Tracking the environmentally responsive intracellular fate and autophagic interactions of mesoporous silica nanoparticles via gap spectral detection.

Understanding the complex intracellular fate and autophagic mechanisms of environmentally responsive biomaterials is essential for advancing targeted disease therapies. However, monitoring these dynamic interactions in living cells is frequently hindered by the spectral crosstalk of traditional fluorescence microscopy and the requirement for specialized multichannel GaAsP/PMT detector arrays with high digitization depth. Herein, we develop a gap spectral detection method that captures spatial and spectral information across the visible range using a general confocal microscope. By implementing defined interval gaps, this approach eliminates excitation laser interference, enabling high-fidelity, six-color live-cell 2D and 3D imaging. We utilized this method to systematically track the intracellular routing of mesoporous silica nanoparticles (MSNs), which utilize the acidic pH of lysosomes as an environmental trigger for the proton sponge effect. Our analysis revealed that MSNs induced cellular autophagy and dynamically altered the organelle interactome. Notably, following the proton sponge effect, MSNs escaped from lysosomes after 6 h and subsequently increased their co-localization with the Golgi apparatus and endoplasmic reticulum. These findings demonstrate that our gap spectral imaging technique provides an accessible and ultra-multiplexed analytical tool for evaluating the intracellular fate of responsive biomaterials, thereby facilitating the rational design of nanomedicines.