Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions, and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly deteriorates resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets and therefore allows their locations to be pin-pointed with precision that achieves the Cramer-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures from mitochondrial networks and nuclear pores in mammalian cells, to amyloid β plaques and dendrites in brain tissues, and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extra-cellular targets deep inside tissues.