NetNotes


a laser scanning DIC modality that relies on linear polarization of the laser. 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 some fluorescence intensity depending on the fluorophore. Craig Brideau craig.brideau@gmail.com


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 uses 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 (2012), NT Shaked, Z Zalevsky and LL Satterwhite, eds, Elsevier. Of course, if the excitation light is linearly polarized and its polarization plane is parallel to the eigenpolarization of the DIC prism, then the beam will not split and there will be 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 the PSF accordingly. Usually, the fluorescent light is not polarized, and we cannot avoid splitting of the emission beam by the DIC prism. Terefore, the prism should be taken out. To answer the question about a quarter waveplate, it is necessary to know its type (model), location and orientation. Michael Shribak mshribak@mbl.edu


Tank you for all the interesting and useful feedback and links. I


went back to the microscope, and it was indeed the DIC prism (I should have thought about this). From one of the references, I understand now that the reason Nikon did not put a quarter waveplate in place is probably because we have a multiphoton laser. Te program PSFj seems to do the trick, thanks for the suggestion! Herlinde De Keersmaecker herlinde.dekeersmaecker@ugent.be


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 the DIC prism, the polarized beam causes PSFs which are elongated in one lateral dimension. Tis 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. Andreas Bruckbauer a.bruckbauer@imperial.ac.uk


We’d like to add that asymmetrically shaped PSFs are not


uncommon, even without accidentally having a DIC slider in the optical path. Te possibility to produce a theoretical PSF with exactly the same image parameters as a sub-resolution bead facilitates quality control measurements of an experimental PSF. Our Huygens Professional soſtware (available for a fee - sorry) can produce a high- quality theoretical PSF based on the same parameters as the bead image. FWHM measurements are performed on-the-fly and can also be measured in all XYZ directions. Subsequently, experimental PSFs can be


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derived from bead images using our Huygens PSF Distiller. Te Distiller also reports the FWHM measurements of the experimental PSF. Lastly, images recorded with a system can be corrected by deconvolving them with this distilled PSF. Vincent Schoonderwoert vincent@svi.nl


In 2010 I found that one of our confocals had a PSF off by a


few degrees. However, in more than 10 years we have not detected any problems, including with 3D reconstructions of biological samples, and I say “we” liberally because no other user, out of hun- dreds, has noticed the problem. Certainly, the users who insist on using the 10X NA 0.3 lens would never notice this. Michael Cammer michael.cammer@nyulangone.org


One Versus Two Disks for Spinning Disk Confocal


Microscopes Confocal Listserver We are considering a new spinning disk confocal live imaging HCS


system. Some come with Yokogawa (2 disks) and some with single disks. I understand the advantage the dual disk provides in terms of concentrating the excitation light through the bottom disk onto the sample, but is a laser- based single-disk spinning disk confocal still a good solution? Does anyone have a single-disk confocal they can comment on? Tanks. Irit shoval irit.shoval@biu.ac.il


We have the Crest V3 from Crest Optics (single disk) and we


also have a Yokogawa X1 (double disk) which has pinholes closer to each other than the newer W1 version. We are happy with both, and the advantages/disadvantages are not caused by the number of disks but more the microscope and the camera they are attached to. 1 versus 2 disks: 1. More powerful lasers are needed with the single disk design; 2. The system you choose should have an illumination homogenizer. Otherwise, the corner of each image will be darker, which will be visible when tiling. Sylvie Le Guyader sylvie.le.guyader@ki.se


I’ve worked with both the X1 and W1 instruments, and while


I really liked the X1 design (no dust issues), the W1 delivers much better images. Te increased pinhole spacing makes a difference. Crest was going to offer a single-disk setup with the W1-like pinhole arrangement, but I haven’t heard about it recently. I haven’t tested the Crest V3 extensively, but in some Crest designs there might be an issue with very dim samples. Te autofluorescence from some elements common to the powerful excitation beam and the emission light might contribute to background (=noise). Te best way is to demo the instrument, it’s quite likely that it will perform just fine. Zdenek Svindrych zdedenn@gmail.com


We just posted to bioRxiv a manuscript (https://www.biorxiv.


org/content/10.1101/2021.09.04.458950v1) on a “do-it-yourself spinning disk” that may be of interest to some readers of this list. (Tis is not a commercial post.) It is also somewhat relevant to the original inquiry about single versus dual layer disks. Te short of it is that for those with a background in home-built instruments, one can design, purchase, and assemble a single-layer spinning disk module inexpensively ($1,000-$7,000 in our case) that can be integrated with an existing microscope setup that already includes lasers, chassis, objective lenses, filters, camera, etc. Te disk pattern can be easily customized for a variety of objective lenses (air, oil, water, different magnification and/or NA) and applications (cells, tissue, expansion, sm-mRNA FISH, STORM, DNA PAINT). As stated by others, the


www.microscopy-today.com • 2021 November


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