Simultaneous Real-Time Three-Dimensional Localization and FRET Measurement of Two Distinct Particles

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most occur in three dimensions, simultaneous measurement three-dimensional is necessary. However, precise localization real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles dimensions with an accuracy (2.35σ) tens nanometers exposure 100 ms measure their real-time using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, tracked vesicles containing t-SNAREs or v-SNARE observed FRET simultaneously during single-vesicle fusion time, revealing nanoscale motion single vesicle fusion. Thus, study demonstrates our microscope can provide detailed information about locations, motion, processes.