SMARTQ: single-molecule amyloid fibRil tracking and quantification. A method for accurately imaging, tracking, and quantifying the growth of individual amyloid fibrils using TIRF

We introduce Single-Molecule Amyloid fibRil Tracking and Quantification (SMARTQ), a method to collect high-quality total internal reflection fluorescence (TIRF) microscopy amyloid growth data, followed by automated tracking and quantification of a large number of individual amyloid fibrils using classical image analysis. SMARTQ automatically tracks and categorizes the thousands of fibrils recorded in a single TIRF experiment and then analyzes and compares their growth rates. The script for this operates with three major steps: first, all images are normalized with regards to intensity, stabilized to remove translational shift, and a median filter is applied to improve contrast. Second, all fibrils are tracked through the addition of nodes to high-intensity areas that are then connected through a scoring algorithm to create “growing” fibrils. Third, all tracked fibrils are cleaned by removing leaves (false positives), and a modifiable fibril length threshold is set to only analyze fibrils above a certain length. The result is then exported both as graphics and as multiple data tables. Using TIRF image data for the fibril growth of two variants of the functional amyloid protein CsgA from E. coli, we validate SMARTQ by comparison with manual analysis.