Operating F20/F30 with SerialEM

Transcription

1 Chen Xu $BrandeisEM: ~emdoc-xml/en_us.iso8859-1/articles/operating-f20-or-f30/article.xml, :42:20 xuchen Exp$ This is a quick check list for the Tecnai F20 or Tecnai F30 users who already have basic knowledge of Tecnai FEG microscope. I list here the steps that I would perform if I am on the scope with cryo sample and plan to collect single particle data using SerialEM. A pdf version of this document (article.pdf) is also available. 1

2 Table of Contents 1 Check E-logbook to see if there have been any problems Login to Tecnai Computer Check vacuum status Check settings for H.T., Gun Lens and Extracting Voltage Cool down the scope Check CryoBox Position Put in a single tilt holder with a standard grid Eucentric height, Eucentric focus and Pivot Points Check coma-free and beam conditions for high-res imaging Start up CCD or 3rd party software interfaces Set Bias for 4-port CCD Prepare Gain Reference Files Setup Low-Dose in SerialEM Cryo Holder In Stop LN2 bubbling in the holder dewar Eucentricity, without wobbling the stage if possible Obtain Full Grid Overview Resume Low-Dose Mode Mark Interested Points Check your main Macro Acquire at all points Finishing Up Check E-logbook to see if there have been any problems It is not a bad idea to see if there is any problem or issue from last user that should have been logged on the E-logbook. 2 Login to Tecnai Computer Start up Tecnai User Interface (TUI). 3 Check vacuum status The vacuum status should show "Column Valves Closed". IP3, IP1 and P3should all be in good ranges. 2

3 4 Check settings for H.T., Gun Lens and Extracting Voltage Make sure they are in desired values, and set them to otherwise. 5 Cool down the scope Add LN2to the big cold trap dewar. This is important for cryo session. 6 Check CryoBox Position It is a good time to make sure the CryoBox is at IN position. When it is IN, the "aperture" indicator points to position "1", close to horizontal direction. This is absolutely needed for a serious cryo session. 7 Put in a single tilt holder with a standard grid Currently, the standard grid is a holey grid with Au particles deposited on. Ask Chen if there isn t one available. 8 Eucentric height, Eucentric focus and Pivot Points This is to tune miscroscope, mainly to set beam tilt pivot points to the right level. This should be in the order : 1. Eucentricity 2. precisely focus 3. adjust pivot points using Multi-function knobs. You might want to check rotation center at this point too. 9 Check coma-free and beam conditions for high-res imaging This is relatively more important for single particle user. Tomography user may skip this step. Come-free aligning. This should be done at the highest magnification of SA range. Check the shape of beam cross. This should be done at a mag higher than SA range. The bright spot should be sharp and close to the center of the whole crossed beam. Otherwise, perform gun alignment procedure. This step might also concern tomography user, as if the gun tilt is off too much the beam intensity cross gradient could cause sharp border between montage pieces. Check FFT of the image from a patch of gold particles on grid. 3

4 10 Start up CCD or 3rd party software interfaces Operating F20/F30 with SerialEM On F20: DigitalMicrogaph TIA SerialEM On F30: Filter Control DigitalMicrogaph SerialEM. 11 Set Bias for 4-port CCD This is done with NO beam, and from CCD s native controlling software interface. For FEI Eagle CCD camera that was on Tecnai F20, it is from CCD Camera All Bias. For Gatan US4000 CCD that was on F30 and now moved to F20, DigitalMicrogaph Camera Set Bias. 12 Prepare Gain Reference Files This is done with proper beam and NO specimen. Either specimen rod is in retracted position or there is a hole or broken area that is large than the CCD area. As we mainly use SerialEM for this preparation task, this is done from SerialEM Camera Prepare Gain Reference. For the new generation direct detector camera such as Gatan K2 Summit camera and FEI Falcon camera, the procedure to prepare gain reference is more complicated. Therefore, it is perhaps not a bad idea to use the native controlling software to prepare the gain reference. You can ask SerialEM to use that. This is done by SerialEM Camera uncheck Normalize here. 13 Setup Low-Dose in SerialEM A typical setup like this might work for you too. View: mag = 1700x, binning = 4, area = full. Focus: mag = 100kX - 200kX, binning = 4, area = half or quarter-wide. Trial: mag = The same as Focus(check box "keep Focus and Trial the same"), binning = 4, area = full. The beam size should slightly smaller than CCD area so the edge of the beam can be seen on T image. This is needed for "CenterBeamFromImage" routine to work nicely. Record: mag = 59,000x, binning = 1 or 2, area = full Set proper offsets for View mag, first defocus and then IS. The IS offset only works for the specific mags of R and V. If down the road, you change the mag of V or R, you need to recheck this. 14 Cryo Holder In Now the scope and CCD are ready, it is time to handle the real cryo sample now. I would wait until IGP1 < 10 before opening the cryo shutter. 4

5 15 Stop LN2 bubbling in the holder dewar Allow 30+ minutes for holder temperature to settle. And then quiet the bubbling. 16 Eucentricity, without wobbling the stage if possible. If possible, try not to use procedure that involves stage wobble. The main reason is that wobbling the stage will likely introduce LN2 bubbling again in the dewar of 626 side-entry cryo holder. The quickest way is perhaps to run Z_byVmacro in Low-Dose mode. Example 1. Z_byV MacroName Z_byV ############################## # Z_byV.txt # by Chen Xu, Feb 8, 2013 ############################## # # a macro to adjust the eccentric center using beam tilted pairs. # It uses Autofocus to measure the focus and adjust Z instead. # # assume the Eucentric Focus has been calibrated for the mag you use. #================================== # remember the starting focus for V #================================== GoToLowDoseArea V ReportFocus DEF = $ReportedValue1 #==================== # set objective lens #==================== SetEucentricFocus NormalizeLenses 2 Delay 1 #=========== # Adjust Z #=========== Loop 2 Autofocus -1 1 ReportAutofocus t = -1 * $reportedvalue1 MoveStage 0 0 $t echo --> Z moved $t micron EndLoop #================================== # set back original defocus of View #================================== 5

6 SetStandardFocus $DEF 17 Obtain Full Grid Overview Go out of Low-Dose mode, go to low mag like 120x or 140x, Obj. Aperture out, set C2% to 100. Set binning_r = 4, Exposure = 0.3 second. Take a R shot to check if the image has complete full area, not partially blocked by CryoBox. It is a good time to check if the cryobox is centered. Navigator -> Open Navigator -> Montaging and Grids -> Setup Full Montage Response to the dialog window and define filename. Click on "start" on Montage control panel. Make the final overview to Map. Close the stack file. Change mag back to above LM range so that Obj Lens can warm up. Note: As you can see, it would be nice if we can do the last three steps automatically so that we don t have to wait for the montage to finish to increase mag. Indeed, we can by taking advantage of macro. After defining the filename for the montage, the stage will go to (0,0) by itself. You can then add current stage position as a point item and flag it with "A", followed by running a very simple macro from Navigator -> Acquire at Points. Example 2. TimeForCoffee.txt MacroName TimeForCoffee # warm up obj lens after map is done. Vmag_LD = 2300 M NewMap SetMag $Vmag_LD 18 Resume Low-Dose Mode Recheck low-dose condition, after Obj lens temperature settles down from previous step. With current geometry of the grid, set "Focus" position. Double check if the V and R mags are aligned still. Don t forget to put back Obj Aperture in and center it. Check the Obj stigmation at this point. 19 Mark Interested Points Go to one of the meshes picked from grid atlas map. And you might want to "align" the images between V (e.g. 3000X) and mag at which the grid overview map is created (e.g. 140X) using "shift to marker" under navigator. 6

7 Adjust to eucentric hight using macro "Z_byG". Same as before, the F should be copied as V beam temporarily. Take a V shot and make it into map. Add interested points as point item. And flag all of them as "A" - Acquire. 20 Check your main Macro Make sure the main Macro and all other are loaded in Macro editors. Here is an example. Example 3. LD.txt MacroName LD #main macro for data collecting in LD mode ## positioning - goto a spot RealignToNavItem 1 Copy A P ## Center beam T CenterBeamFromImage T ## Adjust Z to Eucentricity Call Z_byG ## refine X Y Call AlignToP ## now autofocus G G ## Drift Control Call Drift ## Take shot R S #=== end ==== Run this macro for one point (highlighted in navigator window). If it works fine, then the hard part is done. Congratulations! 21 Acquire at all points Navigator -> Acquire at Points, and define the macro, in this case "LD", as the main task. That s it. Sit back to watch. 7

8 22 Finishing Up Close Column Valves! Close the cryo shutter on the holder Take out cryo holder from scope Take out clamp ring and the grid from holder Warm up the holder Cryo Cycle Log the session 8