Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Oil-vs-water-objectives-tp7579114p7579137.html
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>OK, so let's get into this in (minimal) depth.
>
>The reason an oil, or water, objective has a
>higher resolution than a water one is that the
>wavelength of light in oil, or water, is shorter
>than in air so resolution is automatically
>better. However the homogeneous medium has to
>go all the way to the objective, otherwise the
>acceptance angle is reduced by the refractive
>index change - and since both effects depend
>directly on the refractive index the loss
>exactly cancels out the gain. (See Chapter 1 of
>Optical Imaging Techniques in Cell Biology).
>
>Now if we have an oil immersion objective of
>(say) NA 1.0, it will still have that NA even
>imaging into water because the change in
>refractive index means that the lens can collect
>a wider angle and that will compensate exactly
>for the longer wavelength (as Steffen says).
>However this cosy relationship breaks down once
>we reach the critical angle, since rays leaving
>the sample in a downward direction are obviously
>never going to reach the objective. Likewise,
>in confocal illumination, rays beyond the
>critical angle will experience total internal
>reflection and will never reach the sample.
>Therefore an oil immersion lens cannot have an
>NA of more than 1.33 when imaging into water,
>and every oil immersion lens of NA greater than
>1.33 will have an effective NA of 1.33 when
>imaging into water.
>
>I don't want to write pages on this, but I think
>if you just draw it out with a pencil and paper
>it will be quite clear. There is neither magic
>nor deep physics involved!
>
> Guy
Very clear.
But what I would like to add is that, even before
you reach the critical angle for TOTAL internal
reflection, the interface will still reflect some
fraction of the incident light. This fraction
increases as you get closer to the critical
angle. As a result, it doesn't make much sense to
make an NA 1.33 water lens. Not only would the
correction collar be very finicky, but the rays
between 1.20 and 1.33 would be increasingly
attenuated by "non-total" reflections taking
place at all (3?) of the flat, horizontal
glass-water surfaces.
Cheers,
Jim P.
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:
[hidden email]] On
>Behalf Of Steffen Dietzel
>Sent: Friday, 5 October 2012 10:29 PM
>To:
[hidden email]
>Subject: Re: Oil vs water objectives
>
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>On 04.10.2012 18:20, Cammer, Michael wrote:
>> Also, while people have generated various test samples of
>collagen and latex and acrylic and fat globules
>and glass beads and carrageen gum and dissolved
>Scotch tape glue etc., the only test sample that
>really answers the question is the sample itself.
>
>Again, I agree, in particular if you look at
>tissues with more or less unpredictable optical
>properties. Still, bead preparations allow to
>demonstrate the general effect very clearly and
>thus convincingly. They also allow
>quantification and sometimes hard numbers help
>to convince people. Some person might argue that
>they don't care about resolution since s/he is
>planning to use a 1 µm pixel size anyway. But
>you still can convince these people if you can
>show them that they will also loose a lot of
>intensity.
>I use such a demonstration to argue for the
>usage of 170 µm coverslips and a good embedding
>medium.
>
>On 05.10.2012 02:40, Guy Cox wrote:
>> Nobody seems to have mentioned so far that the NA of an oil
>objective will NOT be 1.4 if it is imaging a sample in water.
>The maximum it can be is 1.33 - the refractive index of water.
>...
>> So the oil objective has little or no advantage in NA
>
>Is that really true? If there is oil between the
>coverslip and the oil objective, but water
>immersion for the water objective, this should
>make a noticeable difference in effective
>acceptance angle, should it not?
>(Assuming the object is rather close to the coverslip).
>
>Steffen
>
>
>--
>------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle
>Medizin (WBex) Head of light microscopy
>
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