Intravital Imaging of Liver Function using multiphoton microscopy: The liver is a favorable organ for intravital microscopy. After anesthesia and laporatomy, the liver can be gently pulled from the abdomen and placed in the view of a microscope lens. Confocal and multiphoton fluorescence microscopy increases effective resolution compared to widefield microscopy. Red/intrared excitation used in multiphoton microscopy allows deeper tissue penetration with less photodamage and more efficient collection of fluorescence in a scattering environment. Intravital confocal/multiphoton micrographs rival in quality and resolution the corresponding images collected from cell culture monolayers. Figure 1 shows a rat liver labelled with rhodamine 123 and PI fluorescence and imaged with 820-nm light from a Chameleon Ultra Ti-Sapphire pulsed laser (Coherent Inc., Santa Clara, CA) on a Zeiss LSM 510 NLO inverted laser scanning confocal microscope under diferent treatments. The intravital multiphoton microscopy images show bright fluorescence of rhodamine 123 in hepatocytes whose punctuate pattern denotes polarization of individual mitochondria. Cytosolic and nuclear areas had little fluorescence.
Figure 1. Intravital images of rat liver with multiphoton excitation. For these experiments rat liver transplantations were performed. At 4 h postoperatively, liver grafts were visualized by intravital multiphoton microscopy. Shown are representative overlay images of green rhodamine 123 and red PI fluorescence collected from livers after sham operation (A) and liver grafts after vehicle (B), minocycline (C) and NIM811 (D) treatment. Punctate staining of rhodamine 123 denoted polarization of individual mitochondria, whereas dim diffuse cellular staining indicated mitochondrial depolarization (white arrows). PI nuclear staining signified loss of cell viability (yellow arrows). Bar is 30 µm. (From Hepatology 2008; 47:236-46)