Image Aligning Tool window

This tool allows for manual alignment of images in image stacks with timelapse image series that drift too much and cannot be aligned automatically. The tool can export the alignment data in the format readable by MicrobeTracker and can export shifted phase contrast or fluorescence images.

Basic Operation

• Load images by clicking Load phase button.

• Pick an object in the images that does not move (a non-motile cell or a dust particle). Scroll through different frames and click on the object. You do not need to click on it on every frame, clicking before and after big jumps would be sufficient. A cross appears in the image in each point you click on.

• Click Align button to align the images using the picked points (labeled with crosses) as the initial guess for the alignment.

• Save the alignment data by clicking Save and use this data later in MicrobeTracker.

• In MicrobeTracker load the alignment data by clicking Load button on the Alignment panel and proceed to cell detectin. Do not realign the images.

Buttons and Controls

• Help - call this help window.

• Load Phase, Load signal 1, Load signal 2, stack - load images the same way you would do in MicrobeTracker.

• Align - align frames using clicked points positions as the original guess. If these points are not available, do automatic alignment, similarly to that in MicrobeTracker.

• Points - produce the alignment from clicked points, without doing automatic alignment. The alignment is done in the way so that the clicked points remain in place, and the frames are shifted accordingly.

• Load, Save - load or save alignment data.

• Reset - reset the alignment to none.

• Panel 1 - information on the images loaded, current frame, and cursor position.

• Panel 2 - image zoom, message output, and contrast controls, which operate similarly to those of MicrobeTracker.

• Panel 3 - selection of the image: either original or shifted (by Align or Points buttons) and of the channel (i.e. Phase, Signal 1, or Signal 2).

• Panel 4 - parameters of the program, similar to those in MicrobeTracker. aligndepth indicates to the number of frames used in alignment, it should be intreases for long slowly-drifting series. bgrErodeNum is used for background subtraction and indicates the width of the area around cells (or other dark objects) in pixels that needs to be excluded from background calculation.

• Panel 5 - background subtraction panel similar to that of MicrobeTracker. You can subtract background and save the images. The images will be saved in the mode they are displayed in, i.e. the currently displayed channel (Phase, Signal 1, or signal 2) and currently displayed shifted or non-shifted images.