comparison of cover slips and mounting media

[milton charlton]

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Dear confocal cognoscenti,
 
Has anyone compared confocal images obtained with #1 (0.13-0.17mm) and
#1.5(0.16-0.19mm) cover slips using 40X(1.25NA) and 63X(1.32 NA) oil
objectives or similar?  
 
Mounting media:  Is there any consensus on the best mounting medium that
combines high refractive index (near RI= 1.5) with preservation of
fluorescence?
 
Thanks

Milton P. Charlton, Professor
Physiology Department
Medical Science Building #3308
University of Toronto
1 King's College Circle
Toronto, ON, M5S1A8
Canada
tel:  [phone]
fax:  [phone]
[hidden email]



 

[Cromey, Douglas W - (dcromey)]

http://www.olympusmicro.com/primer/anatomy/coverslipcorrection.html

This might be of help.
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 Milton Charlton
Sent: Friday, October 07, 2011 9:50 AM
To: [hidden email]
Subject: comparison of cover slips and mounting media

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Dear confocal cognoscenti,
 
Has anyone compared confocal images obtained with #1 (0.13-0.17mm) and
#1.5(0.16-0.19mm) cover slips using 40X(1.25NA) and 63X(1.32 NA) oil objectives or similar?  
 
Mounting media:  Is there any consensus on the best mounting medium that combines high refractive index (near RI= 1.5) with preservation of fluorescence?
 
Thanks

Milton P. Charlton, Professor
Physiology Department
Medical Science Building #3308
University of Toronto
1 King's College Circle
Toronto, ON, M5S1A8
Canada
tel:  [phone]
fax:  [phone]
[hidden email]



 

[James Pawley]

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>http://www.olympusmicro.com/primer/anatomy/coverslipcorrection.html
>
>This might be of help.
>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"

Thanks for the suggestion Doug. I like the Applet at

http://www.olympusmicro.com/primer/java/aberrations/slipcorrection/index.html

However, as to their instructions, I believe it to be almost
impossible to detect and correct SA using any object other than a
very small luminous point. As soon as you have a small, "real"
object, it almost always changes in Z and as the best-focus plane
changes as you move the collar, you end up looking at different focus
planes. In addition, you always see a superposition of in-focus and
out-out-focus features.

Z-symmetry is all.

The only other method for correcting SA is to make a confocal X-Z
reflection image of a mirror surface (TURN YOUR laser and PMT WAY
DOWN!). As with the point object, you look for z-symmetry in the
intensity plot along a line scanned in z, across the mirror surface.

But this only shows that you have corrected SA up to this mirror
surface. The interesting part of your specimen is unlikely to be on
this surface. Small beads are good, but it takes practice and (if you
do it in Widefield) good fast sCCD or EMCCD cameras or dark-adapted
eyes.

JP

>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On Behalf Of Milton
>Charlton
>Sent: Friday, October 07, 2011 9:50 AM
>To: [hidden email]
>Subject: comparison of cover slips and mounting media
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear confocal cognoscenti,
>
>Has anyone compared confocal images obtained with #1 (0.13-0.17mm) and
>#1.5(0.16-0.19mm) cover slips using 40X(1.25NA) and 63X(1.32 NA) oil
>objectives or similar?
>
>Mounting media:  Is there any consensus on the best mounting medium
>that combines high refractive index (near RI= 1.5) with preservation
>of fluorescence?
>
>Thanks
>
>Milton P. Charlton, Professor
>Physiology Department
>Medical Science Building #3308
>University of Toronto
>1 King's College Circle
>Toronto, ON, M5S1A8
>Canada
>tel:  [phone]
>fax:  [phone]
>[hidden email]
>
>
>
>

--
James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726
Phone: [phone]

[Martin Wessendorf-2]

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Dear Dr. Charlton--

On 10/7/2011 11:49 AM, Milton Charlton wrote:

> Mounting media:  Is there any consensus on the best mounting medium that
> combines high refractive index (near RI= 1.5) with preservation of
> fluorescence?

We use DPX for most applications.  It dries hard, has an index of
refraction near that of glass and is compatible with Cy2, Cy3 and Cy5.
Photobleaching is acceptable except with high-NA oil lenses and
widefield illumination; I've had very few problems with such lenses and
confocal microscopy.  I have tissue that I stained and mounted in DPX
almost 20 years ago that I still use regularly for teaching.

It has one disadvantage: you have to dehydrate your tissue through
alcohols, which can shrink it in the z-axis.

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]