Effects of cell state and staining on femtosecond laser nanosurgery

Femtosecond laser nanosurgery enables precise manipulation of subcellular elements to study regeneration. However, currently it is not frequently employed—probably because of its unknown consequences on the whole cell level. To better understand the associated biological response of the cell, especially in the context of different cell states and cell staining, we manipulated C2C12 myoblasts and myotubes, which were either unstained (nicotinamide adenine dinucleotide signal) or stained with MitoTracker Red. Both signals overlap well and stain similar areas in untreated cells. We chose 3 different cutting lengths and performed surgery in the cytosol along the major cell axis. The cuts resealed within several minutes independent of the cutting length. We analyzed cell area, perimeter, major and minor axis on long term. We observed significant changes in the cell area and perimeter, dependent on the staining and more pronounced in differentiated myotubes. We conclude, that laser parameters must be chosen carefully, depending on the staining of the cell, its (differentiation) state, and the extent of the cut region, such that unwanted cell responses can be avoided. Laser manipulation of C2C12 myotubes with small ablation (0.8 μm) and large ablation (3.0 μm). While small damages recover, larger damages lead to elimination from the syncytium. Scale bar: 20 μm.