BPS2026 - An integrated platform for microsecond-precision control of single-molecule imaging microscopes

Our laboratory aims to advance single-molecule FRET (smFRET) imaging through the development of custom instruments and software solutions to examine time-dependent changes in molecular composition and conformation to gain insights into biological function and regulation (Juette, M. et al., Nat. Methods 2016). Here, we report custom image-acquisition software (FLASH) that interfaces with platform components—including microfluidic pumps and high-speed CMOS cameras—to enable robust, reproducible microscope control, including advanced illumination schemes such as alternating laser excitation (ALEX). The requirement for high-speed performance has been met by relocating the triggering logic from software to a dedicated microsync device that operates independently of the host computer to ensure reliable signal timing. This microsync device is built on the Arduino Due platform, based on a 32-bit ARM microcontroller with a real-time event scheduling system, provides a cost-effective solution for controlling multiple peripheral components with microsecond precision. FLASH combined with our custom analysis software pipeline, SPARTAN, offers a comprehensive and accessible solution for precise and robust single-molecule investigations that promises to expand the breadth and scope of scientific inquiry on this frontier of biophysics research.