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

Abstract

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.

Cellular macromolecules-tethered DNA walking indexing to explore nanoenvironments of chromatin modifications

“…In principle, they can directly estimate the proximity of biomoleculs by fluorescence colocalization without biochemical recognition reactions such as DNA proximity. However, this resolution can vary considerably in optically complex samples, and the z -axis resolution is still limited 53 . Furthermore, registration errors from sample drift especially in multichannel imaging often lead to incorrect colocalization analysis 54 .…”

Super-resolution Microscopy with Single Molecules in Biology and Beyond–Essentials, Current Trends, and Future Challenges

“…Methods have been described to retrieve the PSF in situ and should be considered. 160 In any case, disagreement between theoretical and experimental PSFs cause poor fitting results in e.g. PSF localization, which deteriorates the quality of the reconstruction.…”

Chemical Analysis of Single Cells and Organelles

“…With similar intentions to achieve high localization precision, Xu et al presented a different approach called in situ point spread function retrieval (INSPR) SMLM. 73 The system showed the ability to construct an in situ 3D response of single emitters directly from single-molecule blinking data sets. The authors demonstrated the use of this method in imaging various organelles in mammalian cells (e.g., mitochondrial networks and nuclear pores) as well as in tissue (e.g., amyloid-β plaques and dendrites in brain tissues) at a depth of <20 μm, achieving 7–12 nm lateral and 21–45 nm axial precision in localization.…”