JEOL 1400 (SOP)
JEOL 1400 (SOP)
Both 1400 microscopes are operating at 120 kV and can be used for negative stain as well as cryo-EM. Images can be recorded with the TVIPS camera. Before using the microscopes, any new user has to undergo a standard training. The goal of this training is for the user to become independent enough to do the screening and data collection on the JEOL1400’s by themselves. The facility staff will decide at which moment this happens. Once independent, the user can work in out of business hours without observation. However, every user is free to ask at any time for help and advice from the facility staff.
Start After ACD Heats Up
1) Remove the ACD spiral heater from the ACD and store it in the back of the microscope.
2) Switch on HT by pushing the HT ON button. Wait until the readout reached 120 kV.
3) Insert the CL aperture by turning the metal lever to the left
4) Insert the OL aperture of choice (can also be done later).
Holder insertion
Important: Before inserting the holder ensure that the O-rings are clean. Small contaminants can be removed with the fingers (there is always some grease on your hand). Large contaminants should be removed with tweezers.
Diagram for specimen holder insertion
- Align the pin of the holder with the hole in the Airlock system.
- Insert the holder until the end. The sensor will give a clicking sound.
- Switch lever on the airlock to pump.
- Wait until Specimen Chamber vacuum indicator turns green (Evac. ready) and the valve V8 and V21 closed (black)
- Rotate the holder clockwise till the resistance and guide the holder into the next lock. Important: The vacuum will suck on the holder. Do not just let it fall into the lock.
- Rotate the holder further clockwise until the next resistance and guide the holder into the microscope column. The flap on the holder will be aligned with the slit on the airlock. Important: The vacuum will suck on the holder. Do not just let it fall into the column.
Checking grid quality in low Mag
1) If not yet done, remove objective lens (OL) aperture using the big dial (Out position is indicated with the red dot on the aperture).
2) Go in low Mag mode by hitting the “Low mag” button on the right hand panel.
3) Move stage with trackball if desired. Find the area you want to image.
4) Go back to Normal mode by hitting the “Mag1” button (right hand panel)
Initial setup
1) Move stage with the trackball or the nanospace map to a suitable square. Ideally a black feature is close by.
2) Increase magnification (right hand panel; magnification knob) to working magnification (e.g. 60k). Adjust the brightness (left hand panel: brightness knob) so that the beam stays visible. Important: Turning counter-clockwise should make the beam more narrow and more intense. Turning clockwise should spread the beam and make it less intense. If not, you are on the wrong side of the crossover point.
3) Ensure that the condenser lens (CL) aperture is in (lever is to the left)
4) Center the beam with the beam shift X & Y knobs (green dials, hand panels).
5) Minimize beam until crossover point.
6) Center the beam with the beam shift X & Y knobs (green dials, hand panels).
7) Slightly spread the beam (clockwise!). If the beam is not round condenser lens stigmators have to be adjusted: Switch on CL Stigmators (TEM center). Use multifunction X & Y dial (yellow dials) to obtain a round beam shape. Switch off CL Stigmators (TEM center)
8) Spread the beam until it reaches the edge of the phosphor screen
9) If the beam did not spread evenly, adjust the position of the CL aperture using the small dial in the front and the small dial on the right of the CL aperture.
10) Repeat centring of the beam and centring of aperture until stable.
11) If not done yet, insert the OL aperture by turning its big dial. (usually the second smallest aperture is used, indicated by second smallest white dot on the dial)
12) Switch to diffraction mode using the diff button (right hand panel).
13) The OL aperture (the black shadow) should be centred around the bright diffraction spot on the phosphor screen. Adjust its position (the shadow) using the small dial in the front and the small dial on the right of the OL aperture. Important: You move the aperture. Not the beam.
14) Go back into normal magnification mode by pressing the “Mag1” button (right hand panel)
15) If the OL aperture is heavily misaligned it can be roughly aligned in normal magnification mode at low (<2000x) magnifications.
16) Hit Standard Focus (right hand panel).
17) cryo user continue with specific measuring section.
18) Insert small phosphor screen and adjust beam intensity using the brightness knob (left hand panel) for imaging. The current density (read in TEM center) should be below 10 pA and above 5 pA. Keep in mind that the beam diameter needs to be broader than the CCD detector. Keep in mind that the beam current should be 5-7 uA above dark current (increased beam current will increase current density)
19) Raise phosphor screen (“F1” right hand panel)
20) On camera computer select camera 2 and switch on continuous view (400 ms exposure time).
21) If the Thon rings are strongly visible, the z-height has to be better adjusted (TEM center “+z” and “-z”) adjust the height until the Thon rings disappear.
22) Once the optimal z-height is reached use the defocus dial (right hand panel) to go to fine eucentric height (no Thon rings visible at all)
23) Hit the “Mag1” button (right hand panel) to reset the displayed defocus value to zero.
24) Change the defocus (always underfocus, negative values) with the defocus dial (right hand panel) until you see 1-2 Thon rings. If the Thon rings are not round: a) Switch on OL Stigmators (TEM center). b) Use multifunction X & Y dial (yellow dial, coarse mode) to obtain a round shape of the Thon rings. c) Switch off OL Stigmators (TEM center). Hint: If there is a lot of astigmatism visible, start at higher defocus values, make a rough correction and finally go to lower defocus to make a final adjustment.
Removing the holder
Note: If you finished the negative stain session you should leave the holder in the microscope
Note: If you finished your cryo session you have to start a warm up cycle on the holder once you removed the holder.
Diagram for specimen holder insertion
- Pull out the holder until you feel the resistance. Important: Do not hold your hand against the airlock.
- Turn the holder counter-clockwise until you feel the resistance.
- Pull out the holder a small amount and turn it completely counter-clockwise. You will hear a clicking noise. Important: If you pull too much you will be located behind the sensor. Let the holder move back in slightly until you hear the click. Do not force the holder out!!!
- Switch the lever on the airlock system from Pump to Air.
- After venting (noise can be heard) remove the holder completely.
Performing the ACD heat-up
Do this procedure only if you are the last user of the day.
1) Switch off HT by pushing the “HT OFF” button (TEM center). Wait until the HT readout is 0.
2) Ensure that the holder is removed from the microscope.
3) Remove the CL aperture by turning the metal lever to the right.
4) Remove the OL Aperture (indicated with the red dot on the dial).
5) Insert the ACD spiral heater into the ACD. Ensure the plug is properly inserted.
6) In the TEM center software go to “Control” –> “Maintenance” –> “ACD”
7) The Window ACD controller opens. Click “ON. Button turns green.
8) Ensure that you did all the previous steps and click “OK” on the confirmation message. Heat up will last ~2h and stops automatically.
SerialEM guidelines for JEOL 1400
Important: Never save any calibrations as administrator. This would affect everybody!
Initial Setup
1) Create your own SerialEM setup (one time setup, Skip)
a) On the Desktop open folder “User_SerialEM_files”
b) Right click and select “new Folder”
c) Go to folder “Marcus” and copy three files. SerialEM.exe, SerialEMsettings.txt, SerialEMsettings.bak.txt
d) paste these files into your directory
e) right click on SerialEM.exe
f) left click on properties
g) Change in the line “Start in” the folder location from “Marcus” to “Your name”
h) confirm with “OK”
Start-up SerialEM
Important: Never save any calibrations as administrator. This would affect everybody!
2) Ensure that the EMMenue software is closed
3) Open serialEM with the executable file in your personal directory. (there is no password. Just click OK)
4) Activate MDS mode by clicking “LowDoseMode” in the pink window
5) Select any of the Modes (View, Focus, Trial or Record) by clicking the corresponding button in the pink window.
Setup of beam and camera conditions
6) In low dose control (pink window) select Record mode (Rec.). Use the hand panels to center the beam
7) In low dose control (pink window) activate “continuous update” and adjust beam brightness and magnification as needed with the hand panels.
8) De-activate “continuous update”
9) Make a single view image by clicking View in the camera window (dark green).
10) In low dose control (pink window) select “Focus” in “Define position of area”. A marker will appear on the view image showing you the location of the focus beam. You can change the position with a left mouse click onto the position of choice.
11) In low dose control (pink window) select “None” in “Define position of area”
12) In low dose control (pink window) go to Focus mode (Foc.). Activate “Set” under “additional beam shift”. Use the phosphor screen and the beam shift dials of the hand panels to center the focus beam.
13) De-activate “Set” under “additional beam shift”
14) In low dose control (pink window) activate “continuous update” and adjust beam brightness and magnification as needed with the hand panels.
15) De-activate “continuous update”
16) In low dose control (pink window) go to Trial mode (Tri.). Activate “Set” under “additional beam shift”. Use the phosphor screen and the beam shift dials of the hand panels to center the trial beam.
17) De-activate “Set” under “additional beam shift”
18) If needed: In low dose control (pink window) activate “continuous update” and adjust beam brightness and magnification with the hand panels. Afterwards, de-activate “continuous update”
19) Confirm that the beam in Record mode is still centered. If not center the beam and redo steps 12,13,16,17.
20) If needed, do the steps 12-15 from Focus mode also for View mode.
Making a grid atlas
21) Ensure that in view mode (2.5k magnification) you can see the square
22) In the task menu click Tasks –> Eucentric rough. The program will tilt the stage and estimate the eucentric height.
23) Open the template grid atlas by clicking in the task menu “File –> Open Old…”
24) Select under the image folder –> BECM –> atlas80mag.mrc and click “open” (Or use one of your old atlas files)
25) Confirm the next window with “OK”. If you get a warning message about different imaging mode, just disregard/click ok.
26) In the task menu click navigator –> open
27) Ensure that the OL aperture is removed.
28) in the script dialog box select “MakeGridAtlas”
29) In the open window go to the image directory –> Your folder. Create a new sub-folder, open it and click “OK”.
30) In the next window, ensure that you are in your folder and name the file “Atlas.mrc”. Click “Save”
31) Wait until acquisition is finished.
32) In the pop up window ensure that you are in you working directory and select as file name “navigator.nav”. Click “save”
33) The navigator window now contains one blue item which is your atlas. If you double click on the item it will open in the SerialEM display
34) Reinsert the OL aperture.
Aligning View mode and the atlas file
35) Ensure that the stitched Atlas is loaded on the screen of SeriaEM.
36) Find a recognizable feature on this map (visible at 2.5k Mag)
37) In the navigator window click “add points”
38) click with the left mouse button on the feature in the Atlas map
39) In the navigator window click “stop adding”
40) In the navigator window highlight the feature point you added and click on “Go To XYZ”
41) In the LowDose Control window (pink) select View mode (Vie.)
42) Lower the phosphor screen and center the stage with the trackball on top on you feature. (This feature should usually be close to where you start looking)
43) Make a View image in SerialEM by clicking “View” in the “camera and script panel” (Dark green)
44) Make a left click on your feature in the image you took. Ensure that the corresponding point is still highlighted in the navigator window and in the Menu select “Navigator –> Shift to Marker”
45) Confirm that the measured offset is reasonable and apply it with OK. Now all coordinates in the navigator shifted to the correct register.
Making square montages
46) In the navigator window click “add polygon”
47) on the grid atlas define the four corners of a square (4 left mouse button clicks)
48) In the navigator window click “stop Adding”. The polygon is visible as a green labeled item.
49) With the polygon line highlighted in the navigator window select in the menu Navigator –> Montaging & Grids –> Setup Polygon Montage
50) If a notification window appears click “ok”
51) In the Montage Setup Window ensure that minimum overlap is bigger than 30%. If correct, click “ok”
52) In the File properties window click “OK”
53) Ensure that you are in your working directory and select a filename for your square montages (e.g. squares.mrc)
54) In the navigator window click “add points” and select the squares of interest. Important: The marker needs to be in the centre of the square
55) When finished click in the navigator window “stop adding”. Your points are available wit a red label.
56) select the square you want to collect in the navigator window and push on the keyboard “a” to select the target for acquisition.
57) In the menu select “Navigator –> Acquire at Items”
58) Ensure that “rough eucentricity” and “Acquire map image or montage” are selected. Confirm with “Go”.
59) The script will measure the eucentric height of the square and make a montage. The navigator window displays the estimated completion time. Wait until finished.
60) The new item will be available with a blue label and double click will open the map.
Setup semi-automated imaging
61) With a square map open. Click in navigator window on “add points”. Select all holes you want to measure and finish by click in in the navigator window “Stop Adding”
62) In navigator window click the checkbox “collapse group”. All items that were selected in one go are bundled. For full view uncheck this box again.
63) With the group highlighted push on the keyboard “a” to select all group items for acquisition.
64) Open a new file for your record images by selecting in the menu “File –> Open New”
65) For 16 bit data select ” save as integers and truncate above 32767″. For Windows visible images select save as Bytes.
66) For both options select “save images as Series of TIFF files”.
67) Confirm with OK.
68) Ensure you are in your data folder and define the file name (i.e. My_awesome_images). Click “Save”
69) Check the imaging conditions for SPA by selecting in the menu “Script –> Edit 1”. In this script you define the defocus, no of images per stage motion etc. Choose the values to your ling.
70) In the menu go to “Navigator –> Acquire at items”
71) Ensure that rough eucentricity is NOT checked and enable “run Script collectSPA”. Confirm with “GO”
72) The navigator window shows the estimated time of completion.