NetNotes


straight down. Tis keeps a bit liquid nitrogen in the dewar which also stabilizes the temperature. If the cryo and room temperature holder crash the vacuum too oſten then it might be time for service. Especially the room temperature holder should be “safe” for the vacuum. Generally, you get more crashes with the cryoholder (it’s heavier, less balanced, and is normally used cold and with short pumping times) but again if the vacuum crashes more than 50% of the insertions then something is wrong. Tobias Furstenhaupt furstenh@mpi-cbg.de


Our L120C had a similar vacuum crash problem when a


user inserted the Gatan 626 cryo-holder using the traditional procedure and we have changed one step from the previous insertion procedure which most people use. It seems to drop the column vacuum crash rate quite bit with 30 to 60 second airlock pumping time and we did not see any association of crystallized ice with the 626 insertion. I would like to share our experience with you. We rotated or reset the compustage back to 0o


level for at least 10 seconds before the airlock pumping time ended, then rotated the 626 holder for the rest of the ∼90o


while maintaining the 626 holder at the same tilted to the


insertion position. When the o-ring seal is not perfect, rotating the compustage back to 0o


and maintaining the holder position


might generate a minor leak to the airlock. Tis modified pro- cedure keeps the airlock pumped until rotating the holder for the last 90o


. Dan Shi dan.shi@nih.gov I have two 626 holders and a 910 (3-grid) cryoholder. to maintain liquid N2 1-minute pump, return the stage to 0o


None cause the TF20 vacuum to crash. I do not change o-rings oſten (once a year if that), don’t use vacuum grease (it collects dust), and pump for 60 secs in the airlock. I fol- low a common procedure: insert with the stage at -60o in the cryoholder dewar, wait for the tilt holding the dewar


tilt


steady, and then rotate the dewar upright - all while keep- ing the relative motion slow between the o-ring and the air- lock. Some thoughts on what sounds like a real pain: 1. I use ethane:propane to freeze. It stays liquid and I expect it evapo- rates in the airlock before entry into the column. Ethane ice may instead reach the column. Tis is easy to do: fill the cup to 2/3 with ethane, top it off with propane, and do the plunge as usual. Te ethane/propane mix can become jelly-like, so I bubble a bit of propane in to warm it up and it becomes clear. I drilled holes in the styrofoam platform so that liquid nitro- gen surrounds the brass cup, but that’s not really necessary. 2. Check the o-ring for cuts. If you replace the o-ring (make sure it is the correct size), and if the vacuum breaks again, check it again for new cuts. If you see damage to the surface, there may be something sharp inside the cryo-transfer station or inside the airlock that is ripping the o-ring. Try inserting the holder into each (at room temp) in turn and inspect the o-ring aſterwards with a magnifying glass. 3. My turbo pump was replaced a couple of years ago and the spin-up time on the new pump was much shorter. We hadn’t noticed how the pump had degraded over time because it was very gradual. I’m not sure if this affected the quality of the vacuum, maybe just caused a long (5–6 minute) wait for spin-up that is now 2–3 minutes. James Conway james.conway@pitt.edu


We have also had persistent vacuum break problems with our 626 holder in our Tecnai F20 over the past two years.


2020 May • www.microscopy-today.com I used to do the following prior to every session: Remove


o-ring from holder. Clean o-ring off with kimwipes and fin- gers (I didn’t bother to use any solvent). Clean o-ring groove with kimwipes. Blow away fibers with compressed air. Lightly grease o-ring. Inspect o-ring and o-ring groove under dissect- ing scope and carefully remove any fibers prior to re-installa- tion onto the holder. Don’t scratch the o-ring groove. In fact, don’t touch it with forceps. Use compressed air. Inspect for any nicks or flat spots in the o-ring. If so, then consider a replace- ment. If there is a scratch in the o-ring groove, then you might need a new holder (or maybe not). Also make sure you have the right-sized o-ring. At some they changed the size slightly, such that CM200 o-ring was a bit fatter than the one for the CM12 stage. David Stokes stokes@nyu.edu


Chrétien denis.chretien@univ-rennes1.fr Te modified procedure from the traditional one for our


to -60o


and it will inevitably lead to a vacuum leak (very reproducible!). To limit this issue, we slightly liſt the cryo-holder station with a rectangular grid box to avoid LN2


One frequent issue with cryo-holders: do not fill the cryo- holder station with too much LN2


contacting the o-ring. Denis


L120C is: 1. Start the turbo pump and wait until it reaches 100% speed. Set “airlock pump time” to 30–60 seconds. 2. Set the alpha at -55o


at least 10 seconds before the airlock pump time expires. 5. , you may freeze the O-ring


About 50% of the time inserting the holder breaks the vacuum. I thought it was unique to this holder, but with the Elsa holder that we recently acquired it is even worse: vacuum breaks about 90% of the time. We’ve changed o-rings and had the airlock disassembled and cleaned, but the problem persists. Tat being said, it never affected ice quality, and the temperature usually doesn’t go above -165°C. When the vacuum recovers it’s usu- ally at 25–30 log or so and back to <10 within a half hour. It’s really annoying though. In my experience, if the temperature doesn’t rise above -150°C you should be ok. It helps to make sure the tip is very cold by submerging it completely in LN2 before inserting. Terje Dokland dokland@uab.edu


We also have suffered for years from vacuum breaks. We


tried EVERYTHING. Te most effective thing was to (believe it or not) change the o-ring every single session. Recently we discovered this was due to the fibers and other “dirt” on the o-rings aſter inserting and removing the holder from the microscopes,


transfer stations or pumping station. We


embarked on a “cleaning program” where we gently swab all the above-mentioned devices. It’s now slowly getting better. We are still changing the o-rings, but we hope it will soon no lon- ger be necessary, once all the “stuff” that accumulated over the years is cleaned out from those places. Sharon G. Wolf sharon. wolf@weizmann.ac.il


and click on “set alpha” to tilt the com-


pustage. 3. Insert the 626 holder to the engaging position and select the cryo-ST holder from the screen. 4. Hold the cryo- holder and click on “set alpha” to rotate the compustage back to 0o


Wait until the red light on the compustage is off, rotate the cryo- holder to the insertion position and smoothly insert the holder. 6. Feel free to breathe. Dan Shi dan.shi@nih.gov


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