Motion Correction

1. From the initial GUI, click on the Motion Correction button to open the Motion Correction GUI.
2. On the Motion Correction GUI, click the Add Files button to choose any number of videos you would like to process. If you close the file selection window and decide to add more files later (e.g. from a different folder), they will appear at the bottom of the list. If you accidentally add a same file multiple times, it will not be added to the list again, and a warning window will pop up. Compatible file types include multi-page 8- or 16-bit TIFF and AVI files. Files need to be single-channel images. If your data is of an incompatible file type, it can likely be converted to TIFF or AVI with free software such as ImageJ, available at https://fiji.sc/.
3. To remove a file that has been added to the Files to Process list, select the file with the mouse and click the Remove button.
4. Choose whether to use non-rigid motion correction or not. Non-rigid Motion Correction splits the field of view into a number of overlapping patches to correct for within-frame motion artifacts. While this can give more accurate results, it is considerably slower than rigid motion correction.
5. Grid Size defines the size of each patch in pixels when using non-rigid motion correction. This option is not available if Non-rigid Motion Correction is not selected.
6. Upsampling Factor defines the upsampling factor for subpixel registration. Higher values helps reducing the smearing effect while being slower. Usually no need to change.
7. Max Shift defines the maximum rigid shift in pixels allowed in each direction. Consider increase this value when there is large amplitude of motion.
8. Initial Batch Size defines the number of frames from the beginning used for calculating the initial template. Change this to a lower value if you run into memory issues.
9. Bin Width defines the number of frames of each bin, over which the registered frames are averaged to update the template. Change this to a lower value if you run into memory issues.
10. The Save Settings button allows the user to manually save all settings under a specific file name of one’s choosing. Settings are saved as MAT files. These include all settings in the Settings section. Note that EZcalcium will automatically save the settings into a MAT file (see below), so manually saving the settings is usually not necessary.
11. The Load Settings button allows one to load all saved settings in future sessions. You can load the automatically generated MAT file (which is the same file that saves ROI Detection and ROI Refinement results) to check the settings used to generate the results.
12. Once all settings are configured, click the Run Motion Correction button. Progress of motion correction will be displayed in MATLAB command window.
13. After motion correction is completed, the new file generated will be added to the bottom of the Processed Files list. Original files will not be overwritten. A new TIFF file will be created with '_mcor' appended to the filename, and a MAT file with the same file name as the new TIFF file will be created containing automatically saved settings.
14. The Open button will open the selected file in the default program set by the operating system.
15. Clear List clears the entire list of Processed Files.