Live cell imaging is useful for understanding a large range of cellular functions. Recent developments, such as the availability of green fluorescent protein, have put live cell imaging into the hands of all cell biologists revolutionizing this aspect of structural cell biology. Unfortunately, the software tools needed to analyze these images have not advanced as rapidly as the methods. The long-term goal of this proposal is to develop software that enables more biomedically relevant knowledge to be extracted from live cell images by a larger number of scientists yielding a greater understanding of fundamental biomedical processes. In this Phase I SBIR, we aim to design a software package, named Molecules in Motion (MiM), that allows analysis of live cell and single molecule light microscopy images by novice users. MiM will provide several innovative features compared to existing software, including detailed user guidance and extensive quantitative capabilities. The user guidance features will help the user perform the image processing and quantitative steps needed, by aiding in their choice of what operation to perform, explaining the results of the operation, and suggesting which operation to perform next. MiM will present the data analysis tools to the user in a language and format that a biologist understands. This guidance feature is unique and not present in any other live cell image analysis package. The other innovative feature, quantitative analysis, will emphasize the features extracted from the data rather than simply analyzing raw image data. This reduction of data from images to feature metrics will provide users with new insights into their data. The result of this Phase I SBIR work will be a specification for the MiM software package. Furthermore, one aspect of the software will be designed and implemented with user guidance as a prototype to demonstrate project feasibility. Consultants will evaluate this prototype to validate its effectiveness and utility as an easy to use and productive live cell imaging analysis tool.