of your objective.
eg. recently for a Leica WI lens we used approx. 30mm internal
diameter tubing. Cut a thin slice of this tubing so that you have a
ring, you can then fit this around the top of your objective, and have
it just a little higher than the edges of the objective so that it
forms a "moat", for those lenses that don't have a "moat". For lenses
that do have a moat, allow the tubing to be just slightly proud of the
moat. It should not be so large as to get in the way of the stage or
your specimen.
A trick taught to me by my colleague Robin Cole from the Ludwig: If
the tubing is a little too large in diameter, you can shrink a small
section by tightening a hose clamp around it (not when it is on an
objective). Then heat the tubing (and clamp) with a heat gun (or very
hot water might work too). Remove from heat, once cooled down to room
temp., remove the hose clamp. Voila! Smaller tubing.
Once fitted around the top of your objective, using an 18 Gauge
hypodermic needle and a drilling motion with thumb and forefinger,
drill two small holes through the very top of the tubing that now
forms a moat. These holes will accommodate your re-watering pipette
and suction tube. The pipette should be inserted so that it is just
adjacent to the flat part of the objective that has the top lens. In
other words you don't need it right next to the lens, a little way
back so that it does not get banged against the coverslip. Capillary
action will ensure the water will go between lens and coverslip.
Sometimes you may want to use a little double sided tape to keep
everything out of the way.
Rig up a gravity feed with a 50ml syringe (without plunger) and 3-Way
stop cock. I also use a 2ml syringe (with plunger) in the other port
of the stop cock to prime the system, and for short term experiments
eg, 2 hours you can use this to add water manually every 30 min. For
longer term experiments eg. overnight, use gravity to do the work.
The "failures"are bent and useful. Take one of these and insert
through the other hole drilled into the moat, and connect to a
peristaltic pump. Any excess flow onto the lens will spill into the
moat, and as it builds up will be harmlessly sucked away via the pump
into a beaker. In this way you don't need to carefully regulate flow
rates.
I have a PowerPoint describing this better and will send it via email
to anyone who asks.
I was going to write this up as a technique paper years ago, but the
release of oils the same refractive index as water, and later
commercial re-watering systems stopped me from doing so.
I guess J. Microsc. may still be interested as a short Tech note. What
do people think? Email me if you want the Re-watering gizmo How-to
PowerPoint.
We have successfully used this on time-lapse experiments at 37Deg C
for three days continuous. Topping up the 50ml syringe each morning
just to be sure.
Cheers Steve
Stephen H. Cody
Imaging Research Fellow & Manager
Monash Micro Imaging @ AMREP
Monash University
6 Floor Burnet Tower
Alfred Medical Research & Education Precinct
89 Commercial Rd, Melbourne, Australia, 3004
New email address:
[hidden email]
www.microimaging.monash.org
Phone (Monash): (613) 990 30142
Phone (BakerIDI): (613) 8532 1580
On 24 September 2010 23:03, Sylvie Le Guyader <
[hidden email]>
wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear all
>
>
>
> We have a 60x water objective NA1.2 by Nikon that we would like to use to
> screen 96 and 384 well plates. Does anyone know of a water refill system
> that would fit on this objective? Or has anyone tried the Olympus or the
> Leica water refill device? Do they work well? Would any fit our objective?
>
>
>
> Thanks for your help!
>
>
>
> Med vänlig hälsning / Best regards
>
>
>
> Sylvie
>
>
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader
>
> Live Cell Imaging Unit
>
> Dept of Biosciences and Nutrition
>
> Karolinska Institutet
>
> Sweden
>
> office: +46 (0)8 608 9240
>
> mobile: +46 (0) 73 733 5008
>
>
>
--
Stephen H. Cody