TF20 User Manual
Simons Electron Microscopy Center
(SEMC) at the New York Structural
Biology Center (NYSBC)
Page 1: ...TF20 User Manual Simons Electron Microscopy Center SEMC at the New York Structural Biology Center NYSBC ...
Page 2: ...ession 3 End of Day Checklist 4 Cryo Screening with Tietz TVIPS F416 5 Cryo Data Collection with the DE20 9 Dry Pumping Station User Guide 15 Gatan Elsa cryo holder manual 17 Gatan CT 3500 Oxford holder manual 19 Cryo Holder Insertion Retraction Guide for F20 22 F20 Alignments 25 Troubleshooting 29 ...
Page 3: ... 120 kV 0 03349 Å 200 kV 0 02508 Å 300 kV 0 01969 Å Lens magnification range LM 21X 2100X M 1700X 3500X SA 65 000X 280 000X Objective lens Cs 2 1 mm Aperture 1 2 3 4 Condenser 50 um 70 um 100 um 150 um Objective 40 um 70 um 70 um 100 um Selected area 40 um 70 um 100 um 200 um ...
Page 4: ...yo Holder Insertion Retraction Guide for F20 for detailed instructions b CHECKLIST before insertion 1 turbo is on and ready yellow 2 objective aperture is out 3 flu screen of microscope is down 4 shield of cryo holder is closed 5 airlock pump time 120s 6 reset holder 7 alpha 60 5 Insert cryo holder into EM 6 After insertion wait for IGP1 to reach 10 before opening the column valves 7 Select the ca...
Page 5: ...ctive aperture is out e high mag lenses on send scope to EN FA FC preset anything in the SA mag range f click Cryo Cycle on microscope PC 240 minutes 4 Remove cold trap from the stand when IGP1 99 a You can keep the remaining LN2 inside the cold trap off the coils for tomorrow s user 5 Bake out the cryo holder a See Dry Pumping Station User Guide for cryo holders for detailed instructions 6 Log of...
Page 6: ...on_client depending on your access type on camera microscope computer a TVIPS camera control on monitor input DVI 2 3 Open Leginon program on leginon PC a In terminal type betaleginon if using EMG Leginon_client or nccatleginon if using NCCAT Leginon_client 4 Setup Leginon session settings a Select create new session next b Select holder Elsa or TF20 cryo holder_AMI 2 c In session description type...
Page 7: ...nd spot size until you achieve target dose of 40 70 e A2 with 1 sec exposure iv For the FA FC preset you can make this beam a bit brighter and smaller v Open the Edit box and apply intensity from scope and beam shift from scope vi Repeat for FA and FC preset 11 Get eucentric height and focus a On a square of non interest b Z_focus node simulate target icon c Recommended record the eucentric height...
Page 8: ...est a Using acquisition mouse select holes of interests on the carbon every 2x2 holes away from each other b Using focus mouse select area of carbon towards the center of the hole areas you selected in the step above this is where leginon will acquire the eucentric height of the square c Click Submit target d Click Submit queued targets when you are done with every hole target 19 In Exposure_Targe...
Page 9: ...Last updated 10 29 2020 8 ...
Page 10: ...ill occur At 50 000X this corresponds to 45 electrons Å2 for a 2 second exposure 1 54 Å pixel 1 After step 6 of F20 User Guide starting your session 2 Open DE20 software to insert and cool down camera NOTE can only open MicroManager or Leginon client server at one time DE 20 camera control on monitor input DVI 1 a Close Leginon client on camera computer b Open DE server GUI OLD on camera computer ...
Page 11: ...he Edit box and click apply beam shift from scope ii Repeat step i for square and hole preset iii Center and adjust intensity of FA FC EN preset beam 1 Send scope to EN preset and lower flu screen 2 Center beam using left trackball 3 Open the Edit box and apply beam shift from scope 4 Apply the same beam shift from scope for FA and FC preset 11 Get eucentric height and focus a Send scope to square...
Page 12: ...ter if objective aperture is in or out Must be done over vacuum a EN preset gains i Prepare beam for target dose 30 60 electrons Å2 SS 5 beam diameter 2X bigger than second circle on flu screen ii Dark reference 1 Close column valves and flu screen down 2 In correction node settings see LEFT image below dark and both channels ...
Page 13: ...set gains SS 5 beam diameter 2X bigger than second circle on flu screen target dose 20 30 electrons Å2 i Repeat step 13a using settings from RIGHT image below 14 Set up Ice Thickness a Go to vacuum area b Send scope to EN preset make sure beam is centered c Simulate target in exposure node d Record the mean value and input into Ice_Thickness node under ALS box NOT ZLP e ALS coefficient with 100 um...
Page 14: ...arget 20 In Exposure_Targeting node select exposure targets a Using acquisition mouse select the center of the holes of interests on the ice Select up to 4 holes that are closest to the center of the screen use toggle center crosshair tool to find where center is This ensures that you do not collect images with a lot of image shift b Using focus mouse select area of carbon towards the center of th...
Page 15: ...e20framealignment or ssh zeus h Paste the command from step 25e 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: ...ilize the V1 V2 module gently apply force to pull out cryo holder a Transfer cryo holder from pumping station into cryo transfer station 8 Place black blanking plug into opening where cryo holder was Inserting holder into dry pumping station and warming it up 1 Make sure all valves on pumping station are closed If there are 2 holders make sure the second holder has V4 and V3 closed in that order 2...
Page 17: ...one close V1 9 Open V3 then open V4 of holder 1 10 Open V3 then open V4 of holder 2 11 On Fridays start the ZEOLITE CYCLE for 2 hours If for any reason you cannot finish warming up the 626 and or Elsa holder and open V3 and V4 to pump down the jacket of the holder by the end of your session please send an email to emg nysbc org OR slack in EMG channel 626 and or Elsa is still warming up right now ...
Page 18: ... holder from dry pumping station f Transfer Elsa holder to the Elsa cryo transfer station 2 Cool down the Elsa holder in Cryo Transfer mode should be done on the pumping station desk a Attach the Elsa holder to the Elsa temperature controller using the 698 cord thick grey cord Make sure to connect red dots to one another b Switch to Cryo Transfer mode and start c Slowly add liquid nitrogen to the ...
Page 19: ...of the holder b Insert holder into the dry pumping station and turn it on c Once MDP status and System Status turn green open V2 and then V1 d Attach the holder to the Elsa temperature controller e Run the Warm up cycle This takes 2 hours 8 On Fridays pump down the dewar open V3 and V4 If an overnight collection was done and you want to use the holder the next day on the next day If you have time ...
Page 20: ...ygon tubing from holder and re connect to silver stopping plug near V1 V2 d Turn the pumping station off and wait for the green MDP ready light to turn off e Using one hand to stabilize V1 V2 vacuum area gently apply force to pull out cryo holder Open the holder entry valve on the workstation and insert the holder f Immediately upon removing cryo holder store in cryo transfer station g Place blank...
Page 21: ...remove the clip ring if it has been over tightened Rotate counterclockwise to remove clip ring off the tip of the holder and clockwise to secure the clip ring in place d When unscrewing the lid off the gridbox you will need to use a pair of forceps to hold the box down station was originally designed for square boxes 4 Transfer cryo holder into the TEM a Same insertion mechanism as 626 holder b Th...
Page 22: ...ted This step takes 30 min 7 Bake out the holder 50 C a After warm up the holder should be baked out with dewar jacket pumped down close V1 open V2 V3 and V4 You will need to attach the silver black red adapter to the end of the holder to open V4 b Connect the holder to the temperature controller cord See image c You should see a value in the DISPLAY window 20 C or so If not turn POWER OFF and the...
Page 23: ... orange while warming up and yellow when ready 2 Set holder alpha value at 60 degrees Goniometer will rotate 3 Pre pump airlock Takes 4 seconds 4 Position styrofoam box below goniometer to catch LN2 5 Remove holder from the transfer station and rotate the holder so that the holder top is at the 3 o clock position Immediately insert holder going straight into the goniometer with holder at 3 o clock...
Page 24: ...h Tension and Operate are still yellow did you crash the vacuum Will be grey if vacuum crashed 13 Turn turbo pump off 14 Do not open the column valves until IGP1 Column 10 If you are ever in doubt close the column valves and contact an EMG staff member immediately Room Temperature Holder Insertion 1 Make sure IGP1 20 High Tension ON column valves closed reset holder objective aperture out flu scre...
Page 25: ...ate of the goniometer and pull holder back until it stops then turn holder clockwise so that the hat rotates from the 12 o clock to the 5 o clock position 3 Pull holder straight out of the column 4 Transfer holder immediately to the transfer station 5 If vacuum spikes and column pressure crashes contact an EMG staff member immediately 6 Turn turbo on to prepare for next insertion ...
Page 26: ...er condenser 2 aperture image g Click next repeat steps b e 2 Gun Shift Tune Gun Gun Shift a Condense beam then center beam using MF X and Y b Repeat for next step 3 Spot size dependent gun shift Tune Gun Spot size dependent Gun Shift a Similar to step II focus beam and center using MF X and Y for all spot sizes 4 Condenser Astigmatism Tune tab Stigmator inset Condenser a NOT tune alignments stigm...
Page 27: ...via Beam_Tilt_Image node b Click on Simulate target c Use click tool to ensure that 11 Preset alignments a Find feature that is visible at medium mag b Center feature on the flu screen using FA preset c Under navigation node acquire image make sure the feature is centered d Send scope to HL preset and take a picture using image shift center the feature using the click tool e Go to preset manager a...
Page 28: ... on flu screen ii Check en presets and in the Correction node settings choose the camera of interest and set the appropriate camera configuration binning and exposure time iii Dark reference 1 Close column valves and flu screen down 2 In correction node settings see LEFT image below dark and both channels 3 Acquire you should see black image ...
Page 29: ...age vi When done with en preset gains make sure to apply that same brightness beam diameter to the en preset in Preset_Manager b FA FC HL SQ GR preset gains SS 5 beam diameter 2X bigger than second circle on flu screen target dose 20 30 electrons Å2 i Check FA preset especially camera size and binning and in the Correction node settings choose the camera of interest and set the appropriate camera ...
Page 30: ... when IGP2 10 d Manually set HT to 80 kV e Click HT to turn on i Wait till measured HT is 80 kV 2 minutes f Click Operate to turn FEG on 2 minutes i Emission should be 10 uA after any increase in HT wait a few minutes for emission to recover and stabilize g 80 200 kV 10 kV steps i Free high tension true ii 10 000 eV steps iii Select iv At 200 kV should read 7 uA emission under HT FEG 75 uA h Make ...
