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metadata for the stacks into STAR fi les FOCUS can also be used for 2D crystal sessions, where it can do the entire 2D crystal processing automatically (the full functionality of the 2dx soſt ware package is included in FOCUS). T is allows direct 3D reconstructions during the 2D crystal data collection session. FOCUS maintains a batch queue, with which is runs all processes above in parallel. We have installed this on an Ubuntu PC with 2 ×12 cores (= 48 threads) 256 GB RAM (of which 96GB are used as a RAM disk. More would make life easier.) 70 TB HD (RAID5) 2 × GTX1080 GPU We also use a 40” 4k monitor, which is great to display the images full-screen in full quality. We have one such system per automated cryo-EM microscope, which is able to keep up with single particle data collection of 40-frame 8k movies at a rate of 90 movies per hour (Fourier cropping, driſt correction with MotionCor2 on 5 × 5 patches, CTFFIND4, gautomatch, etc.), and is thereby on par with data collection. FOCUS is described in Biyani et al., J Struct Biol , 2017 ( ). It is also available on bioRxiv at . T e soſt ware is open-source and freely available in precompiled versions for Linux and OS X. A manual in wiki form is available, all at http:// . Nikhil Biyani, Ricardo D. Righetto, Robert McLeod, Daniel Caujolle-Bert, Daniel Castano-Diez, Kenneth N. Goldie, and Henning Stahlberg. Henning Stahlberg Wed May 10

TEM: repair recommendations

We have a Zeiss CEM 902 Transmission Electron Microscope that is currently displaying the following error code: 3-1: Output voltage of high-voltage measuring tube below 0.1V (broken measuring line, defective measuring instrument or contaminated measuring tube does not ignite). Zeiss no longer supports this instrument so we are looking for recommendations for maintenance and repair. Any suggestions? Amy Replogle Fri May 12 When does the error code light up? Only aſt er quite a while when the EM was running? I heard of a problem that in rare cases the vacuum becomes too good (e.g., better than 10 -7 ) and thus the voltage exceeds the minimal value which is necessary. T e people helped themselves via a needle valve continuously admitting a smallest amount of air. Peter Heimann Sat May 13 First, it would be useful to know if the error comes from a Pirani or a Penning tube. Can you be more specifi c? Can you relate the error code with the location / kind of the measuring tube? If Penning, I would fi rst try to clean it. If the Penning is too contaminated, it looks like the vacuum is ultra-good. Procedure would be to set the part of the vacuum system the tube is fi xed at on air, then switch off the microscope completely, since there might be high voltage on the tube. You can send me an image of the measuring tube(s) and I can check if I have a working one to send over. Stefan Diller diller@ Sat May 13

TEM: question regarding K-kit

Has anybody here used K-kit to analyze liquid samples under TEM? Is the K-kit useful for liposome too? What kind of specimen holder is needed? Will the FEI double tilt holder work? RaviT akkar Fri May 19

We have worked with the K-kit system in our facility as well as working with others who are using the K-kit for a number of liquid systems. In general, if a K-kit loaded with organic substance that without staining, the imaging results mostly would suff er low contrast under TEM. T e liposome is just one of the examples. A liposome has an aqueous solution core surrounded by a thin hydrophobic

2017 September • 67

membrane, in the form of a lipid bilayer. Due to the contrast issue, the TEM images for some smaller sizes of liposomes are easily looked blurry, and poor for being clearly identifi ed. Per our actual test results, we suggest that Cryo-TEM can be a better option for those smaller liposomes. However, one also can use K-kits for the observations of some larger liposomes under TEM. Since K-kit is mounted on a standard copper grid for TEM observation, it’s surely compatible with most kinds of TEM Holder. Will FEI Double tilt holder works? Some kinds of FEI double tilt holders require using a small screwed cover ring to lock the copper grid fi xed at the holder’s front stage. It usually needs to use a dedicated tool which with a small hexagonal head to fasten them together. In this case, due to K-kit body on the copper grid is too thick, it will cause the tool to fail when screwing down the cover ring. Gene Rodek Mon May 22

SEM: air-sensitive sample

I have a user with a sample that must be kept under an Argon atmosphere until placed in the SEM exchange chamber, and I have never had to deal with this situation before. I am wondering if the following procedure will work, or if there are any issues with it. Our Hitachi S4700 SEM has a separate exchange chamber prior to the Sample chamber. We propose to tape a fl exible glove bag around the frame of the SEC, containing the samples and mounts. We would fi rst evacuate the bag using the rotary pumps and then seal the exchange chamber. T en, we will fl ush the glove bag several times with argon using the glove bag ports only. Once the argon atmosphere is present, the samples will be removed from their carrier, placed on the SEM mount in the glove bag, then into the exchange chamber, and evacuated. Once vacuum is reached, the sample will be put into the sample chamber. I hope that all the argon will be gone by then. Aſt er viewing, there is no current plan to try to keep

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