Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?

Posted by James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-40X-N-A-1-4-Plan-APo-as-replacement-for-63X-tp7582324p7582377.html

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Hi all,

Good discussion and many important points have been raised.

I would like to add a couple more.  The larger
field of the 40x might make it easier to find the
cell you are interested in but remember all
objective lenses are optical compromises. The
manufacturers try to correct for chromatic and
spherical aberration, flatness of field, coma,
astigmatism, distortion etc, by judicious choices
of the shape, material and position of the 10-20
elements in each objective. But they are never
quite successful. True, they can transmit an
image of a point object that looks a lot like an
Airy figure, but it's central peak brightness is
seldom close to 60 times greater than the
brightness of the first bright ring, as the
mathematics suggests it should be.

OK, you know all this. But is it might be
relevant here because it is just a lot harder to
correct for all these things over the larger
field of view of the 40x. It may be easier to
understand this if you have a chance to take a
good look at figures 11-9 and 11-10 in Chapter 11
of the Handbook (by Rimas Juskaitis) . Here you
will be able to get some idea of the loss of
resolution that occurs as one moves off 5 and
10mm off-axis in the intermediate image plane,
with any objective. And you will see how much
more severe this effect is on low-mag, high-NA
objectives. Part of the loss with the 10x 0.5NA
shown here is due to the fact that it is not a
plan- objective (so there is also a focus shift)
but the image shoudl still be symmetrical (and it
isn't). From 11-9 you can see that another reason
is vignetting. The glass is just not big enough
to transmit all the high-NA rays once one gets
off axis

So my message is that with the 63x and even more
so with the 40x NA 1.4s, use the wide field of
view for surveys, but plan to do any high
resolution work in the center of this field of
view.

And yes, at a given NA, the diameter of the back
focal plane is inversely proportional to the
magnification. So, as long as the laser beam has
been spread out enough to fill the BFP
(uniformly!) you should get about 6.25x more
light from the 40x as the 100x. However, you
probably won't quite do this, not only because it
is hard to make a large uniform laser beam but
because the transmission losses of the the
objectives may differ and, more importantly, more
light will probably be lost to vignetting in the
40x.

Happy summer,

Jim Pawley

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>Hi,
>
>The 40x/1.4 also has some drawbacks. It has a
>working distance of only 130 microns (40x/1.3
>Apo: 210 microns, 63x/1.4 Apo: 190 microns, even
>a 100x/1.4 Apo has 170 microns). Not a problem
>when you're using cell monolayers (provided
>they're on the coverslip), but in 2P or with
>cleared samples, it might become a factor.
>
>More importantly, our 40x/1.4 Apos are a bit
>worse than the 63x/1.4 Apos when it comes to
>chromatic aberration. I generally do not suggest
>it for colocalization studies. Of course you can
>correct with a calibration slide, but this is
>always more accurate if the error is as small as
>possible to begin with. In my opinion, chromatic
>aberration is the only reason to have a 100x
>objective on a confocal, because it tends to
>have the smallest lateral chromatic shift.
>
>Anyway, I've tested the 40x/1.4 objective for
>transmission of laser power (with a powermeter),
>and found the power (very roughly in the focal
>plane) to be 2.1 times higher with the 40x/1.4
>Apo as compared to a 63x/1.4 Apo. I think this
>is mostly because of the much larger rear
>aperture. Because of the powermeter I used there
>is an uncertainty as to the validity of these
>results. I will repeat this, as soon as I have
>the microscope slide power meter head from
>Thorlabs.
>
>After adjusting the AOTF to achieve the same
>excitation power for both objectives, the 40x
>was not brighter anymore. In fact, it was a bit
>darker, although that might have been true for
>that individual objective only. So my impression
>is that the 40x will only provide stronger
>excitation, at the same AOTF setting, than a
>63x. This is nice, but hardly remarkable. In my
>opinion, the rise of NA from 1.3 (previous 40x)
>to 1.4 is counterbalanced by the reduced working
>distance and the non-optimal chromatic
>aberrations. It's a nice enough objective, but
>far from being a gamechanger.
>
>Disclaimer: Of course, it was only one objective
>for the transmission testing, and four for the
>tests of chromatic aberration, in which one was
>okay, two were so-so (but worse than 63x/1.4 in
>general), and one sucked the big one.
>Note that all brightness tests were done with
>PMT point detection. For cameras, magnification
>plays an important role with respect to
>resolution as well as brightness, and it's a
>whole different matter.
>
>Best,
>Tobias
>
>
>BTW: Why "new"? We're talking about product
>number 420762-9900-000
>(https://www.micro-shop.zeiss.com/?s=118967537d39b70&l=en&p=us&f=o&a=v&m=s&id=420762-9900-000),
>right? Not all that new, if you ask me.
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Steffen Dietzel
>Sent: Wednesday, July 16, 2014 7:03 PM
>To: [hidden email]
>Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
>
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>Am 14.07.2014 16:28, schrieb Feinstein, Timothy:
>>  You are correct that even in the best case a traditional optical
>>  microscope can only separate point sources > 100 nm apart.
>
>Except of course the two point sources are in different color channels.
>Then you can get the two much closer together
>and still measure the distance between their
>intensity centers very accurately. In the
>two-digit nm range, depending only on your
>positioning accuracy.
>Provided you correct for chromatic aberration.
>
>Cheers
>Steffen
>
>
>--
>------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle
>Medizin (WBex) Head of light microscopy
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
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>Marchioninistr. 27,  München-Großhadern

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