Immersion Objectives

[Vonderheit, Andreas]

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Dear list,

I learned and I thought that the different types of immersion media are there to get the best refractive index match to the media or cells behind the cover glass, in order to minimize spherical aberration.
Now some microscope companies say that this discussion is tedious since the error of the lenses/objectives are higher than the effect of the refractive index match, is that true?

Totally understandable: when I use a mounting medium, which would be close to the refractive index of the used oil (refractive index : 1.52) and the cover glass (refractive index: 1.52), this would be a perfect match.

But what happens, when I am using a mounting medium based on glycerol (refractive index: 1.47), still the cover glass has 1.52. Should I use a glycerol objective and glycerol as an immersion medium, or an oil objective with oil as immersion medium?

I thought when I do live microscopy in an aqueous solution (refractive index of water: 1.33) the best choice would be a water-immersion objective.

Now one company also offers silicone-oil-immersion objectives, the silicone oil has a refractive index of 1.4, which would be closer to the refractive index of the sample which they claim to be at 1.38.

I have the feeling that I opened the box of Pandora and all that I wanted was the best solution to a setup, where one of my users wants to look at C.elegans in aqueous solution, which objective type should be used?

Thanks, best regards
Andreas

[Guy Cox-2]

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Andreas,

  Always look for the best refractive index match.  Be aware that some 'glycerol' mounting media are NOT pure glycerol but more like 50%.  I have demonstrated experimentally that in this case you can do better with a water immersion lens setting the correction collar as if for a very thick coverslip, than you can with an oil objective.  IF all oil immersion objectives were equipped with correction collars the situation could be different.   Let's be clear that they should be - oil varies hugely in refractive index with temperature.  If you are looking at living samples in aqueous media you should always use a water (coverslip) lens - at least they always have correction collars.

                The corollary is that you do need to learn how to use a correction collar - it's a skill, and not a difficult one, but it does need to be mastered.  Simply setting it to what 'should' be the correct value never works.  

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox.  2nd edition 2012    CRC Press
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vonderheit, Andreas
Sent: Tuesday, 31 July 2012 7:19 PM
To: [hidden email]
Subject: Immersion Objectives

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Dear list,

I learned and I thought that the different types of immersion media are there to get the best refractive index match to the media or cells behind the cover glass, in order to minimize spherical aberration.
Now some microscope companies say that this discussion is tedious since the error of the lenses/objectives are higher than the effect of the refractive index match, is that true?

Totally understandable: when I use a mounting medium, which would be close to the refractive index of the used oil (refractive index : 1.52) and the cover glass (refractive index: 1.52), this would be a perfect match.

But what happens, when I am using a mounting medium based on glycerol (refractive index: 1.47), still the cover glass has 1.52. Should I use a glycerol objective and glycerol as an immersion medium, or an oil objective with oil as immersion medium?

I thought when I do live microscopy in an aqueous solution (refractive index of water: 1.33) the best choice would be a water-immersion objective.

Now one company also offers silicone-oil-immersion objectives, the silicone oil has a refractive index of 1.4, which would be closer to the refractive index of the sample which they claim to be at 1.38.

I have the feeling that I opened the box of Pandora and all that I wanted was the best solution to a setup, where one of my users wants to look at C.elegans in aqueous solution, which objective type should be used?

Thanks, best regards
Andreas

[Johannes Helm]

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Dear Andreas,

Pandora's box, indeed.

You write:

> I learned and I thought that the different types of immersion media are
> there to get the best refractive index match to the media or cells behind
> the cover glass, in order to minimize spherical aberration.

This is an issue, which is frequently being asked during lectures, and,
historically, you are wrong, in modern times you are - sometimes - right.
Unfortunately, a long answer:

A relevant aspect - besides what you have mentioned - in this immersion
story is:

The cover slip is an important optical element in the ray path of the
microscope. Life scientists frequently think this would not be true or
even ridiculous, but it is an important issue. On most objectives, you are
going to find an indication for which thickness of cover slip they are
constructed - i.e. 170 micron of BK7 glass - and you are going to
experience spherical aberration if the thickness of the cover slip you use
does not match the thickness for which the objective optics has been
designed.
The degree of image distortion by spherical aberration is dependent on the
numerical aperture of the objective. While many objectives of small
magnfication factors and low numerical aperture will have a dash, "-",
instead of the "0,17" indicating the 0,17mm of the standard coverslip
thickness because the quality of the image created by these lenses,
indeed, does not suffer considerably if "wrong " cover slips are being
used, modern high quality lenses at higher numerical apertures -
specifically "dry immersion lenses" or "air immersion lenses" - will even
feature a user adjustable compensator in order to correct for deviations
in cover slip thicknesses. In 1949 / 1950, Grey published a series of
papers on the design of a catadioptric lens, in which he in one figure
quantified the amount of image distortion induced by deviations of
thicknesses in cover slips as a function of the NA of dry lenses. Result:
At an NA of 0.9 on an air immersion lens, the image will be totally
blurred if the thickness of the cover slip deviates by as little as 10
microns (sic!) from what it should be. See:
1)
Grey DS, Lee PH (1949) A new series of microscope objectives:
I. Catadioptric Newtonian systems, J Opt Soc Am
39:719-723
2)
Grey DS (1949) A new series of microscope objectives: II.
Preliminary investigation of catadioptric Schwarzschild systems.
J Opt Soc Am 39:723 728
3)
Grey DS (1950) A new series of microscope objectives: IlL
Ultraviolet objectives of intermediate numerical aperture.
J Opt Soc Am 40:283-290

and specifically Figure 3 on p. 285 of the third publication in this series.


To produce cover slips in large numbers of 170microns in thickness so that
the thickness deviates considerably less than 10 microns from the nominal
value is a techological challenge this day today, let alone during the 40s
and 50s.

However, if one introduces a transparent "flexible" medium, i.e. a liquid,
between front lens and upper surface of the cover slip, and makes sure
that the refractive index of that liquid is close to that of the cover
slip, then one has created a system in which the coverslip thickness also
is "flexible". Given the fact that in one cover slip the thickness will
not stagger considerably across the distance of the field of view of an
objective - it will rather be too thick or too thin by a value more or
less invariant over the entire field of view - the combination of cover
slip and immersion medium - both featuring nearly the same refractive
index - will establish a solution to the problem. What the lens designers
will do is to design their lenses for a cover slip thickness of, e.g.,
260micron, but they will write on the lens "170 micron" and "Oil
immersion". Un-knowingly the "cheated" user will automatically adjust for
the correct thickness of the liquid layer simply by focussing until the
image has its best quality.

In the historical context, cedar wood oil had been the immersion medium of
choice since it has across the entire visible wavelength range a
refractive index which deviates - as far as I know, please correct me if I
am wrong - not any more than in the third decimal from the BK7 glass
refractive index.

When UV microsocpy became important, in biology with the advent of DNA
research, Zeiss developed the Ultra Fluar lenses designed for cover slips
made from quartz or fused silica. Fortunately, glycerine showed to have a
suitable refractive index for quartz in the UV and visible wavelength
ranges.

The lenses David Grey - see above - had designed, were dry immersion
lenses at NA 0.8. They were built by Bausch & Lomb as condenser -
objective twins and in order to use them properly, one had to use the
proper slides made from quartz and cover slips made from quartz. This was
expensive stuff, since all the glasses were hand made!

Also, in the historical context, when these immersion lenses were made, a
microscopic preparation was "something thin". One would not want to
penetrate millimeters deep into tissue at that time. As a consequence, you
will find, e.g., old Zeiss 63X/1.4 Oil immersion lenses, which do not have
the "0.17" engraved onto their barrel but rather a dash, "-", because of
the fact that the true cover slip thickness anyway would be un-important
for the user. For calculation purposes, the lens designer considered the
specimen as something "infintely thin" (physicists - I am one myself -
like to use this termini, "infinitely thin", instead of "thickness 0").

If you intend to focus deep into biological tissue, then you are, indeed,
in deep trouble if you are a perfectionist. The refractive index measured
as a function along the several mm of focus depth into your preparation or
measured across the field of view is something really "chaotic", and no
lens designer can ever compensate for that.

The hi-tech and hi-cost solution for this problem is adaptive optics.

Best wishes,

Johannes

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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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>
> Dear list,
>
> I learned and I thought that the different types of immersion media are
> there to get the best refractive index match to the media or cells behind
> the cover glass, in order to minimize spherical aberration.
> Now some microscope companies say that this discussion is tedious since
> the error of the lenses/objectives are higher than the effect of the
> refractive index match, is that true?
>
> Totally understandable: when I use a mounting medium, which would be close
> to the refractive index of the used oil (refractive index : 1.52) and the
> cover glass (refractive index: 1.52), this would be a perfect match.
>
> But what happens, when I am using a mounting medium based on glycerol
> (refractive index: 1.47), still the cover glass has 1.52. Should I use a
> glycerol objective and glycerol as an immersion medium, or an oil
> objective with oil as immersion medium?
>
> I thought when I do live microscopy in an aqueous solution (refractive
> index of water: 1.33) the best choice would be a water-immersion
> objective.
>
> Now one company also offers silicone-oil-immersion objectives, the
> silicone oil has a refractive index of 1.4, which would be closer to the
> refractive index of the sample which they claim to be at 1.38.
>
> I have the feeling that I opened the box of Pandora and all that I wanted
> was the best solution to a setup, where one of my users wants to look at
> C.elegans in aqueous solution, which objective type should be used?
>
> Thanks, best regards
> Andreas
>

--
P. Johannes Helm

Voice: ([phone] (office)
Fax: [phone] (office)

[Stanislav Vitha]

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Hallo Andreas,

Since this is a confocal microscopy list, I assume your are trying to focus more
than ~ 10 micrometers beyond the coverslip.
You are correct that the best would be to have the refractive indices of the
speciment and the immersion medium matched.
So, glycerol immersion objective for samples mounted in glycerol, oil immersion
objective for samples mounted in oil (n=1.515),  2,2-thiodiethanol  and similar,
and water immersion for samples that are essentially water.
Most glycerol and water immersion objectives that are designed for coverglass
use will have a correction collar for coverglass thickness adjustment. Or they
may have a correction collar for temperature settings (refractive index of
media varies with temperature), which does the same thing, i.e., introduces
certain amount of spherical aberration opposite of what the coverglass (or
temp change) would cause.

So if you have a specimen and the immersion medium perfectly matched and
set the correction collar to minimize spherical aberration (maximum signal), you
get the best resolution and signal intensity throughout the thickness of the
sample (I am ignoring other factors, like light absorption, scattering, local
inhomogenities in refractive index of the sample, ..).

Quite often, the sample is not perfectly matched to the immersion medium. For
instance  imaging skin with a water immersion objective. The skin has higher
refractive index than water, so the deeper you image, the worse the effect of
RI mismatch. You can use the coverglass thickness correction collar and
maximize the signal (minimize spherical aberration) for a given depth in the
sample. When you change focus, however, the correction is no longer optimal.

Booth MJ, Wilson T (2000) Strategies for the compensation of specimen-
induced spherical aberration in confocal microscopy of skin. J Microsc 200: 68-
74

If you only have oil immersion objectives and need to image aqueous samples,
it is possible to corerct for spherical aberration by using thinner coverslips and
immersion medium of higher RI than standard oil.   This may be practical only if
the objective has a adjustable aperture to lower it NA, since for high NA, the
required R.I. of the immersion oil is too high (to be practically available).
 
See
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
and the comments by Sheppard J Microsc 200: 177-178

>Now one company also offers silicone-oil-immersion objectives, the silicone oil
has a refractive index of 1.4, which would be closer to the refractive index of
the sample which they claim to be at 1.38.

I have tested the two silicon immersion objectives from Olympus. Compared to
the 60x/1.2 water immersion, the 60x/1.3 Silicon immersion lens was not only
brighter (as expected, given the higher NA) but also allowed deeper imaging
into plant tissue (none of my users brought animal tissues for the demo, so I
cannot comment on those samples).  
When imaging plant protoplasts, with the water immersion objective the signal
started to drop off half-way through the protoplast and it was necessary to
increase detector gain or laser power. The silicon immersion lens was able to
image the through the entire protoplast (about 50 um sphere) without
adjustments.
 
here is a link to our web page with an example image, it has a short through-
focus movie as well.
http://microscopy.tamu.edu/picture-of-the-month.html

No commercial interest, just a happy tester.

>I have the feeling that I opened the box of Pandora and all that I wanted
was the best solution to a setup, where one of my users wants to look at
C.elegans in aqueous solution, which objective type should be used?
>

The effect of spherical aberration caused by RI mismatch is going to be less
dramatic with lower NA objectives.
A low-mag, low-NA water immersion or even dry objective may be OK for
imaging the whole C.elegans. It depends what resolution you need.

Try imaging with what you have and see, then ask you microscope rep for few
objectives to try. At least in my geographical area, all of the major microscope
reps are very good and willing to bring an objective for a test.

Yet another option is one of those multi-immersion objectives. Our 2-photon
microscope has a Zeiss 25x/0.8  water/glycerol/oil immersion lens. It is quite
nice. It has a correction collar for immersion medium with and without
coverglass.

Best regards,
Stan Vitha
Microscopy and Imaging Center
Texas A&M University