Novel ultracompact multiphoton sub-20 femtosecond near infrared MHz laser scanning microscopes and conventional
250 fs laser microscopes have been used to perform high resolution multi-photon imaging of stem cell clusters as well as
targeted intracellular nanoprocessing and knock-out of living single stem cells within a 3D microenvironment. Also
lethal exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive.
Mean powers in the milliwatt range for 3D nanoprocessing and microwatt powers for two-photon imaging were found to
be sufficient. Ultracompact low power sub-20 fs laser systems may become interesting tools for nanobiotechnology such
as optical cleaning of stem cell clusters and optical transfection.
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 μs 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.
We demonstrated that the dispersion of scanning microscope optics (including a Zeiss 40x/1.2 Apochromat objective)
can be compensated by means of chirped mirrors over a bandwidth of 170 nm at 800 nm. The interferometric
autocorrelation trace recorded at the focus of the microscope objective with a two-photon diode indicated a pulse
duration of < 12 fs. The propagation time difference of the system can be minimized by proper choice of the
components, enabling sub-12-fs pulse delivery with a completely filled 40x/0.8 Zeiss Achroplan water immersion