Inputs needed:

1. Directory with motion-corrected images. If you generated your images using TomoNet, the motion-corrected images are under directory MotionCorrection/MotionCor/corrected_images. You can also use other MotionCor2 output locations if you did motion correction separately.

Output you will get:

Root directory

— Recon:

— ts_tlt: output of tilt series (*.st) generation, and its associated *.rawtlt files.

— history_record.txt: This text file records the operation history. Each line is associated with one tomogram. If you need to redo tilt series generation, please delete lines in this file or delete the file entirely before rerunning TS generation.

—eTomo: reconstruction results using etomo

Explanation for each of the required fields in the GUI (see Fig. 2).

• “Motion Corrected Images Folder”: the path to the directory that stores all your motion-corrected images. If you used MotionCor within TomoStudio, the directory is “MotionCorrection/MotionCor/corrected_images” No default value.

• “Filename filter text”: The constant part of the filename of your target images. For example, if your tilt images are named “20221027_HCMV_130_00020_30.0.mrc,” then your “Filename filter text ” could be “20221027_HCMV” (HCMV data collected only on 2022/10/27) or “HCMV” (all HCMV data despite the collecting time) or “20221027” (all data collected only on 2022/10/27), etc. No default value.

• “Delimiter”: it separates the indexes of your file name. For example, if your naming convention is “HCMV-#-#-#”, your delimiter is “-”. Default: ‘_’

• “Tilt info Index”: the position/index in the naming that tells this image’s tilt angle. For example, in “20221027_HCMV_130_00020_30.0.tif” the tilt info index is 5 because the tilt angle 30.0 is the 5th item separated by the delimiter ‘_’. No default value.

• “Base Name Index”: This is the constant part (defined by index range) of the file name that defines one tomogram. For example, in a tomogram containing tilts of “20221027_HCMV_130_00xxx_xx.tif”, the index should be “1-3” because 20221027_HCMV_130 represents the constant part of the filename for one tomogram and has the index range “1-3”. No default value.

• “Target Base Name”: The program will rename the output with the format as follows:

  [target base name]_[index].st. Default: TS

  • This new name format will make the tomogram management easier in the next few steps.

• “Images file suffix”: Suffix of the motion-corrected image filenames you want to use (another filter for the file type). Typically these are the options: *.mrc. No default value.

• “cpu(s) #”: number of CPUs you wish to use for processing. Default: 8

• “Flip Images?”: If the user wants to flip images before 3D reconstruction (Horizontally over the X-axis, Vertically over the Y-axis). Default: No

• “Min Tilt Number”: the minimum number of tilts in a tomogram that is acceptable for you. Usually, during image capturing, tomograms will have different tilt numbers due to technical challenges. If a tilt series has too few tilt numbers, there will be information loss and more severe missing wedge artifacts. You are always suggested to click the check button to determine how many tomograms are expected to be generated with the current parameters set. Default: 30

• “Remove Dup”: In some cases, more than one image will be captured for the same tilt angle due to failures, such as bad tracking. In this case, we want to remove the earlier failed duplicates. Default: Yes

• “Add New Data”: Check if you want to add new data to existing data. Default: Yes

  • If set to No, all existing data will be overwritten, and the index will restart from 1.

Fig. 2: TS Generation tab with example parameters.