Membrane Kymograph Generator: A cross-platform GUI software for automated generation and analysis of kymographs along dynamic cell boundaries

SummaryThe plasma membrane and accompanying cortex serve as one of the major hubs of the signal transduction and cytoskeletal activities that collectively regulate numerous cell physiological processes such as migration, polarity, macropinocytosis, phagocytosis, cytokinesis, etc. Yet, dynamically tracking membrane-cortex associated protein or lipid kinetics over time from live-cell image series remains a challenging task, primarily due to the difficulty of accurately extracting and aligning the cell boundary between consecutive frames, as the cell continuously deforms and moves. Here, we present Membrane Kymograph Generator, a cross-platform software that accepts multichannel time-lapse live-cell fluorescent imaging datasets as input and automates the cumbersome, heuristic process of boundary tracking, inter-frame alignment, and intensity sampling along the boundary. The software implements a rotational offset minimization algorithm that circularly aligns boundaries across consecutive frames by exhaustively searching for the optimal angular shift that minimizes point-to-point distances, while handling variations in boundary point counts due to cell shape changes. The software outputs kymographs that represent spatiotemporal dynamics of different membrane-associated proteins or biosensors, allows users to fine-tune visualization parameters through an interactive interface, and provides built-in correlation analysis tools for multi-channel datasets. Furthermore, the software allows advanced programmatic usage for batch processing and further analysis via a native API. Our validation tests demonstrated that the Membrane Kymograph Generator can be used to accurately track, visualize, and quantitate the spatial kinetics of a wide array of membrane proteins and lipid biosensors over extended time periods, in a variety of cell types, including Dictyostelium amoeba, human neutrophils, mouse macrophages, and different mammalian cancer cells. The GUI-based software is user-friendly, does not require any technical expertise from users, and significantly reduces the manual effort and time required for kymograph generation and downstream analysis, while ensuring high accuracy and reproducibility. Availability and ImplementationMembrane Kymograph Generator is a free and open-source software, licensed under GNU General Public License 3.0 or later. This software is cross-platform: It can be graphically installed on both x86-64 and AArch64/ARM64 computers, running either Windows, macOS, or any standard Linux distribution. The software is distributed as single installer files (and portable executables) targeting specific hardware architectures and operating systems, and hence, it can be installed natively without any dependency resolution. The source code, detailed documentation, specific installers, portable binaries, and test data are freely available at https://github.com/tatsatb/membrane-kymograph-generator. Additionally, since the software is written in Python, it can also be installed inside any Python environment using PIP package manager (package ID: https://pypi.org/project/membrane-kymograph) and can also be interacted via a built-in Python API.