Event-triggered MINFLUX microscopy: smart microscopy to catch and follow rare events

Abstract MINFLUX microscopy is a powerful microscopy method allowing for the characterization of molecular organization and dynamics with single nanometer spatial resolution and sub-hundred microseconds temporal resolution. However, acquisition times often span minutes to hours as a single fluorophore is measured at a time. Applying it to study cellular processes in living cells therefore requires careful consideration of where and when to apply MINFLUX data acquisition, a consideration where manual control limits its potential applications. Here, to overcome the limitations of acquisition speed, acquisition initiation, and data throughput, we present a smart microscopy method that uses confocal imaging as a monitoring method, runs real-time image analysis, and only applies MINFLUX data acquisition exactly where and when deemed necessary based on the analysis outcome. The method, event-triggered MINFLUX, is controlled through a custom-written and open source Python widget that automatically controls a commercial MINFLUX microscope. We apply this method to investigate molecular membrane dynamics and organization during three different cellular events: two-dimensional lipid dynamics at caveolae; three-dimensional membrane topography during dynamin-mediated endocytosis; and three-dimensional membrane fluidity and topography during budding site formation of HIV-1 proteins. Thanks to rapid event detection and minimal regions of interest the method provides data that would be unfeasible or impossible to acquire through manual control of the microscope. Competing Interest Statement The authors have declared no competing interest.