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

Abstract

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 the in vivo environment. We developed an evaluation method combining longitudinal imaging analysis in vivo with simultaneous manipulation on single cell level. An abdominal imaging window was implanted in vivo in 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 imaging in vivo on 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 μJ 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 microscopy in 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.

Fig 1

Schematic depiction of the planned procedure: (A) Mouse with implanted abdominal imaging window (AIW) and liver directly under the AIW. (B) MPM-image of the healthy liver (green: Rhodamine 6G, blue: FITC-Dextran) after application of a targeted femtosecond laser pulse. (C) MPM-image of a follow-up analysis of the manipulated region.

Fig 2

Comparison of different excitation wavelengths for imaging: [left] MPM images of the same region at the same depth in the liver at different imaging wavelengths (green = Rhodamine 6G, blue = FITC-Dextran; scale bar 50 μm). [right] Diagrams considering penetration depths of different imaging wavelengths into hepatic tissue stained with FITC-Dextran (blue, graphs A and B) and Rhodamine 6G (green, graphs C and D).

Fig 3

Microscopical images of the targeted ablation of ZMTH3 cells: (A) Expressing a mitochondrial-tagged red fluorescent protein (green) and Calcein AM (blue) using varying pulse sequences. (B) Dependence of the ablation efficiency on the pulse number and pulse energy based on 10 targeted cells with a sigmoidal fit function. With 10 pulses, all cells were ablated for pulse energies of more than 0.2 μJ. (C) Dependence of the ablated area on the pulse energy.

Fig 4

Single cell ablation ex vivo: (A, B) Visualisation of cell damage after application of the amplifier laser system. (C) Evaluation of the targeting efficiency.

Fig 5

In vivo ablation of single cells in the liver: (A) Images acquired before ablation and at various time points after ablation showed fast regeneration of the targeted tissue. (B) The ablated area first expanded and later receded again.