Xenbase Image ID: 159293

Figure 1. Filopodyan, a highly customizable pipeline for detection, tracking, and analysis of protrusions in time-lapse datasets. (A) Image of a Xenopus retinal ganglion cell growth cone with filopodia (left) is processed with a Laplacian of Gaussian (LoG) filter for edge contrast enhancement (middle) and binarized with a chosen threshold method (right). (B) Parameters for boundary mapping are customizable; colored lines show the different cell boundaries detected by using three different values of LoG σ with the Renyi entropy thresholding method. (C) Iterative erosion and dilation (ED) of the thresholded mask (from A, right) removes protrusions (left), and the difference between these two masks yields a mask with protrusions alone (center), leading to a fully segmented image (right; protrusions in cyan, growth cone body in magenta, and its boundary in yellow). (D) The cutoff threshold for segmenting protrusions is fully user customizable by adjusting the number of ED operations. The two panels illustrate the difference upon changing the ED number from 3 to 6, showing additional protrusions (white arrowhead) and shifted position of their bases (black arrowheads). (E) Automated filtering removes false hits that do not satisfy user-defined selection criteria. Detected candidate protrusions include true (black arrowheads) as well as erroneous (white arrowheads) annotations that are removed by the filter. Any false hits that escape automated filtering and incorrect tracking can be corrected manually. (F) A time series of images segmented into body (magenta), protrusions (cyan), their bases (orange), and tips (green). (G) Output tables contain the measurements at each time point (row) of protrusion shape, movement, fluorescence, and coordinates (column) for all detected protrusions (column sections). (H) Descriptive properties of filopodia extracted by Filopodyan plugin and the downstream analysis scripts include length, straightness, direction-corrected measures of tip movement and base movement, tip persistence, and the time spent extending, retracting, or stalling. Bars: (A and C) 5 µm; (B and D–F) 2 µm. Image published in: Urbančič V et al. (2017) © 2017 Urbančič et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

Return to previous page