Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro
Current Protocols, vol. 2, pp. e462
Abstract
Sarcomeres are the structural units of the contractile apparatus in cardiac and skeletal muscle cells. Changes in sarcomere characteristics are indicative of changes in the sarcomeric proteins and function during development and disease. Assessment of sarcomere length, alignment, and organization provides insight into disease and drug responses in striated muscle cells and models, ranging from cardiomyocytes and skeletal muscle cells derived from human pluripotent stem cells to adult muscle cells isolated from animals or humans. However, quantification of sarcomere length is typically time consuming and prone to user-specific selection bias. Automated analysis pipelines exist but these often require either specialized software or 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 organization quantification in a variety of striated muscle in vitro models: Two dimensional (2D) cardiomyocytes, three dimensional (3D) cardiac microtissues, isolated adult cardiomyocytes, and 3D tissue engineered skeletal muscles. Based on an existing mathematical algorithm, this image analysis software (SotaTool) automatically detects the direction in which the sarcomere organization is highest over the entire image and outputs the length and organization of sarcomeres. We also analyzed videos of live cells during contraction, thereby allowing measurement of contraction 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 and contraction characteristics in different types of in vitro models and we provide basic validation and discussion of the limitations of the software tool. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Staining and analyzing static hiPSC-CMs with SotaTool Alternate Protocol: Sample preparation, acquisition, and quantification of fractional shortening in live reporter hiPSC lines Support Protocol 1: Finding the image resolution Support Protocol 2: Advanced analysis settings Support Protocol 3: Finding sarcomere length in non-aligned cells.
Authors 10
-
Jeroen M. Stein Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
-
Ulgu Arslan Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
-
Marnix Franken Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics
Leiden University Medical Center
Affiliation as printed
Department of Human Genetics Leiden University Medical Center Leiden The Netherlands
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
-
Jessica C. de Greef Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics
Leiden University Medical Center
Affiliation as printed
Department of Human Genetics Leiden University Medical Center Leiden The Netherlands
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
-
Affiliation as printed
National Heart and Lung Institute Imperial College London United Kingdom
National Heart and Lung Institute, Imperial College, London, United Kingdom
-
Affiliation as printed
National Heart and Lung Institute Imperial College London United Kingdom
National Heart and Lung Institute, Imperial College, London, United Kingdom
-
Valeria V. Orlova Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
-
Milena Bellin Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology
University of Padua · Leiden University Medical Center · Veneto Institute of Molecular Medicine
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
Department of Biology University of Padua Padua Italy
Veneto Institute of Molecular Medicine Padua Italy
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
Department of Biology, University of Padua, Padua, Italy
Veneto Institute of Molecular Medicine, Padua, Italy
-
Christine L. Mummery Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology
Leiden University Medical Center · University of Twente
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
Department of Applied Stem Cell Technologies University of Twente Enschede The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
Department of Applied Stem Cell Technologies, University of Twente, Enschede, The Netherlands
These authors contributed equally to this work
-
Berend J. van Meer corresponding Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
These authors contributed equally to this work
Cited by 34 stored of 34
No patents citing this paper on Lens.org (checked 2026-10-11).