Revealing the Phase Separation in ER Membranes of Living Cells and Tissues by In Situ NIR Ratiometric Imaging

Biomembranes in the endoplasmic reticulum (ER) play indispensable roles in various bioactivities, and therefore, visualizing the phase separation in ER membranes is crucial for the studies on the fundamental biology of the ER. However, near-infrared (NIR) ratiometric imaging of the phase behaviors of the ER in living cells with different statuses and in diverse tissues has not been investigated. Herein, we developed a polarity-responsive NIR fluorescent probe (DCA) for the visualization of the phase behavior in ER membranes. The probe displayed a large Stokes shift and was highly sensitive to polarity. By direct and native fluorescence imaging at room temperature, the ERo and ERd biomembranes in the ER could be clearly distinguished by dual NIR emission colors. Oxidative damage by H2O2 and homocystein (Hcy)-induced ER stress can efficiently induce the formation of large-scale ERo domains in ER membranes. Moreover, we have also revealed that different tissues exhibited diverse phase behaviors in the ER membranes. The ER membranes in cardiac and skeletal muscle tissues showed no evident phase separation, while large-scale ERo domains existed in the ER of liver tissues and formed at the ER membranes adjacent to lipid droplets (LDs) in white adipose tissues. We expect that the probe could serve as a powerful molecular tool to promote fundamental research studies on ER membranes and relative biomedical areas.