Posted by
Andreas Bruckbauer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-40X-N-A-1-4-Plan-APo-as-replacement-for-63X-tp7582324p7582370.html
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Does the collimator move when changing the objective? Otherwise this would cause a big difference in laser power as you are unferfilling the back aperture and more of the beam passes through. Resolution might then also be worse.
Best wishes
Andreas
-----Original Message-----
From: "Müller,Tobias" <
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Sent: 17/07/2014 17:38
To: "
[hidden email]" <
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Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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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.
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From: Confocal Microscopy List [mailto:
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Sent: Wednesday, July 16, 2014 7:03 PM
To:
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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
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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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