NetNotes


use a strong solvent such as lighter fluid to dissolve the thicker oxidized film followed by a water-based cleaner. Te petroleum cut through the oil and oxidized film (stuck to the glass) and the water-based cleaner dissolved any remaining salts and sugars that may have accidentally deposited from media or handling. It is important to note that when using a petroleum solvent, you don’t saturate the wiping tissue, only make it “damp” and limit the amount of time exposure to the lens. Many times, the user would be surprised at how improved the image was. For wiping paper, I strongly suggest Berkshire, Lensx®


R90 (https://berkshire.com/


shop/cleanroom-wipes/nonwoven/lensx-90/ln90040624p/). It is the soſtest, most absorbent optical wipe I ever used. I have no interest in the company, but I haven’t found anything better for lenses. Dan Fochts dan@bioptics.com


Hi Dan, just to clarify: are you talking about Type F immer-


sion oil from Zeiss, the stuff that is still currently in use? Type F immersion oil can oxidize? We use that exclusively and never no- ticed anything weird when leaving a thin film on the objectives. So far, I am more worried about users scratching the front lens during “cleaning” rather than small amounts of oil remaining. Now you gave me something to worry about. Steffen Dietzel lists@sdietzel.de


Not to be too pedantic but, for cleaning an air lens, the video


(https://youtu.be/Tz4Dy5D6kdw?t=203) shows a straight swipe across to top surface straight across the glass. Te same surface of the cotton swab is used repeatedly. Tis means that any crud on the top surface is dragged across the glass and any dirt collected on the first swipe is rubbed over the lens again on the second swipe and so on. For the air lenses, we clean all the oil or other crud from the top surface around the recessed glass lens. Rotating the swab as wiping around guarantees a clean fresh surface at each location. Ten we clean the recessed glass with a cotton swab. We rotate the swab in the recess as we wipe to guarantee a clean fresh sur- face of cotton. Aſter one rotation wipe, the swab is thrown away. Michael Cammer michael.cammer@med.nyu.edu


I’m a bit alarmed to hear what some of you are dealing with


in terms of oil films and solvents! I’ve just used isopropanol and a premium optical-grade tissue wipes on all our lenses for the last 16 years and have encountered none of these problems. I’ve been exclusively using Cargille oil, which I realize may not meet the higher optical standards many of you are aiming for, so it could be a difference in oil formulation. Just wanted to express my surprise at how vexing this problem is for some. Craig Brideau craig.brideau@gmail.com


Contrast Transfer Function (CTF) in Tilted Images 3DEM Listserver What is the best way to evaluate the CTF of an image that has a sig-


nificant amount of tilt? David Gene Morgan dagmorga@indiana.edu I think any patch-based CTF program, like the one in Cryo-


sparc or gCTF’s local mode (with evenly spaced coordinates instead of real particle locations), would work well. With Cryo- SPARC there’s an easy method to plot the tilt axis; you can also choose a specific number of X and Y divisions for the patches. Daniel Asarnow asarnow@msg.ucsf.edu


Te problem is that the tilt causes the higher resolution Ton rings to become out-of-phase, so the standard programs can’t


64


give a decent estimate of the goodness-of-fit. I vaguely remem- ber that someone had a program that took the tilt into account several years ago, but a quick hunt for it didn’t turn up anything. David Gene Morgan dagmorga@indiana.edu


Te tilt can be considered as described in the CTF correc-


tion section of the paper on the 3D reconstruction of the V1- ATPase: J Struct Biol 135 (2001), 26-37 https://doi.org/10.1006/ jsbi.2001.4395. Maybe this is what you referred to. Essentially the tilt image is divided into equal patches, the CTF determined for each patch, and a plane fit to the CTF values. Te equation of this plane can then be used to determine the CTF values of each boxed-out particle based on its location in the large image and the CTF values corrected individually. Michael Radermacher michael.radermacher@uvm.edu


Both emClarity and cisTEM (recent alpha version) implement


an algorithm that also tiles an image of a tilted specimen but stretches the off-axis power spectra in such a way that the Ton rings add more-or-less coherently. In emClarity, this is based on the tilt-series alignment. In cisTEM, the tilt-axis-angle and tilt-angle are fit as a part of the tilted CTF refinement. Te basic algorithm is described in my thesis (online at Pitt med) and I believe in the emClarity Nature Methods paper. Here’s a movie: https://twitter.com/cryo2go/status/1 282036490378280961?s=20&t=kg1qVYhpGcqQa5z3qyAFAg. You can find some developmental versions of cisTEM and docs here: https://bhimes.github.io/cisTEM_docs/docs/sim/tutorials/tutorials. html. Benjamin Himes himes.benjamin@gmail.com


Tere are two separate problems here. Te easier one is to sim-


ply determine the defocus in various positions of the specimen. If you only want to work with small cut-outs (such as single particles) then you use the normal CTF-correction with the local defocus. Others pointed to soſtware that does that. If you want to work with the entire image (for example for 2D crystals) the problem is more difficult. Essentially you don’t have a CTF in that case (it’s not a transfer function). Look at the paper by Ansgar Philippsen: Ultrami- croscopy 107 (2007) https://doi.org/10.1016/j.ultramic.2006.07.010 if that’s what you’re aſter. All the best, Philip Koeck koeck@kth.se


Just to clarify, the patch methods don’t require particle loca-


tions. Te gCTF method requires particle locations as input but there’s no need for them to be real particles; it too just estimates the CTF on the patches. David, what do you mean by the good- ness-of-fit estimates being incorrect? In these methods a goodness- of-fit measure is used to pick the optimal defocus values in the patch, and these values do give superior reconstruction resolution and appear to accurately define the real tilt axis. Daniel Asarnow asarnow@msg.ucsf.edu


Maybe goCTF is the program you were thinking of? https://


doi.org/10.1016/j.jsb.2018.11.012 and https://www.lsi.umich.edu/ science/centers-technologies/cryo-electron-microscopy/research/ goctf. Guillaume Gaullier guillaume.gaullier@icm.uu.se


If you do subtomogram averaging in Relion-4, estimating


these high-tilt CTFs over the entire micrograph is probably not that important if you optimize defoci in a CTF refinement job aſter averaging. Tere, the entire tilt and sample geometry are taken into account. Sjors Scheres scheres@mrc-lmb.cam.ac.uk


Perhaps CTFTILT? https://grigoriefflab.umassmed.edu/ctf_ estimation_ctffind_ctſtilt Mike Strauss mike.strauss@mcgill.ca


www.microscopy-today.com • 2022 May


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