Leginon

h1. Running the application

h2. Import Notes about Image Intensity Recorded through

Tomography Node

Tomography node saves the images in a different format from other Acquisition nodes. By

default, the flat-field correct CCD counts are multiplied by 10 and converted to signed

16-bit integer before the image is displayed and saved. This makes CCD counts of 3276.8 or

larger overflow to negatives. Other Leginon Acquisition images are saved as float without

manipulation.

To avoid this problem, find out what exposure time corresponds to the fractionated dose

from your tilt angle step and range and total dose and take an image at tomo preset with

such an exposure in Navigation node. You will need to reduce the total dose if a good

fraction of the counts are larger than 3200 even though it would not appear to be saturated

in the float scale without the 10x factor. Alternatively, change the scale factor in

Tomography node.

h2. Multiscale Imaging

*  Preset image shift alignment/beam shift alignment are the same as in MSI

application

*  New dark/bright references should be reacquired for "tomo" preset that acquires the

final data. It is best to do this at the same dose per tomography image calculated from

the total dose, the tilt parameters, and the dose measurement.

*  For best focusing result, perform autofocus at the same magnification as the

tomography data collection, align microscope well at the eucentric focus and the

rotation center and save them before data collection.

h2. Using Tomography Preview

*  Preview targets (pink) can be selected when selecting targets in "Tomography

Targeting"

*  When the targets are processed, targets that are of the type "preview" are

processed before focus and acquisition targets.

*  Tomography Preview node acquires a image at the preview target using "preview"

preset which should be set at minimal dose.

h2. Dose Measurement

If "Measure Dose before collection" is checked in Tomography node, the stage will be

moved to the reference target and a dose image of the "tomo" preset will be acquired (center

512x512 of whateven binning of the preset) before each tilt series if the interval between

the series is longer than the limit time set in the settings of Dose Measurement node. The

measured value will then be used to recalculate the proper exposure time for tomography

imaging.

For this function to behave properly, the followings should be done during

operation:

* One, and only one, "reference" target should be selected in either "Square

Targeting" or "Hole Targeting" or "Tomography Targeting" node. The reference target

should be of either a broken square or a empty hole if no broken square can be

found.

* "Measure Dose" before collection should be selected in Tomography node.

* "Exposure time max/min" in Tomography node should be in a range that can accommodate

the electron beam fluctuation over time.

h2. Align Zero Loss Peak

This function applies only to Gatan energy filter EFTEM. If "Align ZLP before

collection" is checked in Tomography node, the stage will be moved to the reference target

and starts the procedure to align zero loss peak before each tilt series if the interval

between the series is longer than the limit time set in the settings of Dose Measurement

node.

For this function to behave properly, the followings should be done during

operation:

* One, and only one, "reference" target should be selected in either "Square

Targeting" or "Hole Targeting" or "Tomography Targeting" node. The reference target

should be of either a broken square or a empty hole if no broken square can be found.

This is the same reference target used for dose measurement.

* "Align ZLP" before collection should be selected in Tomography node.

h2. Low Magnification Model Fitting

The fitting of optical axis offset does not always works if the offset is so large that

the feature moves out of view with even a small tilt. In such a case, it is worth first

collect a tomography series at a lower magnification to define roughly the model.

At beginning of each session, or forced by the user, the model is initialized. By

default, at the initialization, Tomography node uses past fitting results that show good

agreement with the experimental data at the magnification of the preset used. If a good

model is not found, that from lower magnifications will be used. It is possible to force the

node to use a model fitted at a particular magnification by selecting it in

Tomography/Settings/Model.

Therefore, we recommend that, in case of fitting failure on good contrast images, the

followings should be done:

#  Tomography/Settings/Image Acquisition> change the preset to "hl".

#  Tomography/Settings> adjust Tilt and Exposure parameters to match.

#  Acquire the tiltseries images.

#  If the tracking is good, change the preset back.

#  Tomography/Settings/Model> Initialize with the model of (the mag of "hl"

preset).

#  Acquire the tomo-series.

#  If tracking is good, change back to Initialize with the model of "this preset and

lower" mag.

h2. What is a Good Tilt-Axis Model?

The goniometer-tilt-axis-based tracking model developed by Zheng et. al. corrects the

specimen height (z-axis) by a change of defocus using measured shift of feature shifts in

the images (x and y-axes). The tracking in the x and y directions does not involve the use

of such model, but is done by smooth curve fitting or preceding tilts. Therefore, to judge

the adequacy of the model, one should check the resulting defocii of the images in the

series remain unchanged.

On the other hand, the feature tracking in x and y is likely to fail only if the tilting

does not induce a smooth shift of the imaging feature a sudden drop of specimen position at

a particular tilt angle often throws off the smooth curve fitting. It is possible to reduce

such effect by increasing the number of data points included in the smoothing as set in the

model section of the tomography node settings window. Otherwise, the goniometer need to be

serviced.

[[Troubles with Tomography|Tomography Trouble Shooting]] section covers many of the problems we have encountered.

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[[Set-up Before Running|< Set-up Before Running]] | [[Full Protocol on a F30 with an energy filter| Full Protocol on a F30 with an energy filter >]]