Abstract

Intravital two-photon microscopy enables deep-tissue imaging of subcellular structures in live animals, but its original spatial resolution and image quality are limited by scattering, motion, and low signal-to-noise ratios. To address these challenges, we used a combination of tissue stabilization, denoising methods, and motion correction, together with resolution enhancement algorithms, including enhanced Super-Resolution Radial Fluctuations (eSRRF) and deconvolution, to acquire high-fidelity time-lapse images of internal organs. We applied this imaging pipeline to image genetically labeled mitochondria in vivo, in Dendra2 mice. Our results demonstrate that the eSRRF-combined method, compared to other evaluated algorithms, significantly shows improved spatial resolution and mitochondrial structure visualization, while each method exhibiting distinct strengths in terms of noise tolerance, edge preservation, and computational efficiency. These findings provide a practical framework for selecting enhancement strategies in intravital imaging studies targeting dynamic subcellular processes.