3D Single-Molecule Super-Resolution Fluorescence Microscopy with the Corkscrew Point Spread Function

Abstract

The Moerner Laboratory has been engineering the point spread function (PSF) of a wide-field fluorescence microscope in order to obtain simultaneous X, Y, and Z spatial information from single molecules over a large Z-range. The new PSFs (e.g., the double-helix, tetrapod, and corkscrew PSFs) change their shapes or positions as the single-molecule emitter moves along the optical (Z) axis—thus conveying rich information about the Z position of the molecule. These approaches are relevant both for tracking individual biomolecules at low concentration as well as reconstructing super-resolution images by single-molecule localization microscopy. The corkscrew PSF is special because it behaves like a single Gaussian spot of light that moves along a U-shaped path as the emitter moves along the optical axis. Hence, the corkscrew PSF occupies less space on the camera as compared to our other 3D PSFs and allows us to image a sample more quickly at a higher density of localizations per camera frame.In this work, we use the corkscrew PSF to image a rectilinear array of nanoholes filled with fluorescent molecules to calibrate the three-dimensional response of our microscope. This involves mapping the 3D object space of molecules within a sample to the 2D image space on our camera. By sampling the microscope's response throughout the field of view, we account for field-dependent aberrations that would otherwise cause errors in the position measurements of single molecules. Recent progress in imaging biomolecules with the corkscrew PSF will be summarized.