resolution of Zeiss Water and Glycerine 63X lens

[Xiao Liu]

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Hi, everyone:

I'm looking for a 63X objective for live imaging and its resolution is my major concern.  Zeiss recommands Objective LCI Plan-Neofluar 63x/1.3 Imm Corr DIC M27, saying it has NA = 1.3 in both water and glycerine.  Does it really have NA=1.3 even in water?  

Your reply is highly appreciated.
Xiao

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Xiao Liu
School of Life Sciences
Tsing-Hua University
Beijing, China
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[Pascal Weber]

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What is your goal !

Do you really nead glycerine if yes tehre just one objective. This one
recommanded by Zeiss. But you juste have only 170 micron working distance.

I worked with this one on  a live mouse brain stained with GFP . And we
obtained 700 microns penetration on a two photon microscope.

Objectif W Plan-Apochromat 63x/1,0 M27.

[Guy Cox-2]

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The only way this would be possible is if it were stopped down to reduce its actual aperture in glycerine.  This is technically possible but (IMHO) unlikely.  Glycerol has a refractive index of 1.4722 at 590nm (the closest I can find to the official 550nm).  NA 1.3 therefore corresponds to an acceptance half-angle of 62 degrees.  Water at 550nm has a refractive index of 1.33.  This same half-angle would be an NA of 1.17.  

Looking at it the other way round, to achieve NA 1.3 in water requires an acceptance angle of 77 degrees, which while not impossible is pushing the envelope.

                                      Guy

Optical Imaging Techniques in Cell Biology
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Xiao Liu
Sent: Friday, 16 December 2011 11:09 AM
To: [hidden email]
Subject: resolution of Zeiss Water and Glycerine 63X lens

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Hi, everyone:

I'm looking for a 63X objective for live imaging and its resolution is my major concern.  Zeiss recommands Objective LCI Plan-Neofluar 63x/1.3 Imm Corr DIC M27, saying it has NA = 1.3 in both water and glycerine.  Does it really have NA=1.3 even in water?  

Your reply is highly appreciated.
Xiao

*******************
Xiao Liu
School of Life Sciences
Tsing-Hua University
Beijing, China
*******************
     

[James Pawley]

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>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
>
>Hi, everyone:
>
>I'm looking for a 63X objective for live imaging and its resolution
>is my major concern.  Zeiss recommands Objective LCI Plan-Neofluar
>63x/1.3 Imm Corr DIC M27, saying it has NA = 1.3 in both water and
>glycerine.  Does it really have NA=1.3 even in water?
>
>Your reply is highly appreciated.
>Xiao
>
>*******************
>Xiao Liu
>School of Life Sciences
>Tsing-Hua University
>Beijing, China
>*******************
>    

Well, it may have such an NA in water, but most of the rays
approaching the flat surfaces (two sides of the cover slip and the
front element of the objective) will be reflected when transmitting
these surface, so will not contribute much to the image.

Keep in mind that, when actually tested, using the Methods in Chapter
11 of the Handbook, Juskaitis found that even high quality NA 1.4
objectives only worked "properly" up to 1.35 (The high-NA ray were
indeed propagated by the lenses but had the wrong phase so actually
did not contribute to image sharpness).

In general, when viewing living specimens, "resolution" (that
slippery concept that can only be considered in terms of more or less
contrast) is more likely degraded by aberrations brought on by wrong
immersion media or by the RI variations of the specimen itself than
by the NA marked on the side of the lens.

Cheers,

Jim Pawley
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