Multiphoton intravital microscopy in small animals of long-term mitochondrial dynamics based on super‐resolution radial fluctuations
基于超分辨率径向波动的多光子活体显微术观察小动物长期线粒体动力学
超解像放射状変動に基づく長期的ミトコンドリアダイナミクスにおける小動物の多重光子生体内顕微鏡法
장기 미토콘드리아 동역학을 위한 초해상도 방사 변동 기반 소동물의 다중광자 생체 내 현미경
Microscopía intravital multiphotónica en pequeños animales para la dinámica mitocondrial a largo plazo basada en fluctuaciones radiales de superresolución
Microscopie multiphoton intravital chez de petits animaux pour l'observation à long terme de la dynamique mitochondriale basée sur des fluctuations radiales de super-résolution
Мультифотонная интравитальная микроскопия долгосрочной динамики митохондрий в мелких животных на основе супер-разрешающих радиальных флуктуаций
Saeed Bohlooli Darian ¹, Jeongmin Oh ², Bjorn Paulson ², Minju Cho ¹, Globinna Kim ¹, Eunyoung Tak ¹ ², Inki Kim ³, Chan-Gi Pack ¹ ² Jung-Man Namgoong ⁴, In-Jeoung Baek ¹ ², Jun Ki Kim ¹ ²
¹ Department of Biomedical Engineering, Brain Korea 21 Project, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
² Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
³ Department of Pharmacology, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
⁴ Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo. To accomplish this, a conventional two-photon microscope was equipped with a 3D-printed holders, which stabilize the tissue surface within the focal plane of immersion objectives.
Further computational image stabilization and noise reduction were applied, followed by super-resolution radial fluctuations (SRRF) analysis, doubling image resolution, and enhancing signal-to-noise ratios for in vivo subcellular process investigation. Stabilization of < 1 µm was obtained by suction, and < 25 nm were achieved by subsequent algorithmic image stabilization. A Mito-Dendra2 mouse model, expressing green fluorescent protein (GFP) in mitochondria, demonstrated the potential of long-term intravital subcellular imaging.
In vivo mitochondrial fission and fusion, mitochondrial status migration, and the effects of alcohol consumption (modeled as an alcoholic liver disease) and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally. Suction-based stabilization in two-photon intravital imaging, coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.
