Vortex Interference Enables Optimal 3D Interferometric Nanoscopy

Super-resolution imaging methods that combine interferometric axial (z) analysis with single-molecule localization microscopy (iSMLM) have achieved ultrahigh 3D precision and contributed to the elucidation of important biological ultrastructures. However, their dependence on imaging multiple phase-shifted output channels necessitates complex instrumentation and operation. To solve this problem, we develop an interferometric superresolution microscope capable of optimal direct axial nanoscopy, termed VILM (Vortex Interference Localization Microscopy). Using a pair of vortex phase plates with opposite orientation for each dual-opposed objective lens, the detection point-spread functions (PSFs) adopt a bilobed profile whose rotation encodes the axial position. Thus, direct 3D single-molecule coordinate determination can be achieved with a single output image. By reducing the number of output channels to as few as one and utilizing a simple 50∶50 beam splitter, the imaging system is significantly streamlined, while the optimal iSMLM imaging performance is retained, with axial precision 2 times better than the lateral. The capability of VILM is demonstrated by resolving the architecture of microtubules and probing the organization of tyrosine-phosphorylated signaling proteins in integrin-based cell adhesions.