Graphical User Interface (GUI)

In this manual, we describe the necessary steps that the User needs to follow to analyse a set of tube images. In addition, the different parameters of the GUI are explained and suggested values are proposed. We tried to make the AutoTube GUI intuitive and easy to use. In the following, the most important steps for the correct usage of the software are explained. Note that each of the steps are associated with a number in red in Figure 2.

Step 1: Click on the “Select Folder” button to locate the directory containing the (*.TIF, *.JPEG, *.PNG) files of your experiments. You should select the top directory containing the images. After that, click “Enter”. You’ll next see that the list of image filenames gets displayed.

Step 2: Microscope Setting parameters. These parameters are related to the microscope and are used to convert distances from pixels to um. There are five main parameters:

• Object Magnification: this is the power of the microscope objective. Example values are: 10x, 20x, and 40x.
• Lens Magnification: extra magnification of e.g. 1.25x, 1.6x, or 2x. Leave it at 1 if your microscope does not have any extra lens magnification.
• C-Mount: is usually set to 1. Other values, e.g. 0.45x, may be used if a camera adapter with that power of magnification is in place.
• Camera Pixel Size (CCD): linear size in um of a physical pixel (assumed to be square) in the CCD chip of the camera used. Typical values are in the range 0.5 - 16 um.
• Binning: combines pixels in the camera to one single pixel in the acquired image - typically used under low light conditions. The common options are 1 (1X1), 2 (2X2), or 4 (4x4). Set this value to 1 as default and it represents that each pixel in the camera is mapped to its own pixel in the output image.

Step 3: Pre-processing parameters for the input images. These parameters are responsible for reducing detrimental effects from image acquisition, such as poor contrast, uneven illumination or noise (For more details, please read the Image Pre-processing subsection in the Materials and Methods Section of the paper). There are four main parameters to be tuned:

• Input Colour Channel: the color channel over which the analysis is going to be performed.
• Adjust Intensity: choose whether an intensity adjustment correction shall be performed. You can choose among three different options: auto-contrast operation, global histogram equalization, and adaptive histogram equalization. The auto-contrast and histogram equalisation options are useful when the contrast across the different image regions is homogeneous, otherwise, use adaptive histogram equalization.
• Correct Illumination: choose whether the illumination should be normalized and set the approximate diameter size (in pixels) associated with the circular uneven illumination effect. Note that this parameter is related to the microscope lenses diameter in pixels. The lenses diameter size that we considered for both lymphatic (LV) and blood vessels (BV) was set to 51, and for the acquisition settings of our images, a value in the range 40-60 pixels.
• Correct Noise: choose whether noise shall be removed from the input images. Options include: BM3D or Wiener Filtering. BM3D is a more robust de-noising method that should be preferred and is the default option for image de-noising. The Wiener filter is a less computationally heavy approach, preferred when results should be obtained faster.