Live visualization of dynamic autophagy processes in LC3 reporter mice using enhanced two-photon microscopy.

We developed a computationally enhanced intravital imaging framework for high-resolution analysis of macroautophagy/autophagy dynamics in vivo. By integrating two-photon microscopy with an optimized tissue stabilization method, motion artifacts were minimized, enabling real-time imaging of LC3-positive structures in the liver and skeletal muscle under physiological stress conditions, including starvation, ischemia, and ethanol exposure. Image quality was further improved using a trained denoising model and enhanced Super-Resolution Radial Fluctuations (eSRRF), enabling nanoscale visualization of autophagy-related compartments. To differentiate increased autophagosome formation from impaired degradation, autophagic flux was assessed via chloroquine-mediated lysosomal inhibition, enabling functional interpretation beyond static structural measurements. Together, this pipeline overcomes major technical limitations of intravital imaging and provides a robust framework for integrated structural and functional investigation of autophagy and related intracellular processes in vivo.