Superresolved femtosecond laser nanosurgery of cells

Multiphoton fluorescence microscopy based on femtosecond laser scanning is a powerful technique for three dimensional optical sectioning in life sciences. The method is based on the simultaneous absorption of two or three photons in the focal volume of a high NA microscope objective. The same setup is suited for nanodissection of living cells and subcellular structures. A very small lateral extent of the modified focal volume is required to minimize collateral damage in the vicinity of the laser focus and to improve long-term cell viability. By using high NA microscope objectives and laser pulse energies close to the ablation threshold, the lateral extent of the modified material is limited to less than 1 μm. This diffraction limited resolution can be further improved by techniques generally referred to as superresolution. These are achieved by controlling the phase of the laser beam with a diffractive filter placed at the exit pupil of an optical system. We integrated this technique into nanosurgery of cells, which we demonstrate here for the first time.