Multimodal Nanoplasmonic and Fluorescence Imaging for Simultaneous Monitoring of Single-Cell Secretory and Intracellular Dynamics.

Abstract

Current imaging technologies are limited in their capability to simultaneously capture intracellular and extracellular dynamics in a spatially and temporally resolved manner. This study presents a multimodal imaging system that integrates nanoplasmonic sensing with multichannel fluorescence imaging to concomitantly analyze intracellular and extracellular processes in space and time at the single-cell level. Utilizing a highly sensitive gold nanohole array biosensor, the system provides label-free and real-time monitoring of extracellular secretion, while implementing nanoplasmonic-compatible multichannel fluorescence microscopy enables to visualize the interconnected intracellular activities. Combined with deep-learning-assisted image processing, this integrated approach allows multiparametric and simultaneous study of various cellular constituents in hundreds of individual cells with subcellular spatial and minute-level temporal resolution over extended periods of up to 20 h. The system's utility is demonstrated by characterizing a range of secreted biomolecules and fluorescence toolkits across three distinct applications: visualization of secretory behaviors along with subcellular organelles and metabolic processes, concurrent monitoring of protein expression and secretion, and assessment of cell cycle phases alongside their corresponding secretory profiles. By offering comprehensive insights, the multifunctional approach is expected to enhance holistic readouts of biological systems, facilitating new discoveries in both fundamental and translational sciences.