Combined multiphoton imaging and cell surgery by femtosecond laser pulses

Multiphoton microscopy is a very promising method for 3D imaging of living cells. The fluorochromes are solely excited at the laser focus by multiphoton absorption using near-infrared femtosecond laser pulses. The arising fluorescence serves for a pixel-to-pixel imaging with a resolution in the submicron range. At higher laser powers, the multiphoton absorption creates a micro plasma which induces an outwardly propagating shock wave. The rapidly expanding cavitation bubble causes disruption of the material, with hardly any interaction with the surrounding tissue as the optical breakdown proceeds faster than the thermal conduction. This combination offers the possibility of simultaneous manipulation and analysis of living cells or cell organelles. Manipulation is achieved using laser pulses with energy of a few nanojoules while imaging is done at less than 1 nJ. The obtained resolution allows the precise cutting of single cell organelles without compromising the cells' viability. Thus, the implementation is excellently suited for cell surgery. We conducted ablation of different subcellular structures at different pulse energies within living cells while studying cell viability.