Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging with a Single-Wavelength Excitation in Living Cells

Abnormal pH values in the organelles are closely associated with inappropriate cellular functions and many diseases. Monitoring subcellular pH values and their variations is significant in biological processes occurring in living cells and tissues. Herein, we develop a series of ratiometric fluorescence nanoprobes for quantification and imaging of pH values with a single-wavelength excitation in cytoplasm, lysosomes, and mitochondria. The nanoprobes consist of mesoporous silica nanoparticles assembled with aminofluorescein as the recognition unit for pH measurement and ethidium bromide as reference fluorophore. Further conjugation of subcellular targeting moiety enables the nanoprobes to specifically target lysosome and mitochondria. Confocal fluorescence imaging demonstrated that the nanoprobes could effectively monitor the pH fluctuations from 5.0 to 8.3 in living cells by ratio imaging with 488 nm excitation. Subcellular pH determination and imaging in lysosome and mitochondria could also be achieved in different conditions. The current method can offer a general strategy to determine subcellular analytes and investigate the interactions in biological samples.