DrSTAR: Tracking real-time nanometer axial changes.

Protein interactions with the plasma membrane mediate processes critical for cell viability such as migration and endocytosis, yet our understanding of how recruitment of key proteins correlates with their ability to sense or induce energetically unfavorable plasma membrane shapes remains limited. Simultaneous two-wavelength axial ratiometry (STAR) microscopy provides millisecond time resolution and nanometer axial resolution of protein dynamics at the basal plasma membrane. However, STAR microscopy requires extensive and time-consuming quantitative data processing to access axial (Δz) information. Therefore, addressing questions about the influence of biological and biophysical factors on the interaction between the plasma membrane and protein of interest remains challenging. Here, we overcome the limitations in STAR data processing and present dynamic reference STAR (DrSTAR): a user-friendly, automated, open-source MATLAB-based package. DrSTAR enables processing multiple experimental conditions and biological replicates, employs a novel local background referencing algorithm, and accelerates processing time to facilitate broad adaptation of STAR for studying nanometer axial changes in protein distribution.