An Automated In Vitro Experimental Platform to Analyze Structure, Motion and Forces in Adherent Cells

Abstract

Background Commonly used optical imaging and data analysis tools employ hardware and software technologies that are nearly antiquated. The lag in translation and implementation of advances in electronic devices, computational approaches and mathematical analysis into common experimental cell biology tools is a major constraint on the pace of growth in our knowledge of cellular physiology. Here we present Integrative Toolkit to Analyze Cellular Signals (iTACS) which enhances performance of microscope hardware and enables cellular biologists to readily examine a wide variety of cellular properties. Methods Using μManager - a NIH-ImageJ-based image acquisition application - we have developed an Acquisition and Training Module (AcTrM) which enables users to set up simultaneous measurements of traditional optical properties and a variety of physical properties of adherent cells. AcTrM facilitates user training through brief directions included in the graphical interface. To increase productivity and improve workflow of a shared microscope facility, AcTrM includes the ability to remove the sample from the microscope and resume the measurements at the precise physical spot automatically correcting small errors in position. This feature is particularly important for accurate measurement of physical forces in adherent cells. In addition to AcTrM, we have developed an Analysis and Visualization Module (AnViM) which enables users to quantify a wide variety of properties from the acquired data without mastering the underlying technical background. AnViM can be used in an interactive or batch processing mode on a personal or a cloud computer. It enables quantitative assessment of the properties including cellular shape, size, orientation, speed and direction of motion, tractions exerted on the substrate and on neighboring cells, contractile and shear moments, and similar wide variety of physical properties of the region neighboring each cell. AnViM interface enables the user to provide highly technical input in a more relatable manner. AcTrM and AnViM are the two key components of iTACS and they were primarily programmed primarily using Beanshell scripts and ImageJ macros. Results and Conclusion We collected input from users of various educational backgrounds to optimize the graphical user interface of AcTrM and AnViM. We successfully tested the functionality of iTACS in various experimental situations including rapid response of cells to fluid shear stress or pharmacological compounds, slow response in the context of cellular migration or growth from subconfluent to a confluent monolayer. iTACS has enabled us to identify a mechanistic fingerprint of adherent cellular monolayers and discover fundamental laws that govern mechanical behavior of adherent cells. Such studies require state-of-the-art engineering tools that were so far confined to a very few labs that have the necessary engineering background. iTACS now brings these and other novel engineering tools at the fingertips of experimental cell biologists.