Negatively-Chirped Laser Enables Nonlinear Excitation and Nanoprocessing with sub-20-fs Pulses

摘要

It has long been considered that the advantages emerging from employing chirp pre-compensation in nonlinear microscopy were overweighed by the complexity of prism- or grating-based compressors. These concerns were refuted with the advent of dispersive-mirrors-based compressors that are compact, user-friendly and sufficiently accurate to support sub-20-fs pulse delivery. Recent advances in the design of dispersive multilayer mirrors resulted in improved bandwidth (covering now as much as half of the gain bandwidth of Ti: Sapphire) and increased dispersion per bounce (one reflection off a state-of-the-art dispersive mirror pre-compensates the dispersion corresponding to > 10mm of glass). The compressor built with these mirrors is sufficiently compact to be integrated in the housing of a sub-12-fs Ti: Sapphire oscillator. A complete scanning nonlinear microscope (FemtOgene, JenLab GmbH) equipped with highly-dispersive, large-NA objectives (Zeiss EC Plan-Neofluoar 40x/1.3, Plan-Neofluar 63x/1,25 Oil) was directly seeded with this negatively chirped laser. The pulse duration was measured at the focus of the objectives by inserting a scanning autocorrelator in the beam path between the laser and the microscope and recording the second order interferometric autocorrelation traces with the detector integrated in the microscope. Pulse durations < 20fs were measured with both objectives. The system has been applied for two-photon imaging, transfection and optical manipulation of stem cells. Here we report on the successful transfection of human stem cells by transient optoporation of the cell membrane with a low mean power of < 7 mW and a short us beam dwell time. Optically transfected cells were able to reproduce. The daughter cell expressed also green fluorescent proteins (GFP) indicating the successful modification of the cellular DNA.