Opening long-time investigation window of living matter by nonbleaching phase intensity nanoscope: PINE

Abstract: Fundamental to all living organisms and living soft matter are emergent processes in which the reorganization of individual constituents at the nanoscale drives group-level movements and shape changes at the macroscale over time. However, light-induced degradation of fluorophores, photobleaching, is a significant problem in extended live cell imaging in life science, and excellent prior arts of STED, MINSTED, or MINFLUX intentionally apply photobleaching optics for super-resolution, yet lack extended live-cell imaging capability as fluorophores reach an irreversible photobleaching limit. Here, we report an innovative way of opening a long-time investigation window of living matter by a nonbleaching phase intensity nanoscope: PINE. We accomplish phase-intensity separation to obtain phase differences between electric field components, such that nanoprobes within a diffraction limited region are distinguished from one another by an integrated phase-intensity multilayer thin film (polyvinyl alcohol/liquid crystal). We obtain distributional patterns of precisely localized nanoprobes to overcome a physical limit to resolve sub-10 nm cellular architectures, and achieve the first dynamic imaging of nanoscopic reorganization over 250 hours using nonbleaching PINE. Since PINE allows a long-time investigation window, we discover nanoscopic rearrangements synchronized with the emergence of group-level movements and shape changes at the macroscale according to a set of interaction rules. We believe that PINE and its application presented here provide a mechanistic understanding of emergent dynamics important in cellular and soft matter reorganization, self-organization, and pattern formation.