Frequently Asked Questions about CurveAlign
These two programs were developed with complementary but slightly different main goals. CurveAlign was developed first and had the main goal of quantifying all fiber angles within a region of interest relative to a user defined boundary be it a straight line or a tumor boundary. As our research grew in investigating the role of collagen in cancer progression and invasion we wanted to investigate how individual fiber parameters could influence cancer and other diseases. Out of this need came the development of CT-FIRE to analyze individual fiber metrics such as length, width, angle, and curvature. Besides the relative angle quantification with respect to the boundary, the current version of CurveAlign can be used to extract other collagen fiber features, such as localized fiber density, fiber alignment, and the spatial relationship between fiber and the associated boundary. In addition, the extracted individual fibers extracted by CT-FIRE can be imported into the CurveAlign for additional feature extraction mentioned above. We have future plans to integrate these programs further. For now, CurveAlign should be used for bulk assessment of collagen features including alignment/density and CT-FIRE for individual fiber quantification.
Q2: How do I use CurveAlign?
The manual including a number of tutorials is a good start point for learning the basics of CT-FIRE.
Q3: How do I manually draw boundary in CurveAlign V4.0?
From CurveAign Version 4.0 on, there is a separate button named "BD Creation" to launch the functions to manually or automatically create the boundary. tiff boundary is commonly used as the "Boundary Method" in CurveAlign V4.0. The steps to manually create the tiff boundary file are briefly listed below:
(1) Load one or multiple images into CurveAlign ;
(2) Choose the image to work on if multiple images are loaded;
(3) Click the "BD Creation" button, and in the prompted dialog window, choose the default option, i.e., "Manual Mask", then in the consequent dialog window, choose the default option,i.e., "freehand mode";
(4) Follow the instructions/message displayed in the message window highlighted in green at the bottom of the main GUI to start and finish the boundary creation. For the freehand mode, you can click any point in the image, and then drag to draw each boundary.
(5) When boundary creation is done, the corresponding mask will be automatically saved in a subfolder named "CA_Boundary" with correct file name for later CurveAlign analysis.
(6) To draw boundary for another image loaded, repeat the above steps 2-5. In step (3), all the options are set to be identical for all the loaded images and only need to choose for the first image.
To be noted, the above procedure is used for directly drawing boundary on the image e.g. SHG image to be analyzed. If you need to draw boundary from other images/channels or using other tools such as Fiji, make sure the created boundary file follows the correct naming convention and is saved in the correct folder. You can check the manual for details.
Q4: What are the definitions for orientation and alignment?
(1) Orientation based on relative angle(angle between a fiber and its nearest boundary) when there is a boundary involved, unit is in degrees, ranging from 0-90 degrees. It is the mean value of the relative angles of the fibers with respect to the nearest boundaries.
(2) Orientation based on absolute angle(angle between a fiber and horizontal axis) when there is no boundary involved, unit is in degrees, ranging from 0-180 degrees. It is the mean orientation calculated by circular statistics. Specifically, angles are multiplied by 2 first to be converted to [0 360] range to make the angles circular, then calculate the mean value of the circular angles, lastly the mean value is divided by 2 to yield the orientation.
(3) Alignment is defined as the mean resultant vector length in circular statistics, ranging from 0 to 1, where 1 means all fibers are aligned in one direction while smaller values mean fibers are less aligned. It is called normalized vector sum of orientation vectors. Actually, it is the length of sum of orientation vectors divided by the number of angles. To be noted, alignment calculation is based on absolute angles mentioned in (2).