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Hi,
Leica has a nice motorized DIC with a wheel underneath the objective turret so that the right prism is inserted when DIC is used and the prism automatically removed when normal transmitted light imaging is used. Very useful for a core facility.
I found that without DIC prism, linear the polarised beam causes PSFs which are elongated in one lateral dimension. This was x for our Leica microscopes and y for the Zeiss LSM 780. Measuring the resolution from line profiles in x and y will give quite different results! Rotating the scan field will change the orientation of the long axis of the elliptical PSF.
best wishes
Andreas
-----Original Message-----
From: Herlinde De Keersmaecker <[log in to unmask]>
To: [log in to unmask]
Sent: Fri, 1 Oct 2021 12:38
Subject: Re: Quality control - PSF fitting
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Hi,
Thank you for all the interesting and usefull feedback and links.
I went back to the microscope and it was indeed the DIC prism (I should have thougth about this). From one of the references I understand now that the reason Nikon did not put a quarter waveplate is probably because we have a multiphoton laser.
The program PSFj seems to do the trick, thanks for the suggestion!
I will also look into becoming member from WG 5, I'm already in WG4 and it would be an interesting combination.
Best regards,
Herlinde
Ghent University
Herlinde De Keersmaecker
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Van: Confocal Microscopy List <[log in to unmask]> namens Michael Shribak <[log in to unmask]>
Verzonden: vrijdag 1 oktober 2021 05:44
Aan: [log in to unmask] <[log in to unmask]>
Onderwerp: Re: Quality control - PSF fitting
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Hi Herlinde,
I think that the DIC (Nomarski or Wollaston) prism splits PSFs by shear amount, which is equal to shear angle multiplied by tube length and divided by the objective lens magnification. I measured the shear angles for high contrast, general, and high-resolution Nomarski prisms for Olympus microscopes. For example the shear is 70nm if one use the high-resolution slider Olympus U-DICTHR and 100x objective lens.
More measurement results and computed shears can be found in my chapter “Differential interference contrast microscopy”. In: Biomedical Optical Phase Microscopy and Nanoscopy (eds. N. T. Shaked, Z. Zalevsky and L. L. Satterwhite), Elsevier, (2012).
https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fpublication%2F277814347_Differential_interference_contrast_microscopy&data=04%7C01%7CHerlinde.DeKeersmaecker%40ugent.be%7Cef0b135aab6d40a09ffb08d9848f8936%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C637686574295887151%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=aSH9HSvkiPK7CpkNFFRIFLReaIr0jFbCCfngy8xxa5I%3D&reserved=0
Of course, if the excitation light is linearly polarized and its polarization plane is parallel to the eigenpolarization of DIC prism then the beam will not split and we will have one PSF. However if the polarization is not linear or it is linear but oriented differently then the DIC prism will separate the beam and split PSF accordingly. Usually the fluorescent light is not polarized and we cannot avoid splitting of the emission beam by DIC prism. Therefore the prism should be taken it out.
In order to answer to question about a quarter waveplte it is necessary to know its type (model), location and orientation.
Best wishes,
Michael
---
Michael Shribak, Ph.D.
Marine Biological Laboratory
7 MBL St
Woods Hole, MA 02543
USA
On Sep 30, 2021, at 11:30:21 AM, Craig Brideau <[log in to unmask]<mailto:[log in to unmask]>> wrote:
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Hi Herlinde, from a paper a while back and Dr. Micu and I discovered that
most confocal systems are linearly polarized. I suspect this allows them to
work with various DIC optics, and the Nikon microscopes in particular offer
a laser scanning DIC modality that relies on the linear polarization of the
laser. Here's a link (open access!), skip down to Table 1:
https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.spiedigitallibrary.org%2Fjournals%2Fneurophotonics%2Fvolume-4%2Fissue-2%2F025002%2FEffects-of-laser-polarization-on-responses-of-the-fluorescent-Ca2%2F10.1117%2F1.NPh.4.2.025002.full%3FSSO%3D1&data=04%7C01%7CHerlinde.DeKeersmaecker%40ugent.be%7Cef0b135aab6d40a09ffb08d9848f8936%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C637686574295887151%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=NC%2FTokC3QpHl2U7zCWZ67K2KcrDoUVZMVJZ21rd8Ies%3D&reserved=0
Without taking a deep dive into the physics, I can tell you that linear
polarization causes unusual interference effects that can manifest in an
oblong PSF when the light interacts with an edge or curved surface. The way
to avoid this is to circularly polarize the light such that there is no
directional preference. If you dig a bit deeper into our paper though you
will see that it may cost you some fluorescence intensity depending on your
fluorophore.
Shout out to Clare: I'm on WG1; really enjoying my interactions with the
QUAREP-Limi group.
Hope this helps!
Craig
On Thu, Sep 30, 2021 at 7:09 AM Mitchell, Claire <
[log in to unmask]> wrote:
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Hi Herlinde
- I would concur with Sylvie on the DIC prism, we have a couple of
(spinning disk and widefield) systems that show a significant 45 degree
elliptical PSF in the lateral plane when a DIC prism is inserted.
- We also have a Nikon SoRa system which showed a very small ellipticity
also at 45 degrees using the 100x. The engineer suggested I tweak the
correction collar to minimise, maybe something to try if you have one on
your objective?
- As for software which can fit the elliptical shape. If I understand
correctly and you mean elliptical in the lateral plane then PSFj (
https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fcmongis%2Fpsfj%2F&data=04%7C01%7CHerlinde.DeKeersmaecker%40ugent.be%7Cef0b135aab6d40a09ffb08d9848f8936%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C637686574295897144%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=%2FrzCqwmGysxMhqs7cFA450QTUmtaIt%2BkTA3wJiBMTI8%3D&reserved=0) and PyCalibrate (
https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.psfcheck.com%2Fpsfcheck-processing&data=04%7C01%7CHerlinde.DeKeersmaecker%40ugent.be%7Cef0b135aab6d40a09ffb08d9848f8936%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C637686574295897144%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=Q2%2BWiQhIaVGcT1PSMz6iN9HF49dNJWStyZ279b9Tv5M%3D&reserved=0) can fit this shape, all the
other PSF programs I am aware of only fit in the x and y directions and
won't spot the elliptical problem. If you need full 3D fitting then PSFj
has an option to do this, although I have never used it myself.
Best wishes
Claire
P.S. If you are interested in looking into this more you might consider
joining the working group 5 of Quarep-Limi (https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fquarep.org%2F&data=04%7C01%7CHerlinde.DeKeersmaecker%40ugent.be%7Cef0b135aab6d40a09ffb08d9848f8936%7Cd7811cdeecef496c8f91a1786241b99c%7C1%7C0%7C637686574295897144%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=gKst3pWEItoY9912VKZcSERgFHyCX7CYCuoYAvdFFgM%3D&reserved=0), we are
looking at fitting aberrated PSFs currently!
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On
Behalf Of Sylvie Le Guyader
Sent: 30 September 2021 13:43
To: [log in to unmask]
Subject: Re: Quality control - PSF fitting
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Hi Herlinde
We also have Nikon confocals and also acquire PSFs. Quite a long time ago
we realized that the DIC slider (I assume that this is what you mean?)
under the objective was making the PSF longer and a bit larger but it was
not at 45 degrees.
Our policy since we got to know this was to have no DIC slider by default.
We keep them in an objective box near each microscope and instruct the
users who require DIC how to Koehler and insert all the DIC elements.
Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7C,
14157 Huddinge, Sweden
mobile: +46 (0) 73 733 5008
LCI website
Follow our microscopy blog!
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On
Behalf Of Herlinde De Keersmaecker
Sent: 30 September 2021 13:48
To: [log in to unmask]
Subject: Quality control - PSF fitting
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Hi all,
At our facility, we image subresolution beads on our confocal microscopes
and perform a 3D gaussian fit to determine the centre and FWHM in x, y and
z direction. We noticed that on our Nikon A1R HD microscope, the PSF is
elliptical at 45 degrees when using a 60X oil, NA 1.4 lens. They told me
that this is caused due to the fact that no quarter wave plate is inserted
to get the best brightness and contrast. This raises three questions that I
would like to ask to the community:
1. Do others with a Nikon A1R HD also noticed this effect with high NA
objectives?
2. Can someone explane why the brightness and contrast is better without
the quarter waveplate? I do not have a physics background and this is hard
to follow for me but I would like to understand the reasoning behind.
3. Does someone know a FIJI plugin or other free software package that can
do an (3D) elliptical fit to analyse the PSF for quality control so we can
quantify the difference between the longest axis and shortest instead of
comparing x and y?
Thanks in advance!
Herlinde De Keersmaecker
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