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SEMC TF20 User Manual

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TF20 User Manual
Simons Electron Microscopy Center
(SEMC) at the New York Structural
Biology Center (NYSBC)

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Summary of Contents for SEMC TF20

  • Page 1 TF20 User Manual Simons Electron Microscopy Center (SEMC) at the New York Structural Biology Center (NYSBC)

  • Page 2: Table Of Contents

    Last updated 10/29/2020 Table of Contents F20 Information Sheet F20 User Guide (starting your session) End of Day Checklist Cryo Screening with Tietz TVIPS F416 Cryo Data Collection with the DE20 Dry Pumping Station User Guide Gatan Elsa cryo holder manual Gatan CT-3500 (Oxford) holder manual Cryo Holder Insertion/Retraction Guide for F20 F20 Alignments...

  • Page 3: F20 Information Sheet

    Last updated 10/29/2020 F20 Information Sheet ● Wavelengths ○ 80 kV = 0.04176 Å ○ 120 kV = 0.03349 Å ○ 200 kV = 0.02508 Å ○ 300 kV = 0.01969 Å ● Lens & magnification range ○ LM = 21X → 2100X ○...

  • Page 4: F20 User Guide (Starting Your Session)

    Last updated 10/29/2020 F20 User Guide (starting your session) 1. Make sure microscope is in good condition: a. HT and FEG are on Setup → “High Tension” and “Operate” are yellow b. Column valves are closed Setup → “Col. Valves Closed” is yellow c.

  • Page 5: End Of Day Checklist

    Dry Pumping Station User Guide (for cryo holders) for detailed instructions 6. Log off Leginon workstation 7. Close Leginon client on both camera and microscope 8. Cleanup the bench space for the next user 9. Log any issues into the EM notebook or notify SEMC staff directly...

  • Page 6: Cryo Screening With Tietz Tvips F416

    Select project → “next” e. Image directory (use default… “/gpfs/leginon/username”) f. Connect to clients= tf20-f416 & tf20-d394 → “next” g. C2 size = refer to the white board → “finish” 5. Once Leginon has started up, open the “application”...
  • Page 7 Last updated 10/29/2020 d. Close window 7. “Cycle” presets to minimize hysteresis in the beam a. Under “preset manager” node, select “settings” button and check the “cycle presets” option (see image) b. Send scope to EN preset 10X 8. Open shield of cryo holder. You may see a slight increase in IGP1, but should settle within a few seconds 9.
  • Page 8 Last updated 10/29/2020 c. Click “play” to collect atlas 13. Insert and center objective aperture a. Go to square of non-interest b. Send scope to EN mag c. Lower flu screen and send scope to “DIFFRACTION” mode (right knobset) Should see bright green small beam on the flu screen with diffuse carbon diffraction ring around it D = 460 - 680 mm d.
  • Page 9 Last updated 10/29/2020...

  • Page 10: Cryo Data Collection With The De20

    Leginon client is open; temperature will not be affected) d. “do not autosave” = true 3. Open Leginon client on camera computer (DE20 DVI1), microscope computer (TF20-D394), and main computer (TF20LEGINON) a. In terminal, type betaleginon 4.
  • Page 11 Last updated 10/29/2020 Scope = tf20-d394→ “run” iii. 6. Upload your presets a. Preset manager → “import presets from another session” (* icon) TEM = tecnai; Digital Camera = DE-20 b. “Find” presets from past 20 days; select desired preset (usually from the most recent user) c.
  • Page 12 Last updated 10/29/2020 b. Click “calculate atlas” c. Click “play” to collect atlas 13. Gain references. For gains, it does not matter if objective aperture is in or out. Must be done over vacuum. EN preset gains Prepare beam for target dose = 30 - 60 electrons/Å (SS 5, beam diameter ~ 2X bigger than second circle on flu screen) Dark reference...
  • Page 13 Last updated 10/29/2020 3. Acquire (you should see black image) iii. Bright reference 1. Open column valves and flu screen up 2. In “correction” node → “bright” and “both channels” 3. Acquire (you should see image of the camera sensor) Confirm that correction is done 1.
  • Page 14 Last updated 10/29/2020 Should see bright green small beam on the flu screen with diffuse carbon diffraction ring around it c. Insert objective aperture and center d. Exit “DIFFRACTION” mode e. If you plan to take a break, send microscope to high mag (FA/FC/EN) to turn on the high mag lenses and close the column valves 17.
  • Page 15 24. Start CTF correction (after first movie is done aligning) a. CTF → estimate the CTF → CTFFIND v4 b. Default all settings except preset = en-a c. Run on semc-head (or copy/paste into emgbox05) 25. If you are done for the day…refer to End of Day Checklist...

  • Page 16: Dry Pumping Station User Guide

    Last updated 10/29/2020 Dry Pumping Station User Guide Do not overtighten the valves! Finger- tight only! Normal state at the beginning of the day: V1 = closed V2, V3, & V4 = open Removing holder from dry pumping station: 1. Vacuum should be reading 10 to 10 2.
  • Page 17 Last updated 10/29/2020 Make sure that the 626 holder has a good vacuum. Finally, send message in slack in #EMG channel: “Elsa is warming up now. V1 and V2 open, V3 and V4 of both Elsa and 626 are closed. 626 has a good vacuum and should be ready to go first thing in the morning.”...

  • Page 18: Gatan Elsa Cryo Holder Manual

    Last updated 10/29/2020 Gatan Elsa cryo holder manual The Elsa holder is a specialized holder with a large cryogenic dewar that can maintain temperature for ~8 hours. Elsa holder handling should be done by EMG staff only; this includes loading the sample, insertions, and retractions.
  • Page 19 Last updated 10/29/2020 d. Transfer cryo grid to the tip of the holder. e. Secure the grid with the clip ring. Place clip ring side of the tool onto the grid until you feel a click. While applying a small amount of vertical pressure on the end of the clip ring tool, slowly turn the tool COUNTER- clockwise 1 - 2 turns until the tool moves freely.

  • Page 20: Gatan Ct-3500 (Oxford) Holder Manual

    Last updated 10/29/2020 Gatan CT-3500 (Oxford) holder manual ● This holder takes ~30 min to cool down and should be done while plugged into the temperature controller to monitor cooling. ● It is nearly impossible to recover grids from the CT-3500 holder. Thus, if the grid is good you should plan to collect rather than save the grid.
  • Page 21 Last updated 10/29/2020 b. Care should be taken as LN is added to the filling cup as excess LN will pour out of the back of the transfer station. 3. Transfer cryo grid to the holder. on the 9’o clock a.
  • Page 22 Last updated 10/29/2020 6. Warm up the holder (20°C) a. Holder should be warmed up with tip pumped down (open V1 and V2). b. Plug in holder to the temp controller. You should see a value in the DISPLAY window (- 150°C or so).

  • Page 23: Cryo Holder Insertion/Retraction Guide For F20

    Last updated 10/29/2020 Cryo Holder Insertion/Retraction Guide for F20 ***Cryo holder insertions on the F20 may only be performed by SEMC staff. NO EXCEPTIONS. *** Cryo Holder insertion: 1. Before insertion confirm that IGP1 = 6, column valves closed, holder is reset, objective aperture out, flu screen down, airlock default pump time = 120 sec, turbo on and ready (button will be orange while warming up and yellow when ready).
  • Page 24 Last updated 10/29/2020 7. Wait for 2 minute airlock pump time to count down. 8. After airlock pump time complete, “reset” the alpha value WITH HANDS ON THE DEWAR OF THE HOLDER so that the holder remains stationary a. The stage should rotate back to 0 degrees while the holder top is still at 3 o’clock position.
  • Page 25 Last updated 10/29/2020 Cryo Holder retraction: 1. Close column valves, reset holder, objective out, flu screen down, cryo shield of holder closed. 2. Gently brace against the purple plate of the goniometer and pull holder back until it stops, then turn holder clockwise so that the “hat”...

  • Page 26: F20 Alignments

    Last updated 10/29/2020 F20 Alignments ● F20 alignments should be done by EMG staff ONLY!! ● Alignments can be done with room temperature (RT) holder or cryo holder. Alignments should be done with a grid with carbon support, ideally a cross-grating replica grid. ●...
  • Page 27 Last updated 10/29/2020 Focus beam using “Focus” knob on right knob-set and camera/FFT. Reset defocus to 0 (press R3 on right knobset) 6. Beam tilt pivot point X and Y (“Tune” → “Direct Alignments” → “Beam tilt pivot point”) a. At eucentric height and eucentric focus. b.
  • Page 28 Last updated 10/29/2020 a. EN preset gains Prepare beam for target dose = 30 - 60 electrons/Å (SS 5, beam diameter ~ 2X bigger than second circle on flu screen) – Check en presets, and in the Correction node settings, choose the camera of interest, and set the appropriate camera configuration, binning, and exposure time.
  • Page 29 Last updated 10/29/2020 Bright reference 1. Open column valves and flu screen up 2. In “correction” node → “bright” and “both channels” 3. Acquire (you should see image of the camera sensor) Confirm that correction is done 1. Under “correction” and “both channels” 2.

  • Page 30: Troubleshooting

    Last updated 10/29/2020 Troubleshooting HT should be brought up by EMG staff ONLY!! 1. If the column vacuum crashes and HT and FEG turn off... a. Notify EM staff b. Wait for TMP rollout to recover (~7 minutes) c. Wait for column pressure to recover (IGP < 30) (30 - 60 minutes) Turn HT on when IGP2 <...

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