Live-cell nanoscopy with spontaneous blinking of conventional green fluorescent proteins

Abstract Single-molecule localization microscopy with spontaneously blinking fluorescent tags holds a promise of simplified imaging setup for live-cell application. However, robust blinking has been reported for just a few fluorescent proteins. Here we report on a comparison of spontaneous blinking for three bright green fluorescent proteins, mAvicFP1, AausFP1, and mNeonGreen. mAvicFP1 outperforms other fluorescent proteins in this list in a wide range of camera exposure times and illumination intensities. We establish imaging conditions for live-cell nanoscopy and single-particle tracking with mAvicFP1.