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

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.