Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence

Quantitative assessment of cellular forces and motion advanced considerably over the last four decades. These advancements provided the framework to examine insightful mechanical signaling processes in cell culture systems. However, the field currently faces three problems: lack of quality standardization of the acquired data, technical errors in data analysis and visualization, and perhaps most importantly, the technology remains largely out of reach for common cell biology laboratories. To overcome these limitations, we developed a new experimental platform - Integrative Toolkit to Analyze Cellular Signals (iTACS). iTACS consists of two components: Acquisition and Training Module (AcTrM) and Analysis and Visualization Module (AnViM). AcTrM is based on µManager - an NIH-ImageJ-based microscope control software - and facilitates user self-training and automation of common image acquisition protocols. AnViM is based on NIH-ImageJ and facilitates user-friendly automation of data analysis and insightful visualization of results. These experiments involve culturing adherent cells on hydrogels, imaging fiducial markers embedded in the hydrogel, and finally extracting from these images a comprehensive mechanical characterization of the cells. Currently, iTACS enables the user to analyze and track a wide array of properties, including morphology, motion, cytoskeletal forces, and fluorescence of individual cells and their neighboring region. The quality standardization issue was addressed in AcTrM with, a reference image-guided refocusing technique. The technical issues in data analysis were addressed in AnViM with a multi-pronged image segmentation procedure, a user-friendly approach to identify boundary conditions, and a novel cellular property-based data visualization. AcTrM is designed to facilitate the straightforward transformation of basic fluorescence microscopes into experimental cell mechanics rigs, and AnViM is equipped to enable users to measure cellular mechanical signals without requiring an engineering background. iTACS will be available to the research community as an open-source suite with community-driven development capabilities.