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AutoTomo checklist
Microscope Alignments
Make sure the scope is well aligned. I can not stress this enough. The calibrations for automated tomography are based on the fact that the has scope been aligned. When the
program was configured and calibrated, the microscope was well aligned. If you notice that you are having a lot of difficulty in terms of getting basic features to work such as
using image shift and autofocus routines to work, then most likely you have not aligned the scope well or the calibrations are no longer valid. If this is the case then you should
ask someone to help you and confirm the alignments and calibrations.
Acquiring the FlatFields and Bias images
Please always remember to acquiring new flatfields and bias images before you begin your experiment for the day. Here is a link
that explains how to do so: Acquiring Flatfields and Bias Image
Baking your specimen
Ensure that you have “baked” or “cooked” your area of interest on the specimen. If you do not do this you risk your specimen shrinking as the dataset is being collected.
There are routines available through SerialEM and also customized cooker routines on the JEOL 4000s.
Eucentricity
Eucentricity of the specimen is another very key component in terms of having a successful autotomo run. You should always do a coarse adjustment
manually, either using the stage software to rotate the rod and adjust z, or using any other method you prefer. The Jeol 4000s will not have
as good eucentricity as the other FEI scopes. Its pretty important that you do a good coarse adjustment of the eucentricity before trying
any of the routines with SerialEM.
Image shift check
Reset image shift and lower the microscope’s screen. The reset image shift button is located on the floating panel for image alignment. Center the objective aperture and make
the beam the smallest you are comfortable with and center it---don’t forget to work on the right side of crossover. The beam should be about 1.5-2 cm larger in radius than the outer
circle inscribed on the screen. This will aid in keeping the beam from from entering the images at high-tilts and keep out any image aberrations due to the beam getting too small.
Image check
Using trial images, center your area of interest in SerialEM. It is a good idea to take a record image and see how many counts there are---especially if this is a new sample
type. If the number of counts at zero degrees is lower than the optimal number of counts for an image, it will be even worse at high tilt. This is because at 60°, your
sample is twice as thick as it is at 0°.
AutoFocus setup and check
To calibrate the Autofocus first get the specimen focused using the video rate camera along with the image wobblers. Once this is done then click on the Menu Item "Focus" and then
"Reset Defocus".After this is done then click on "Measure Defocus" and click ok when it is done. Basically these series of commands instructs the program how to focus the image the way
you would like it.
You can use the above floating window to test out the Autofocus. Simply click on "AutoFocus" after you have completed the above steps to see how well it does the autofocus. If you
are happy with the results then you can continue. This is how the TiltSeries controller will AutoFocus your sample throughout the tilt series.
Below is a sample slide to explain how/why Autofocus works within the program:
Beam Setup
There is a tradeoff between specimen damage and signal-to-noise in images and reconstructions. For plastic section work one must consider the constraints of dynamic
range of the camera vs getting a drift-free high exposure image. It is best to play around with how you want the beam to be setup and how much dynamic range you
are getting in an image. It will all depend on your sample. Once you figure this out though you can use the same parameters for future work with that same sample.
Basics of AutoTomography
The goal in acquiring a tilt series is to have every picture centered on the same specimen feature, and at the same focus value.
The basic sequence is:
- Tilt to a new angle
- Correct change in specimen position
- Correct change in specimen focus
- Acquire final "Record" image
Starting the AutoTomo program
Once the beam is setup and your area of interest is centered, you can open up the tilt-series setup/start menu.
These are standard starting points:
To see the above menu click on "Tilt Series" in the menu bar and then "Startup/Setup Tilt Series"
- Under Tilt angle Specifications: Input your tilt-range (typical is -60 to +60). Heavier rods should work better starting on the --60 side. You should have already “walked” your specimen up to your start angle. You have the option of selecting your "Basic Increment" which is typically 1 or 2 degrees. You will want to think about this option because taking 1 degree increments will dose your specimen a lot more then doing 2 degree increments. If you are planning on doing a double tilt then perhaps 2 degree increments is all you will need, if you are not planning on doing a double tilt then 1 degree increments may be the choice for you. For the JEOL 4000s you will want to keep the "Delay Time" greater than 10 seconds.
- Under Size, Image Shift, and re-centering specifications: Your current mag should be displayed under "Magnification". You will also most likely want binning 1 for collecting your data at that size. You will want to use the option of "At Start and when needed track at magnification" and go below two mag indexes. This helps the program track your specimen better. So for example if you are at 20K for acquiring data, then you will want to set it at 12K for tracking.
- AutoFocus control: You will should calibrate the Autofocus outside of the tilt series menu. Please see above for calibrating the AutoFocus. Really at this point you should not change anything in the AutoFocus control. Autofocus every 4-6 degrees and focus every time above 50-55 degrees. The check autofocus option is used if it is an unfamiliar sample. This will tell you if youare having a difficult time getting accurate focus at high-tilt. Autofocus at high-tilt failures usually happen when something comes into the focus area that is too thick at high tilt. Otherwise, if the sample is familiar and there haven’t been problems in the past, skip it since it does take time.
- Initial Actions: No need to refine eucentricity since it was done before. Using the Align to buffer A is a good option for matching and tracking the area that you want. If you see for example a mitochondria or nucleus that is in the middle of the image, then the program will track that throughout the series.
- Tracking Control Parameters: Really you shouldnt have to change much here, but simply accept the defaults that are in place. Use “Repeat record if percentage of field lost is more than X” when your area of interest fills the entire image area. A good place to start is 5%, but you may have todrop to 3%. If the number is very low, your tilt-series will lengthen in time significantly since it constantly has to redo records. If this happens to you, go to thecamera pull-down menu and uncheck post-actions. This eliminates tilting until after a successful record has happened. The percentage you put here is magnificationdependent---the higher the mag, the more often the program will have to back up to do a record. If it backs up a lot, you might want to increase the tilt delay to give the sample more time to settle. You may also consider going down in magnification or using montaging. Track before autofocus. Again, if your area of interest is large or the sample is drifting, you might want to track before and after autofocus, but it’s usually unnecessary.
Just to note, there are many other options in there, but these usually deal with difficult data---like cryo or unusual plastic embedded samples.
- Go to the tilt series menu and check autosave log. The log file is very small so it doesn’t take up a lot of disk space and can only help you in the future. The only crucial thing to remember is to close the log file and open a new one between each tilt-series. After the first saved record of your tilt-series, the program will prompt you to name the log file and then will automatically save the file after each record.
- It’s really a good idea to stick around for the first 5 tilts or so. It’s tempting to get up and leave right away, but many people have had to retake entire tilt-series because of a mistake in the first high tilt or two.
- When your tilt-series is finished, terminate and say yes to the first question. Then you have the option of saving pre-rotated zero tilt images of the a-axis for reference in aligning the b-axis in buffers G and H. This is a good idea if you are going to rotate right away. However, if you are doing serial sections, it’s generally easier to just do all of the a-axes and then rotate and do all of the b-axes.