Superresolution 3D Microscopy of the Nuclear Pore

Abstract

Nuclear pore complexes (NPCs) regulate macromolecular exchange across the nuclear envelope. While 2D super-resolution microscopy approaches have been used in most single molecule nuclear transport studies to date, high resolution spatial and temporal data in 3D are needed to decipher the functional and dynamic properties of the permeability barrier. We report here a microscope system capable of determining the 3D position of individual NPCs with high spatial precision. NPCs in permeabilized HeLa cells are localized via a blinking dye on a nanobody bound to GFP tagged Nup107. A deformable mirror (adaptive optics system) is used to generate an aberration-free point spread function (psf) and then astigmatism is added to encode z-information. A focus-lock system externally integrated into the microscope is capable of stabilizing the coverslip to ≤ 3nm in z during image acquisition. While an aberration-free psf gives a localization precision of < 10 nm in x and y at a photon level of 1000, an astigmatic psf yields z-information at the expense of x and y precision. Due to the inverse relationship between z precision and x and y precision, astigmatism is tuned based on the need of the experiment. With 3000 photons per 10 ms frame, we obtain 8-18 nm precision in x, y and z.