Two-Photon Light-Activatable Fluorophores for Organelle Imaging in Living Cells and Tissue-Level Imaging

Precise spatiotemporal control of bioactive molecule release within cells is crucial for understanding cellular processes. Two-photon photocaging provides a noninvasive powerful method for deep-tissue activation. This work introduces a series of organelle-targeted, two-photon photoactivatable fluorophores (PAFs) based on 4-nitrobiphenyl derivatives, designed for mitochondria, endoplasmic reticulum, and lysosome targeting. These PAFs exhibit high brightness and photostability under both one- and two-photon excitation. Organelle specificity was achieved by conjugating organelle-targeted motifs to PAFs, with low cytotoxicity, excellent membrane permeability, and specific organelle localization in different cells. <i>Ex vivo</i>, these PAFs enabled deep-tissue imaging (up to 100 μm) in mouse liver, kidney, and heart via two-photon excitation, while <i>in vivo</i>, they supported high-resolution, three-dimensional structural imaging of subcutaneous tumors with minimal background. The findings highlight the potential of these PAFs for high-resolution, organelle, and deep-tissue imaging, offering a versatile platform for studying subcellular dynamics with applications in biomedical research.