sample prep for intra-occular lenses

[Cromey, Douglas W - (dcromey)]

I have a new set of users for our inverted confocal that will be bringing intra-occular lenses (of the kind used to fix cateracts).  The lenses have a thin layer of cells on the surface, which is curved.  Right now they are bringing them mounted in buffer as a whole mount with wax to seal the coverslip edges.  They try to compress the lens by putting the slide under a book after it's mounted.

It seems like there ought to be a better way and I'm hoping that someone out there has dealt with this before.

Thanks!
Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"

[JOEL B. SHEFFIELD]

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This is an interesting puzzle.   Those people who are working with curved
surfaces, such as retinas, make a series of radial cuts that allow them to
make flat(er) mounts.  Of course, in this case, I would imagine that the
lenses are also of variable thickness, so there might be an additional
complication.  You should be sure that the cellular surface is closest to
the cover slip so that the layer of cells is more uniform.  Do you know on
which surface of the lens the cells will be found? Or are they on both?

Joel


On Thu, Apr 19, 2012 at 4:41 PM, Cromey, Douglas W - (dcromey) <
[hidden email]> wrote:

> I have a new set of users for our inverted confocal that will be bringing
> intra-occular lenses (of the kind used to fix cateracts).  The lenses have
> a thin layer of cells on the surface, which is curved.  Right now they are
> bringing them mounted in buffer as a whole mount with wax to seal the
> coverslip edges.  They try to compress the lens by putting the slide under
> a book after it's mounted.
>
> It seems like there ought to be a better way and I'm hoping that someone
> out there has dealt with this before.
>
> Thanks!
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Assistant Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/exppath/
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Cromey, Douglas W - (dcromey)]

Joel,

Today was my first experience with the lenses.  

As I understand it, these are the implantable lenses used to fix cataracts.  After a time in the eye, cells grow (fairly flat cells in a layer that appears to be 2-3 cells thick) on the anterior surface (facing out of the eye).  They mount the lens so that the anterior surface is closest to the coverslip.  While I suppose slicing the lens might be an option, my suspicion is that the surface will always be curved in relation to the flat focal plane of the confocal.  

The curved surface itself doesn't bother me a whole lot, but I would like to think that we can nail down the sample prep to something slightly less kludge-y that would give the best images possible.  Based on today's observations, the part of the sample that is farther away from the coverslip has less detail, most likely due to spherical aberration (Thanks Jim P!) because we are imaging into an aqueous solution that is beginning to be a good distance from the coverslip (due to lens curvature).  I should add that my objective lens options are 20x/0.7 dry, 40x/1.25 oil and 63x/1.4 oil.  No water lenses on this particular confocal.  I used the 20x today, mostly for "field of view" reasons.

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joel B. Sheffield
Sent: Thursday, April 19, 2012 1:56 PM
To: [hidden email]
Subject: Re: sample prep for intra-occular lenses

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

This is an interesting puzzle.   Those people who are working with curved
surfaces, such as retinas, make a series of radial cuts that allow them to make flat(er) mounts.  Of course, in this case, I would imagine that the lenses are also of variable thickness, so there might be an additional complication.  You should be sure that the cellular surface is closest to the cover slip so that the layer of cells is more uniform.  Do you know on which surface of the lens the cells will be found? Or are they on both?

Joel


On Thu, Apr 19, 2012 at 4:41 PM, Cromey, Douglas W - (dcromey) < [hidden email]> wrote:

> I have a new set of users for our inverted confocal that will be
> bringing intra-occular lenses (of the kind used to fix cateracts).  
> The lenses have a thin layer of cells on the surface, which is curved.  
> Right now they are bringing them mounted in buffer as a whole mount
> with wax to seal the coverslip edges.  They try to compress the lens
> by putting the slide under a book after it's mounted.
>
> It seems like there ought to be a better way and I'm hoping that
> someone out there has dealt with this before.
>
> Thanks!
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
> Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/exppath/
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Barbara Foster]

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*****

Hi, Joel

Have recently undergone this operation, this question is near and
dear to my heart!

The ultimate answer to your question rests in what they actually want
to see.  However, in general, this sounds like one of those
situations perfectly suited to a confocal serial section with 3D
reconstruction.  We had a situation years ago when I was working for
Sarastro in which a researcher wanted to image the whole cornea.  In
his case, the most useful information came from a Z
section.  However, more recently, when I went to the surgeon, he did
just regular imaging to look at the cells on my cornea.. a situation
which is very analogous to your problem.

I agree that mounting the lens cell side toward the objective makes
the most sense.  Also, mounting in Saline (or eye drops?) will reduce
the spherical aberration.

Good hunting! .... and let us know how it turns out!

Barbara Foster, President and Sr. Consultant
Microscopy/Microscopy Education   P: [phone]   W:
www.MicroscopyEducation.com

We are now scheduling courses through August 2012... and don't forget
to take part in our most recent surveys.  Check
MicroscopyEducation.com for details.

At 07:27 PM 4/19/2012, you wrote:

>Joel,
>
>Today was my first experience with the lenses.
>
>As I understand it, these are the implantable lenses used to fix
>cataracts.  After a time in the eye, cells grow (fairly flat cells
>in a layer that appears to be 2-3 cells thick) on the anterior
>surface (facing out of the eye).  They mount the lens so that the
>anterior surface is closest to the coverslip.  While I suppose
>slicing the lens might be an option, my suspicion is that the
>surface will always be curved in relation to the flat focal plane of
>the confocal.
>
>The curved surface itself doesn't bother me a whole lot, but I would
>like to think that we can nail down the sample prep to something
>slightly less kludge-y that would give the best images
>possible.  Based on today's observations, the part of the sample
>that is farther away from the coverslip has less detail, most likely
>due to spherical aberration (Thanks Jim P!) because we are imaging
>into an aqueous solution that is beginning to be a good distance
>from the coverslip (due to lens curvature).  I should add that my
>objective lens options are 20x/0.7 dry, 40x/1.25 oil and 63x/1.4
>oil.  No water lenses on this particular confocal.  I used the 20x
>today, mostly for "field of view" reasons.
>
>Doug
>
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>Douglas W. Cromey, M.S. - Assistant Scientific Investigator
>Dept. of Cellular & Molecular Medicine, University of Arizona
>1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
>office:  AHSC 4212         email: [hidden email]
>voice:  520-626-2824       fax:  [phone]
>
>http://swehsc.pharmacy.arizona.edu/exppath/
>Home of: "Microscopy and Imaging Resources on the WWW"
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Joel B. Sheffield
>Sent: Thursday, April 19, 2012 1:56 PM
>To: [hidden email]
>Subject: Re: sample prep for intra-occular lenses
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>This is an interesting puzzle.   Those people who are working with curved
>surfaces, such as retinas, make a series of radial cuts that allow
>them to make flat(er) mounts.  Of course, in this case, I would
>imagine that the lenses are also of variable thickness, so there
>might be an additional complication.  You should be sure that the
>cellular surface is closest to the cover slip so that the layer of
>cells is more uniform.  Do you know on which surface of the lens the
>cells will be found? Or are they on both?
>
>Joel
>
>
>On Thu, Apr 19, 2012 at 4:41 PM, Cromey, Douglas W - (dcromey) <
>[hidden email]> wrote:
>
> > I have a new set of users for our inverted confocal that will be
> > bringing intra-occular lenses (of the kind used to fix cateracts).
> > The lenses have a thin layer of cells on the surface, which is curved.
> > Right now they are bringing them mounted in buffer as a whole mount
> > with wax to seal the coverslip edges.  They try to compress the lens
> > by putting the slide under a book after it's mounted.
> >
> > It seems like there ought to be a better way and I'm hoping that
> > someone out there has dealt with this before.
> >
> > Thanks!
> > Doug
> >
> > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> > Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
> > Cellular & Molecular Medicine, University of Arizona
> > 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
> >
> > office:  AHSC 4212         email: [hidden email]
> > voice:  520-626-2824       fax:  [phone]
> >
> > http://swehsc.pharmacy.arizona.edu/exppath/
> > Home of: "Microscopy and Imaging Resources on the WWW"
> >
> >
> >
>
>
>--
>
>
>Joel B. Sheffield, Ph.D
>Department of Biology
>Temple University
>Philadelphia, PA 19122
>Voice: 215 204 8839
>e-mail: [hidden email]
>URL:  http://astro.temple.edu/~jbs

[Gabriel Lapointe-2]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Doug,
If the cells are fixed, mounting them in 97% 2-thioethanol might reduce
the refractive index miss-match and reduce the spherical aberration.

http://www.ncbi.nlm.nih.gov/pubmed/17131355

sincerely,

logo
Gabriel Lapointe, M.Sc.
6651 de Lanaudière, app
3
Montréal (Québec), H2G
3B1
Cell : [phone]
[hidden email]
http://gabriellapointe.ca
logo



On Thu, 2012-04-19 at 14:13 -0700, Cromey, Douglas W - (dcromey) wrote:

> Joel,
>
> Today was my first experience with the lenses.  
>
> As I understand it, these are the implantable lenses used to fix cataracts.  After a time in the eye, cells grow (fairly flat cells in a layer that appears to be 2-3 cells thick) on the anterior surface (facing out of the eye).  They mount the lens so that the anterior surface is closest to the coverslip.  While I suppose slicing the lens might be an option, my suspicion is that the surface will always be curved in relation to the flat focal plane of the confocal.  
>
> The curved surface itself doesn't bother me a whole lot, but I would like to think that we can nail down the sample prep to something slightly less kludge-y that would give the best images possible.  Based on today's observations, the part of the sample that is farther away from the coverslip has less detail, most likely due to spherical aberration (Thanks Jim P!) because we are imaging into an aqueous solution that is beginning to be a good distance from the coverslip (due to lens curvature).  I should add that my objective lens options are 20x/0.7 dry, 40x/1.25 oil and 63x/1.4 oil.  No water lenses on this particular confocal.  I used the 20x today, mostly for "field of view" reasons.
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Assistant Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/exppath/
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joel B. Sheffield
> Sent: Thursday, April 19, 2012 1:56 PM
> To: [hidden email]
> Subject: Re: sample prep for intra-occular lenses
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> This is an interesting puzzle.   Those people who are working with curved
> surfaces, such as retinas, make a series of radial cuts that allow them to make flat(er) mounts.  Of course, in this case, I would imagine that the lenses are also of variable thickness, so there might be an additional complication.  You should be sure that the cellular surface is closest to the cover slip so that the layer of cells is more uniform.  Do you know on which surface of the lens the cells will be found? Or are they on both?
>
> Joel
>
>
> On Thu, Apr 19, 2012 at 4:41 PM, Cromey, Douglas W - (dcromey) < [hidden email]> wrote:
>
> > I have a new set of users for our inverted confocal that will be
> > bringing intra-occular lenses (of the kind used to fix cateracts).  
> > The lenses have a thin layer of cells on the surface, which is curved.  
> > Right now they are bringing them mounted in buffer as a whole mount
> > with wax to seal the coverslip edges.  They try to compress the lens
> > by putting the slide under a book after it's mounted.
> >
> > It seems like there ought to be a better way and I'm hoping that
> > someone out there has dealt with this before.
> >
> > Thanks!
> > Doug
> >
> > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> > Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
> > Cellular & Molecular Medicine, University of Arizona
> > 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
> >
> > office:  AHSC 4212         email: [hidden email]
> > voice:  520-626-2824       fax:  [phone]
> >
> > http://swehsc.pharmacy.arizona.edu/exppath/
> > Home of: "Microscopy and Imaging Resources on the WWW"
> >
> >
> >
>
>

[Mark Cannell]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Having imaged real lenses, I think you need to define what you want to image (i.e. low res or high res over just a bit). A 40x dipping lens has several mm of working distance and even with a #0 coverslip in place gives quite good images. You may not be able to reach the entire lens surface, perhaps you should cut it up (carefully) .

Hope this helps.

Mark




 
On 19/04/2012, at 10:13 PM, Cromey, Douglas W - (dcromey) wrote:

> Joel,
>
> Today was my first experience with the lenses.  
>
> As I understand it, these are the implantable lenses used to fix cataracts.  After a time in the eye, cells grow (fairly flat cells in a layer that appears to be 2-3 cells thick) on the anterior surface (facing out of the eye).  They mount the lens so that the anterior surface is closest to the coverslip.  While I suppose slicing the lens might be an option, my suspicion is that the surface will always be curved in relation to the flat focal plane of the confocal.  
>
> The curved surface itself doesn't bother me a whole lot, but I would like to think that we can nail down the sample prep to something slightly less kludge-y that would give the best images possible.  Based on today's observations, the part of the sample that is farther away from the coverslip has less detail, most likely due to spherical aberration (Thanks Jim P!) because we are imaging into an aqueous solution that is beginning to be a good distance from the coverslip (due to lens curvature).  I should add that my objective lens options are 20x/0.7 dry, 40x/1.25 oil and 63x/1.4 oil.  No water lenses on this particular confocal.  I used the 20x today, mostly for "field of view" reasons.
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Assistant Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/exppath/
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joel B. Sheffield
> Sent: Thursday, April 19, 2012 1:56 PM
> To: [hidden email]
> Subject: Re: sample prep for intra-occular lenses
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> This is an interesting puzzle.   Those people who are working with curved
> surfaces, such as retinas, make a series of radial cuts that allow them to make flat(er) mounts.  Of course, in this case, I would imagine that the lenses are also of variable thickness, so there might be an additional complication.  You should be sure that the cellular surface is closest to the cover slip so that the layer of cells is more uniform.  Do you know on which surface of the lens the cells will be found? Or are they on both?
>
> Joel
>
>
> On Thu, Apr 19, 2012 at 4:41 PM, Cromey, Douglas W - (dcromey) < [hidden email]> wrote:
>
>> I have a new set of users for our inverted confocal that will be
>> bringing intra-occular lenses (of the kind used to fix cateracts).  
>> The lenses have a thin layer of cells on the surface, which is curved.  
>> Right now they are bringing them mounted in buffer as a whole mount
>> with wax to seal the coverslip edges.  They try to compress the lens
>> by putting the slide under a book after it's mounted.
>>
>> It seems like there ought to be a better way and I'm hoping that
>> someone out there has dealt with this before.
>>
>> Thanks!
>> Doug
>>
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
>> Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>>
>> office:  AHSC 4212         email: [hidden email]
>> voice:  520-626-2824       fax:  [phone]
>>
>> http://swehsc.pharmacy.arizona.edu/exppath/
>> Home of: "Microscopy and Imaging Resources on the WWW"
>>
>>
>>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
>

[Guy Cox-2]

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

Well, I've never imaged 'real' lenses, but I did assist with a (very ambitious) PhD project to grow replacement lenses in a culture dish.  Not only did we need to image them, we also had to work out an optic system to show that they did have a focus.  Amazingly, they did!  Not that it was good enough to give anyone very good vision, but as a proof of concept it was incredible.  I would just add that if you do have to use a cover slip you may be able to  buy 'water' ones with approximately the RI of water.  Check the archives, because I do remember that it was discussed here some time ago.  Maybe they were from Olympus?

                                                        Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Friday, 20 April 2012 5:19 PM
To: [hidden email]
Subject: Re: sample prep for intra-occular lenses

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

Having imaged real lenses, I think you need to define what you want to image (i.e. low res or high res over just a bit). A 40x dipping lens has several mm of working distance and even with a #0 coverslip in place gives quite good images. You may not be able to reach the entire lens surface, perhaps you should cut it up (carefully) .

Hope this helps.

Mark




 
On 19/04/2012, at 10:13 PM, Cromey, Douglas W - (dcromey) wrote:

> Joel,
>
> Today was my first experience with the lenses.  
>
> As I understand it, these are the implantable lenses used to fix cataracts.  After a time in the eye, cells grow (fairly flat cells in a layer that appears to be 2-3 cells thick) on the anterior surface (facing out of the eye).  They mount the lens so that the anterior surface is closest to the coverslip.  While I suppose slicing the lens might be an option, my suspicion is that the surface will always be curved in relation to the flat focal plane of the confocal.  
>
> The curved surface itself doesn't bother me a whole lot, but I would like to think that we can nail down the sample prep to something slightly less kludge-y that would give the best images possible.  Based on today's observations, the part of the sample that is farther away from the coverslip has less detail, most likely due to spherical aberration (Thanks Jim P!) because we are imaging into an aqueous solution that is beginning to be a good distance from the coverslip (due to lens curvature).  I should add that my objective lens options are 20x/0.7 dry, 40x/1.25 oil and 63x/1.4 oil.  No water lenses on this particular confocal.  I used the 20x today, mostly for "field of view" reasons.
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
> Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/exppath/
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Joel B.
> Sheffield
> Sent: Thursday, April 19, 2012 1:56 PM
> To: [hidden email]
> Subject: Re: sample prep for intra-occular lenses
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> This is an interesting puzzle.   Those people who are working with curved
> surfaces, such as retinas, make a series of radial cuts that allow them to make flat(er) mounts.  Of course, in this case, I would imagine that the lenses are also of variable thickness, so there might be an additional complication.  You should be sure that the cellular surface is closest to the cover slip so that the layer of cells is more uniform.  Do you know on which surface of the lens the cells will be found? Or are they on both?
>
> Joel
>
>
> On Thu, Apr 19, 2012 at 4:41 PM, Cromey, Douglas W - (dcromey) < [hidden email]> wrote:
>
>> I have a new set of users for our inverted confocal that will be
>> bringing intra-occular lenses (of the kind used to fix cateracts).
>> The lenses have a thin layer of cells on the surface, which is curved.  
>> Right now they are bringing them mounted in buffer as a whole mount
>> with wax to seal the coverslip edges.  They try to compress the lens
>> by putting the slide under a book after it's mounted.
>>
>> It seems like there ought to be a better way and I'm hoping that
>> someone out there has dealt with this before.
>>
>> Thanks!
>> Doug
>>
>> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>> Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
>> Cellular & Molecular Medicine, University of Arizona
>> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>>
>> office:  AHSC 4212         email: [hidden email]
>> voice:  520-626-2824       fax:  [phone]
>>
>> http://swehsc.pharmacy.arizona.edu/exppath/
>> Home of: "Microscopy and Imaging Resources on the WWW"
>>
>>
>>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
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[Sylvie Le Guyader]

Hi Doug

Try 1% low melting point agarose in culture medium. It is still liquid at 37 deg so it works
for live cells and you will have no problem with index mismatch.

Med vänlig hälsning / Best regards
 
Sylvie
 
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Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14183 Huddinge
Sweden
office: [phone]
LCI room: [phone]
mobile: [phone]

>
> > *****
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> >
> > On Thu, 2012-04-19 at 14:13 -0700, Cromey, Douglas W - (dcromey) wrote:
> >
> >> Joel,
> >>
> >> Today was my first experience with the lenses.
> >>
> >> As I understand it, these are the implantable lenses used to fix cataracts.  After
> a time in the eye, cells grow (fairly flat cells in a layer that appears to be 2-3 cells
> thick) on the anterior surface (facing out of the eye).  They mount the lens so that
> the anterior surface is closest to the coverslip.  While I suppose slicing the lens
> might be an option, my suspicion is that the surface will always be curved in
> relation to the flat focal plane of the confocal.
> >>
> >> The curved surface itself doesn't bother me a whole lot, but I would like to think
> that we can nail down the sample prep to something slightly less kludge-y that
> would give the best images possible.  Based on today's observations, the part of
> the sample that is farther away from the coverslip has less detail, most likely due
> to spherical aberration (Thanks Jim P!) because we are imaging into an aqueous
> solution that is beginning to be a good distance from the coverslip (due to lens
> curvature).  I should add that my objective lens options are 20x/0.7 dry, 40x/1.25
> oil and 63x/1.4 oil.  No water lenses on this particular confocal.  I used the 20x
> today, mostly for "field of view" reasons.
> >>
> >> Doug
> >>
> >> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> >> Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of
> >> Cellular & Molecular Medicine, University of Arizona
> >> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
> >>
> >> office:  AHSC 4212         email: [hidden email]
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> >>
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> >> Home of: "Microscopy and Imaging Resources on the WWW"
> >>
> >>