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Negatively-chirped laser enables nonlinear excitation and nanoprocessing with sub-20-fs pulses

: Uchugonova, A.; Müller, J.; Bückle, R.; Tempea, G.; Isemann, A.; Stingl, A.; König, K.


Periasamy, A. ; Society of Photo-Optical Instrumentation Engineers -SPIE-, Bellingham/Wash.:
Multiphoton microscopy in the biomedical sciences VIII : 20 - 22 January 2008, San Jose, California, USA
Bellingham, WA: SPIE, 2008 (SPIE Proceedings Series 6860)
ISBN: 978-0-8194-7035-5
ISSN: 0277-786X
ISSN: 1605-7422
Art. 686015
Conference "Multiphoton Microscopy in the Biomedical Sciences" <8, 2008, San Jose/Calif.>
Conference Paper
Fraunhofer IBMT ()

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 40×/1.3, Pla n-Neofluar 63×/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.