Long-Term Non-Bleaching Nanoscale Imaging by Plasmonic Nanoscope

Abstract

Long-term observation of live cells and activities, including cell dynamics and cellular responses, is a crucial technology in the field of cell studies such as cancer diagnostics and disease therapy. The challenges of long-term imaging fall under four categories: selection of cell type amenable to long-term imaging; precise control of the cellular environment under in-vitro system; imaging system and probe design to overcome photobleaching, phototoxicity, and to achieve high resolution; efficient data extraction and post data analysis process. Here we present a long-term non-bleaching nanoscale imaging method by plasmonic nanoscope. Through plasmonic molecular marker (gold nanorods) labeling and phase intensity separation of nanoprobes, we achieved non-bleaching, nanoscale imaging of biological systems, such as actin networks. While the standard fluorophore-based method limiting the imaging windows to minutes (< 1 hour), using our optimized imaging system, we were able to continuously observe long-term biological dynamics in the window of hours to days as well as achieving sub-10 nm resolution. The innovative nanoscale imaging system utilizing a plasmonic nanoscope has been successfully developed to address challenges encountered in long-term bio-imaging and demonstrated by visualization and quantitative analysis of actin dynamics during the disassembly process by actin binding protein (cofilin). Furthermore, we demonstrated live-cell imaging using nanoscale plasmonic nanoscope which allowed for the exploration of the orientation distribution of extracellular beta-actin network within a cell-division cycle.