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Fill cold trap dewar with liquid nitrogen.
Log in. It will ask you for your pass word. Double click on Tecnai user interface (on the desk top) Note vacuum levels at gun/column-should be log19 or lower, camera log 55 or lower, valve closed (when it is yellow) & HT on (button is yellow). Turn on the filament.

  • B, Taking out Specimen holder from column, pull it out and then -clockwise to close & out (When you take out holder grasp it firmly because the vacuum will suck it back & damage the holder).
    -Place the wand into the holder on the desktop. Use the specimen holder pins to lift metal ring up which normally holds the grid in place. Load your grid & put the pin back. Note: Don't touch anything beyond the o-ring on the wand!

    Check vacuum levels; GUN/COLUMN-log 19 or less is okay. Open Column valve (it make a little noise as the valve opens). You should be able to see the beam. Adjust the intensity with the left intensity knob on the left control pad to condense/spread the beam illumination.

    LEFT TRACK BALL is for beam centering
    RIGHT TRACK BALL is for stage movement

    ADJUSTING Z HEIGHT for each sample.
    - On search menu - open flap (small triangle button on stage menu bar) -set magnification to 10kX - set up wobbler level to 10-15 - click on alpha wobbler. If the image is moving, with the help of the z axis pressure sensitive pad adjust it in such a way that there is the least movement. To make sure the movement is at a minimum, go to a higher magnification and see if wobbling has been corrected. When corrected, click on alpha wobbler again. Note that the stage should come back to tilt = 0 for alpha tilt. If it doesn't, go to the set tab under the search menu, and type 0 for alpha and press the go to button. The stage should return to 0 alpha tilt. Afterwards, push flap back in and select setup tab.

    -Find a suitable specimen area at low magnification (mag knob on right control pad) -Go to mag desired and focus with knob on right control pad. The lower knob is focusing intensity and the upper is focusing. -If hard to focus, check objective aperture alignment, condenser stigmatism and objective stigmatism

    -If the spread of the beam is not uniformly circular, click on alignment tab, and click on condensor stigmator. Set the beam at the crossover point and center - monitor spreading of beam - with intensity knob use multifunctional knobs X and Y to make the beam circular. When done, click on setup menu.

    -Magnify to 12,000X - Condense the beam - Press Diffraction button on the Right Control Pad - if beam is not centered on the objective aperture, correct it using the objective aperture knobs on the column. Press Diffraction button again to get out of diffraction mode.

    -If focusing the image is difficult, there are 2 ways to correct the objective stigmatism.
    When done with stigmation, click on setup menu.

    -Select the area - focus - note the mag and negative number - adjust the intensity settings for one second for normal exposures as judged by inserting the focusing screen. Check camera setting that: auto exposure is selected; press flip out button; choose option; and insert date -scale -etc as you like. Make sure that the setting is set to Kodak 4489 emulsion 2.8 and data intensity 5.7. Flip in the menu and add text (up to 25 characters if desired for photo). Return to setup menu. - cover the screen with the rubbery mat - press exposure button on the control panel (screen lifts up and the computer will only show the progress of the photo) - Desktop reappears - Remove rubbery mat from microscope.

    Go to the setup menu. Leave HT on, Filament on, lower the magnification & spread the beam. Close the column valve, beam cuts off. Take out your sample as described earlier & change to another grid. Remember to check the vacuum levels at the GUN/COLUMN ( less than log 19). To look at another sample, open column valve & beam should be visible.

    Close column valve.

    Leave HT on. Close valve column valve & take out your sample (as above). To log out: NOTICE click on X at right top of desktop. It will ask you to save data -say yes & then later log off from start menu. From start menu, select shut down, then close all programs and log on as a different user, click yes. DO NOT SHUTDOWN THE COMPUTER. Complete the log book & go home.

    Ask Reena or a EML staff member to retrieve your negatives.

    -From the start menu, click programs, Gatan, Digital Micrograph.
    -Make sure these settings are present before taking pictures:
    -Check for proper vacuum
    -Under the camera settings menu, check that temperature is set at -10C
    -Set up the Global information for the run under the 'Microscope Menu' (microscope, specimen, operator, etc), being sure to check the box for ask for magnification before each acquisition. Click on OK.
    -Camera can be set at auto exposure or manual (pick manual for diffraction)
    -For processing images, prepare the gain reference
    a. Remove the specimen out of the field of view and obtain even illumination on the screen. Insert the prism detector.
    b. Select prepare gain reference under camera menu
    c. Enter target intensity 3072 and 4 for average frame settings
    d. Click on OK for each dialog box that comes up.

    -Remove prism and go to an area for a digital picture and focus. Insert the prism when you are ready to collect an image. -Choose one of the above and record the digital picture. Save it in your account directory (E:\users\myname) by going to the Custom menu and selecting save front image as full-res tiff.
    -Note, files will be stored here only weekly. Use the FTP program to transfer the images to another computer, or to an account on the EML server (wilfred.berkeley.edu).
    Press the X button on digital micrograph to leave digital micrograph and return to the Tecnai interface.