Dissertation
Tools for real-time study of bioorthogonal conversions in the living system
Traditional biochemical methods for studying organelles require cell disruption, preventing the real-time observation of dynamic intracellular processes.
- Author
- Y. Wang
- Date
- 25 March 2026
- Links
- Thesis in Leiden Repository
To address this, this research developed a generalizable live-cell bioorthogonal imaging strategy to visualize subcellular nutrient fluxes.We synthesized a panel of organelle-targeted fluorescent probes utilizing the inverse electron-demand Diels–Alder (IEDDA) reaction. These probes consist of a carboxyfluorescein-tetrazine scaffold conjugated to specific organelle-targeting motifs.The tetrazine moiety acts as both a bioorthogonal reactant and an efficient fluorescence quencher. Upon the influx of a trans-cyclooctene (TCO)-tagged nutrient, the IEDDA reaction rapidly consumes the tetrazine, eliminating the quenching effect and triggering a robust fluorescence turn-on. Live-cell microscopy validated that the mitochondrial- and nuclear-targeted probes exhibited highly specific subcellular localization and rapid fluorogenic activation. This spatiotemporal imaging platform enables the direct observation of compartmentalized nutrient uptake in living cells, providing a critical tool for elucidating organellar metabolism in physiology and disease.