Immersion oil

[Knecht, David]

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If you are imaging a sample in aqueous media through glass with a 1.25 NA 100x oil objective, what is the correct refractive index immersion oil to use?  You are going to have refractive index mismatches no matter what, but are some more critical than others?  Is the 1.25 NA objective designed to be used with 1.515 RI oil or should one use a lower RI oil or match the sample by using water?  Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[mmodel]

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I  think if you attempt to use a lower refractive index you will be getting additional aberration from a coverslip. To test the effect of oil, you can attach beads to two coverslips and place then facing each other and separated by a gap of appropriate size (Journal of Microscopy, Vol. 241, Pt 1 2011, pp. 94–100). Then compare the quality of images of the top an bottom beads.

Mike

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Knecht, David <[hidden email]>
Sent: Monday, April 4, 2016 6:40 PM
To: [hidden email]
Subject: Immersion oil

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If you are imaging a sample in aqueous media through glass with a 1.25 NA 100x oil objective, what is the correct refractive index immersion oil to use?  You are going to have refractive index mismatches no matter what, but are some more critical than others?  Is the 1.25 NA objective designed to be used with 1.515 RI oil or should one use a lower RI oil or match the sample by using water?  Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[Craig Brideau]

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Oil objectives always assume you are trying to match to the index of the
coverslip. By using an oil with the same index as the coverslip, you are
essentially removing the coverslip first surface from the light path. That
is, you have a constant medium between the end of your objective and the
inner surface of the coverslip. This assumes your sample is resting
perfectly against the coverslip surface though, so reality varies. If you
have a gap between that inner surface and the sample you now have a layer
of mounting media to get through, with its own index of refraction
difference.

Craig

On Mon, Apr 4, 2016 at 4:40 PM, Knecht, David <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> If you are imaging a sample in aqueous media through glass with a 1.25 NA
> 100x oil objective, what is the correct refractive index immersion oil to
> use?  You are going to have refractive index mismatches no matter what, but
> are some more critical than others?  Is the 1.25 NA objective designed to
> be used with 1.515 RI oil or should one use a lower RI oil or match the
> sample by using water?  Dave
>
> Dr. David Knecht
> Professor of Molecular and Cell Biology
> Core Microscopy Facility Director
> University of Connecticut
> 91 N. Eagleville Rd.
> Storrs, CT 06269
> 860-486-2200
>

[Jeremy Adler-4]

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An 'oil' objective requires a medium with the RI of oil between itself and the coverslip. This provides the RI the objective is designed for until the outer edge of the coverslip. The RI matching should continue beyond the coverslip, and   from here it goes wrong for a water based sample. The degradation increases with the distance from the coverslip to the imaging plane. So if you are interested in how cells desperately cling to coverslips then the mismatch in RIs is not too serious.

But given the time and resources invested in an experiment why not optimize the imaging by using a water objective.

The question can be addressed experimentally and most manufacturers will lend you an objective in the hope that you will be impressed by its performance. An appropriate test sample would be a slide with 200nm fluorescent microspheres on both the coverslip and slide with a spacer between them whose thickness matches the location of your imaging plane. Compare the observed PSFs of the microspheres on the coverslip with the PSFs on the slide and you have an empirical answer.




________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Knecht, David [[hidden email]]
Sent: 05 April 2016 00:40
To: [hidden email]
Subject: Immersion oil

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If you are imaging a sample in aqueous media through glass with a 1.25 NA 100x oil objective, what is the correct refractive index immersion oil to use?  You are going to have refractive index mismatches no matter what, but are some more critical than others?  Is the 1.25 NA objective designed to be used with 1.515 RI oil or should one use a lower RI oil or match the sample by using water?  Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[Lutz Schaefer]

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David,
when using an immersion medium that has a lower index than the objective is
designed for, you are essentially losing effective NA and therefore
resolution. Also you are no longer corrected for spherical aberration (SA)
at the sample side of the cover slip. However, the remaining SA may no
longer be depth variant in case you match the embedding index. Generally its
not a good idea to deviate from the manufacturers design immersion index,
unless you know how to correct for it.

Regards
Lutz

-----Original Message-----
From: Knecht, David
Sent: Monday, April 04, 2016 6:40 PM
To: [hidden email]
Subject: Immersion oil

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If you are imaging a sample in aqueous media through glass with a 1.25 NA
100x oil objective, what is the correct refractive index immersion oil to
use?  You are going to have refractive index mismatches no matter what, but
are some more critical than others?  Is the 1.25 NA objective designed to be
used with 1.515 RI oil or should one use a lower RI oil or match the sample
by using water?  Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

[Stanislav Vitha-2]

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For your situation, there are two practical options:

1) You could use a thinner coverslip and immersion oil with higher
refractive index than standard oil.  Wan DS, Rajadhyaksha M, Webb RH
(2000) Analysis of spherical aberration of a water immersion objective:
application to specimens with refractive indices 1.33-1.40. J Microsc 197:
274-284, see also comments by Sheppard J Microsc 200: 177-178
The problem is that for full correction at 1.25 NA, you may need immersion
oils of very high RI- those may not be available.  

2)
Use a finite-corrected objective on an infinity-corrected system: REIHANI,
S.N.S. & ODDERSHEDE, L.B. (2009). Confocal microscopy of thick
specimens. Journal of Biomedical Optics 14, 030513-030513-030513.
 

You could also change the tube length to compensate for spherical
aberration - on some old compound microscopes with finite optics and a
trinocular tube it is easy, you can move the camera up or down from the
default 160 mm tube lengths position. I am not sure how easy it would be
on some newer models; probably not possible or practical or modern point
scanning confocals.

Stan Vitha
Micrsocopy and Imaging Center
Texas A&M University

On Tue, 5 Apr 2016 01:39:45 +0000, MODEL, MICHAEL
<[hidden email]> wrote:

>
>I  think if you attempt to use a lower refractive index you will be getting
additional aberration from a coverslip. To test the effect of oil, you can
attach beads to two coverslips and place then facing each other and
separated by a gap of appropriate size (Journal of Microscopy, Vol. 241, Pt
1 2011, pp. 94–100). Then compare the quality of images of the top an
bottom beads.
>
>Mike
>
>________________________________________
>From: Confocal Microscopy List
<[hidden email]> on behalf of Knecht, David
<[hidden email]>

>If you are imaging a sample in aqueous media through glass with a 1.25
NA 100x oil objective, what is the correct refractive index immersion oil to
use?  You are going to have refractive index mismatches no matter what,
but are some more critical than others?  Is the 1.25 NA objective designed
to be used with 1.515 RI oil or should one use a lower RI oil or match the
sample by using water?  Dave
>
>Dr. David Knecht