User Instructions for 3200FSC

These are for users who come to use the microscope
Assumed that the microscope has already been aligned and software set up for imaging
Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
In Search mode, lift screen and view display with Fastscan camera
Find area of interest and center on Fastscan camera
Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
Press Mag 1 to reset defocus to 0
Adjust defocus to desired level
Go to MDS Photo Mode
In EM Menu 3 software, advance image number to a fresh image
In EM Menu 3, press "Exposure". A new image should come up
Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
Go back to Search mode.

After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
In Search mode, center an object of interest
Tilt grid to 15 degrees
If you are not eucentric, you will see image shift on the live display camera window
Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement

Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
Bring it back as follows:
1. lower screen to see beam
2. Make sure PLA button is lit
3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)

Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
Go to Focus, then MDS photoset
Lower screen
Use specimen movement to center the object manually
Go back to Search mode
Be sure that PLA button is lit
Use DEF/Stig X and Y to re-center object

Do not worry about astigmatism in focus mode. Because of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
If it is really bad, try recentering Focus using PLA as described above
At a 2 um radius, you often see the edge of the objective aperture in Focus mode

In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
it is particularly important not to adjust magnification or iMDS mode if both programs are running
SerialEM can be used for montaging while in iMDS as follows:

Go to Standard Photoset mode (normally not used at all in cycle)
Press Low Mag button and lower mag to 150X
Center beam with beam shift, adjust brightness and spot size to 5
Quit EM Menu 3 if it is running
Start SerialEM. Should hear a beep. If you get an error, quit and restart.
Set up Record at binning 4, and 0.1 second exposure
Take a test record image to be sure exposure is good
Open a new Navigator window
Choose to make a full grid montage. Change overlap to 15% and choose to ignore pieces to skip.
Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
Once done, choose to make a new map.
If map doesn't look good, try turning off or on "sloppy montage" option and see which is better. Double click map to reload
Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag
Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
Optionally, save navigator file
Quit SerialEM, go back to mag 1 mode
Cycle through hysteresis cycle a few times. You will likely need to realign things.
Restart EM Menu 3 if you were using it
In Search mode, go to saved positions and search there

Do not use energy filter unless you are in SerialEM low-dose mode
Never let SerialEM adjust eucentricity in low-mag mode
For montages, use at least binning 4
Never try to use SerialEM and MDS/iMDS at the same time, except as described above
It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)
If using energy filter, center it at the same mag that record will be taken at
To program SerialEM to use Energy filter, go to Record mode, insert slit, click then unclick "Continuous update of mag and beam"
If energy filter is properly centered, should get around 2 um of image shift at 20K mag before black spot appears
In tilt series, limit image shift to the maximum you have before a black spot appears (around 2um)
The smaller the slit width, the more difficulty you will have keeping SerialEM aligned. 20 eV is recommended

-- BillRice - 28 Sep 2007