Running the application » History » Revision 23
Revision 22 (Anchi Cheng, 08/28/2016 10:07 AM) → Revision 23/25 (Anchi Cheng, 03/27/2018 07:41 PM)
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 (optional) 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. This value is evaluated after integer conversion and scaling. 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. 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. !tilt.png! 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. ______ [[Set-up Before Running|< Set-up Before Running]] | [[Reading the tomography graphs| Reading the tomography graphs >]]