Flowers Blooming on the Crown: A Novel Halo Tag Protein-Conjugated Ratiometric Fluorescent Sensor for Dual Two-dimensional Real-time Detection of Lysosomal and Mitochondrial Polarity

The concurrent detection across multiple organelles can effectively distinguish diverse cellular activities. However, the development of a cellular monitor that can concurrently target multiple organelles continues to present a substantial challenge. Consequently, for the first time, we engineer a dual-fluorescence emission and multi-organelle targeting (OT) protein-conjugated fluorescent sensor, X-TAG-Halo Tag-OT, enabling precise cellular analysis. The ratiometric fluorescence response of X-TAG to polarity variations within an intact range follows the Boltzmann function with high sensitivity. Notably, the X-TAG-Halo Tag-Cox8a construct can concurrently target both lysosomes and mitochondria, producing a visually striking ratiometric fluorescence image of lysosomal “flowers” embedded within a mitochondrial “crown”. By employing the “flowers on a crown” pattern for dual-organelle analysis, it is observed that autophagy facilitates an increase in lysosomal polarity (Δf _Lyso = 0.2836 to 0.2944), while decreasing mitochondrial polarity (Δf _Mito = 0.2697 to 0.2584). In contrast, both lysosomal and mitochondrial polarity are diminished during apoptosis (Δf _{Lyso, Mito} = 0.2835 to 0.2734, 0.2688 to 0.2665). Furthermore, the crucial organelles involved in the aforementioned cellular activities are also identified with high sensitivity by X-TAG-Halo Tag-Cox8a. X-TAG employs nitrogen condensation to enhance n→π* and π→π* transitions for ratiometric fluorescence emission and also endows it with the capability to bind to OT-fusion Halo Tag proteins via a nitrogen-containing Halo Tag ligand, thereby facilitating interactions with multiple organelles. This versality suggests significant potential for developing dual multidimensional fluorescent sensors, providing new insights into the mechanisms of various diseases.