Longitudinal imaging and femtosecond laser manipulation of the liver: How to generate and trace single-cell-resolved micro-damagein vivo

The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in thein vivoenvironment. We developed an evaluation method combining longitudinal imaging analysisin vivowith simultaneous manipulation on single cell level. An abdominal imaging window was implantedin vivoin Balb/C mice for recurrent imaging after implantation. Intravenous injection of Fluorescein Isothiocyanate (FITC)-Dextran was used for labelling of vessels and Rhodamine 6G for hepatocytes. Minimal cell injury was induced via ablation with a femtosecond laser system during simultaneous visualisation of targeted cells using multiphoton microscopy. High-resolution imagingin vivoon single cell level including re-localisation of ablated regions in follow-up measurements after 2-7 days was feasible. Targeted single cell manipulation using femtosecond laser pulses at peak intensities of 3-6.6 mu J led to enhancement of FITC-Dextran in the surrounding tissue. These reactions reached their maxima 5-15 minutes after ablation and were no longer detectable after 24 hours. The procedures were well tolerated by all animals. Multiphoton microscopyin vivo, combined with a femtosecond laser system for single cell manipulation provides a refined procedure for longitudinal evaluation of liver micro-regeneration in the same region of interest. Immediate reactions after cell ablation and tissue regeneration can be analysed.