Semi-automated Analysis Software for a Novel Biochemistry Assay

Some of the work done in the Baker lab at the University of Nevada, Reno has been directed towards the analysis of muscle tissue in a single molecule arrangement. The process is a novel high-throughput single molecule binding assay or SiMBA for short. This assay is performed by binding single-headed skeletal muscle myosin to a cover slip and looking at how the fluorescently labeled actin filaments bind to these myosin as imaged with a fluorescence microscope. The conditions are varied and the effects on actin-myosin binding kinetics are observed. In order to analyze the binding times and unbound times, a researcher must observe the interactions and manually collect the data. This particular method of data collection is tedious and time consuming, thereby making this portion of the experiment the "rate limiting" factor for producing results in a timely manner. As this can take many hours to analyze a one minute long movie, an automated or semi-automated solution would be beneficial to this assay. This paper presents the design and implementation of a semi-automated solution for identifying and tracking a variable number of objects that exhibit a multitude of behaviors, and extracting the specific behaviors of motion and stagnation as well as the duration of these behaviors.