*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****
Hi Carl
Sounds really good! I'd love to get that PP presentation if you do not mind!
Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
14183 Huddinge
Sweden
office: +46 (0)8 608 9240
LCI room: +46 (0)8 608 9248
mobile: +46 (0) 73 733 5008
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Carl Boswell
> Sent: 29 September 2010 19:14
> To: [log in to unmask]
> Subject: Re: water refill device for WI objective
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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" <[log in to unmask]>
> To: <[log in to unmask]>
> Sent: Tuesday, September 28, 2010 8:00 PM
> Subject: Re: water refill device for WI objective
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:
> [log in to unmask]
>
> www.microimaging.monash.org
> Phone (Monash): (613) 990 30142
> Phone (BakerIDI): (613) 8532 1580
>
>
>
>
> On 24 September 2010 23:03, Sylvie Le Guyader <[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
> > *****
> >
> > 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
|
