Abstract

Abstract In this study, we introduced a novel dual‐laser multi‐color imaging system. Integrated with a multi‐channel filter wheel, this system compared three spectral decontamination algorithms (nonnegative matrix factorization [NMF], RCAN, and PICASSO) showcasing its efficacy in achieving four‐color imaging with only two laser sources. Combined with a reliable image reconstruction algorithm, the spatial resolution of four channels super‐resolution four‐color images reached 130, 125, 133, and 132 nm, respectively. Lipid droplets, mitochondria, lysosomes, and nuclei from the mouse hepatocytes (AML12), human neuroblastoma cells (SH‐SY5Y), mouse hippocampal neuronal cells (HT‐22), and immortalized murine bone marrow‐derived macrophages were imaged. At the same time, the chromatin condensation, nuclear contraction, DNA fragmentation, apoptotic body formation, as well as the fusion of Mito and Lyso involved in mitochondrial autophagy were observed in HT‐22 and SH‐SY5Y cells suffering oxidative stress. Our multi‐color SIM imaging system establishes a powerful platform for dynamic organelle studies and other high‐resolution investigations in live cells.