Sarcomere Organization Texture Analysis Tool
A detailed article about the usage, validation and background of this method can be found here.
Sarcomere characteristics and organization are crucial features of the contractile apparatus in cardiac and skeletal muscle cells. They change during development and disease. Assessment of sarcomere distance, alignment and shortening provides insight into disease and drug responses in muscle cell models ranging from cardiomyocytes and skeletal muscle derived from human pluripotent stem cells (hPSCs) to adult muscle cells isolated from animals. However, quantification of sarcomere length is typically time-consuming and prone to selection bias. Automated analysis pipelines exist but these often require either specialized software and programming experience. In addition, these pipelines are often designed for only one type of cell model in vitro. Here, we present an easy-to-implement protocol and software tool for automated sarcomere length and organisation quantification in a variety of in vitro models: two dimensional (2D) cardiomyocytes, three dimensional (3D) cardiac microtissues, isolated adult cardiomyocytes and a reporter hiPSC line expressing fluorescent sarcomeric α-actinin. This image analysis algorithm automatically detects the direction in which the sarcomere organisation is highest over the entire image and outputs the length and organisation of sarcomeres taking all pixels into account. Moreover, we have adapted this algorithm to analyse videos of live cells during contraction. This provides additional output parameters like fractional shortening, contraction time, relaxation time and beating frequency. In this protocol, we give a step-by-step guide on how to prepare, image and automatically quantify sarcomere characteristics in different types of in vitro models and we provide basic validation and discussion of the limitation of our method.
Download the entire SotaTool folder to your hard-drive. Start the .exe file within the main folder to open the graphic interface. Enter the input folder and the resolution of the input images and click Start. For troubleshooting, read the online protocol.
Stein, J. M., Arslan, U., Franken, M., de Greef, J. C., E. Harding, S., Mohammadi, N., Orlova, V. V., Bellin, M., Mummery, C. L., & van Meer, B. J. (2022). Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro. Current Protocols, 2(7). doi: 10.1002/cpz1.462