Localizing and tracking of single molecules with a MINFLUX-microscope for various applications

Abstract

Recently MINFLUX microscopy was first published, a technique that localizes single fluorescent molecules by probing with a structured illumination excitation beam featuring a central intensity minimum. Single nanometer precision can be achieved by adaptation of the scan pattern geometry rather than solely relying on an increased number of acquired photons. The technique was transferred from highly specialized optical setups to a general purpose MINFLUX microscope featuring a common fluorescence microscope body. 2D imaging at 1 nm precision and 3D imaging in cells at 2.2 and 1.6 nm lateral and axial precision, respectively, was accomplished, attaining formerly published results. The imaging performance of the microscope was investigated. Live background correction facilitates imaging in cells in higher background situations. For the frst time, 3D MINFLUX tracking was experimentally demonstrated. Sinusoidal bead trajectories induced by a piezo stage and single fluorescent molecules diffusing in supported lipid bilayers were tracked in 3D at nanometer precision with 2.8 and 2.1 kHz time resolution, respectively. Super-fast 2D tracking of single fluorescent molecules diffusing for several seconds over a micrometer-range was performed. Tracks featuring localization precision of <13 nm at 4.5 kHz and <24 nm with up to 14 kHz time resolution were recorded using only 30 and 9 photons per localization, establishing a new spatio-temporal tracking benchmark for fluorescence microscopy. An analysis framework was developed to extract diffusion coeffcients from recorded tracking data. Simulations were employed to investigate the tracking performance of the microscope over a variety of diffusion coeffcients using different measurement parameters. Diffusion coeffcients above 1 µm²/s are within trackable range. Improving the scanner performance of the microscope will shift this limit to higher values.