Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images.

The periodically arranged thin filaments within the striated myofibrils of skeletal and cardiac muscle have precisely regulated lengths, which can change in response to developmental adaptations, pathophysiological states, and genetic perturbations. We have developed a user-friendly, open-source ImageJ plugin that provides a graphical user interface (GUI) for super-resolution measurement of thin filament lengths by applying Distributed Deconvolution (DDecon) analysis to periodic line scans collected from fluorescence images. In the workflow presented here, we demonstrate thin filament length measurement using a phalloidin-stained cryosection of mouse skeletal muscle. The DDecon plugin is also capable of measuring distances of any periodically localized fluorescent signal from the Z- or M-line, as well as distances between successive Z- or M-lines, providing a broadly applicable tool for quantitative analysis of muscle cytoarchitecture. These functionalities can also be used to analyse periodic fluorescence signals in nonmuscle cells. Lay description Striated muscles, such as skeletal and cardiac muscles, are composed of contractile units (sarcomeres) that contain specialized protein arrays (thin filaments) with uniform length. We have developed a software program that uses a mathematical technique (Distributed Deconvolution) to allow a user to measure the lengths of thin filaments using fluorescence images collected from a microscope. This paper walks users, step-by-step, through the workflow necessary to use our program and apply it to their own muscle samples. We then explain how to interpret the data generated by our program in the context of the structural features of sarcomeres. We also explore the diversity of other biological applications of our software.