water refill device for WI objective

[Sylvie Le Guyader-2]

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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: [phone]

mobile: [phone]

 

[Boswell, Carl A - (cboswell)]

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The Olympus FV10i:
http://www.olympusamerica.com/seg_section/product.asp?product=1064 does what
you are looking for.  Maybe you can get some info from them on their source
of hardware/setup.
c

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Sylvie Le Guyader" <[hidden email]>
To: <[hidden email]>
Sent: Friday, September 24, 2010 6:03 AM
Subject: water refill device for WI objective


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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: [phone]

mobile: [phone]

[Sylvie Le Guyader-2]

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Hi Tom

Thanks for your suggestion. We use the Zeiss oil for water lens routinely
and it works very well as long as one doesn’t try to image a whole multiwell
plate. Oil (normal or for water lenses) is too viscous for large areas. It
collects at places and goes missing at others. We have tried this a lot
without success. The only option we have found to combine higher throughput
and high resolution is to use a water objective. It works really nicely with
glass bottom plates but now we also acquire time lapses, so I guess the only
way to go is to have a water refill system. Hence my query... Does anyone
have any experience with this type of protocols?

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Sweden
office: [phone]
LCI room: [phone]
mobile: [phone]

> -----Original Message-----
> From: Donnelly, Tom [mailto:[hidden email]]
> Sent: 24 September 2010 19:11
> To: '[hidden email]'
> Subject: RE: water refill device for WI objective
>
> Sylvie;
>
> I have often thought the oil that Zeiss offers that has the same
refractive index of
> water might be a good solution here.  Once the plate is coated ( probably
do that
> before starting scan) it would seem that little additional oil would be
needed.
>
> I have no experience with this and have no idea if it is compatible with
Nikon

> objectives, but just a thought.
>
> Tom
>
> Tom Donnelly
> Applied Precision, Inc.
> 1040 12th Ave. N.W. Issaquah, WA 98027-8929
> [phone]       [phone] fax
> [hidden email]
> http://www.appliedprecision.com
>
>
>
> -----Original Message-----
> From: Sylvie Le Guyader [mailto:[hidden email]]
> Sent: Friday, September 24, 2010 6:04 AM
> To: [hidden email]
> Subject: 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
> *****
>
> 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: [phone]
>
> mobile: [phone]
>
>
>
>
> This email message, together with any attachments, is for the sole use of

the
> intended recipient(s) and is the confidential information of Applied
Precision Inc. If
> you are not the intended recipient, your review, use, disclosure, copying
or
> dissemination of this email message or its attachments, or the information
> contained therein, is strictly prohibited. If you are not the intended
recipient or if you
> think this email was sent to you in error, please notify the sender by
reply email and
> delete this message and its attachments, as well as all copies, from your
system.

[Kurt Thorn]

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  I believe Sutter was working on a custom water pump system to refill
water on an objective, but I'm not sure if it came together or not.

I've also wondered if silanizing the bottom of the plate to make it more
hydrophobic would give a more uniform oil distribution.

Kurt

On 9/27/2010 6:49 AM, Sylvie Le Guyader wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Tom
>
> Thanks for your suggestion. We use the Zeiss oil for water lens routinely
> and it works very well as long as one doesn’t try to image a whole multiwell
> plate. Oil (normal or for water lenses) is too viscous for large areas. It
> collects at places and goes missing at others. We have tried this a lot
> without success. The only option we have found to combine higher throughput
> and high resolution is to use a water objective. It works really nicely with
> glass bottom plates but now we also acquire time lapses, so I guess the only
> way to go is to have a water refill system. Hence my query... Does anyone
> have any experience with this type of protocols?
>
> Med vänlig hälsning / Best regards
>  
> Sylvie
>  
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader
> Live Cell Imaging Unit
> Dept of Biosciences and Nutrition
> Karolinska Institutet
> Sweden
> office: [phone]
> LCI room: [phone]
> mobile: [phone]
>
>> -----Original Message-----
>> From: Donnelly, Tom [mailto:[hidden email]]
>> Sent: 24 September 2010 19:11
>> To: '[hidden email]'
>> Subject: RE: water refill device for WI objective
>>
>> Sylvie;
>>
>> I have often thought the oil that Zeiss offers that has the same
> refractive index of
>> water might be a good solution here.  Once the plate is coated ( probably
> do that
>> before starting scan) it would seem that little additional oil would be
> needed.
>> I have no experience with this and have no idea if it is compatible with
> Nikon
>> objectives, but just a thought.
>>
>> Tom
>>
>> Tom Donnelly
>> Applied Precision, Inc.
>> 1040 12th Ave. N.W. Issaquah, WA 98027-8929
>> [phone]       [phone] fax
>> [hidden email]
>> http://www.appliedprecision.com
>>
>>
>>
>> -----Original Message-----
>> From: Sylvie Le Guyader [mailto:[hidden email]]
>> Sent: Friday, September 24, 2010 6:04 AM
>> To: [hidden email]
>> Subject: 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
>> *****
>>
>> 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: [phone]
>>
>> mobile: [phone]
>>
>>
>>
>>
>> This email message, together with any attachments, is for the sole use of
> the
>> intended recipient(s) and is the confidential information of Applied
> Precision Inc. If
>> you are not the intended recipient, your review, use, disclosure, copying
> or
>> dissemination of this email message or its attachments, or the information
>> contained therein, is strictly prohibited. If you are not the intended
> recipient or if you
>> think this email was sent to you in error, please notify the sender by
> reply email and
>> delete this message and its attachments, as well as all copies, from your
> system.
>

[Stephen Cody-2]

*****
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:
[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: [phone]
>
> mobile: [phone]
>
>
>

--
Stephen H. Cody

[Boswell, Carl A - (cboswell)]

*****
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 [phone]
----- 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


*****
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:
[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: [phone]
>
> mobile: [phone]
>
>
>

--
Stephen H. Cody

[Neeraj Gohad-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Stephen,

The system sounds very interesting, could you please send me the PPT file as well. You could surely write a tech note on this in either J. Microsc or even Microscopy Today.

Thanks,

Neeraj.


Neeraj V. Gohad, Ph.D.
Postdoctoral Fellow
Okeanos Research Group
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: [phone]
Fax: [phone]

Website: http://www.clemson.edu/okeanos 





-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Tuesday, September 28, 2010 11:00 PM
To: [hidden email]
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:
[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: [phone]
>
> mobile: [phone]
>
>
>

--
Stephen H. Cody

[Sylvie Le Guyader-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
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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: [phone]
LCI room: [phone]
mobile: [phone]
 

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Carl Boswell
> Sent: 29 September 2010 19:14
> To: [hidden email]
> 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 [phone]
> ----- 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
>
>
> *****
> 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:
> [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: [phone]
> >
> > mobile: [phone]
> >
> >
> >
>
>
>
> --
> Stephen H. Cody

[Dan Focht]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Interested parties


I just received information from a company that makes a device for this application.  
They were not responding to this thread but after a review of their web site I saw that they have addressed this problem.
I have never used it but I did look into the technology.
Apparently it uses a new kind of micro-pump that uses Piezo deflection of a membrane to induce nanoflow.
It looks good and I am waiting to get a demo of this in my lab.
It looks to me that this is the company that makes a water replenishment system for Leica.
They might have a device for other manufacturers objectives but I don't know.
I am still looking into it.

Further info:
http://www.bartels-mikrotechnik.de/images/stories/components/download/Leica_Microsystems_Water_Immersion_Micro_Dispenser.pdf


Dan




On Oct 1, 2010, at 9:37 AM, Sylvie Le Guyader wrote:

*****
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: [phone]
LCI room: [phone]
mobile: [phone]

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Carl Boswell
> Sent: 29 September 2010 19:14
> To: [hidden email]
> 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 [phone]
> ----- 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
>
>
> *****
> 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:
> [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: [phone]
>>
>> mobile: [phone]
>>
>>
>>
>
>
>
> --
> Stephen H. Cody

Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler, PA 16002
www.bioptechs.com
P: [phone]
F: [phone]
[hidden email]

[Ian Dobbie]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Stephen Cody <[hidden email]> writes:

> 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.

[snip detailed description]

> 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.

I think this would be a very useful paper to have available. I think it
would be well worth your time (and great for the rest of us!) doing a
quick write up with a few diagrams/pictures to make clear exactly how it
works.

Thanks,

Ian

[Csúcs Gábor]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I'd also highly appreciate it,

Gabor

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Stephen Cody<[hidden email]>  writes:
>
>> 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.
> [snip detailed description]
>
>> 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.
> I think this would be a very useful paper to have available. I think it
> would be well worth your time (and great for the rest of us!) doing a
> quick write up with a few diagrams/pictures to make clear exactly how it
> works.
>
> Thanks,
>
> Ian

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: [phone]
Mobile: [phone]
Fax: [phone]
e-mail: [hidden email]