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Femtosecond fiber lasers for nonlinear microscopy. — During the last couple of years, we have been developing a pulsed Yb-fiber oscillator and amplifier system with a variable repetition rate in the 1 to 36 MHz range in collaboration with our industrial partner, R&D Ultrafast Laser Ltd. First we used this laser in our novel, hand-held 3D nonlinear microscope system (FiberScope) for applications in dermatology and nanomedicine. The laser delivers ~0.4 ps pulses at around ~1030 nm, which makes it an excellent candidate for two-photon imaging of GCaMP in neuroscience as well. GCaMP is a genetically encoded calcium indicator, and its main advantage is that it can be genetically specified for studies in living organisms. In the absence of calcium, this protein is in a poorly fluorescent state, but after Ca2+-binding, it is brightly fluorescent. In Figure 1, we show 3D reconstructions of 2PE auto-fluorescence images of brain slices comprising GCaMP in its certain neurons in the “poorly fluorescent state”. The high quality of the images results from the fact that our fiber laser was operated at a relatively low, ~6 MHz repetition rate.

Figure 1. 2PE auto-fluorescence images of GCaMP expressing neurons excited by our mode-locked Yb-fiber laser oscillator - amplifier system operating at ~6 MHz repetition rate (central wavelength ~1030 nm, pulse duration: ~0.4 ps, average power on sample: ~14 mW). Left: 3D reconstruction of z-stack images with dimensions of 600 µm x 600 µm. Right:  3D reconstruction of z-stack images with dimensions of 50 µm x 50 µm.

Coherent anti-Stokes Raman spectroscopy (CARS) microscopy. — Last year, CARS imaging of the CH2 bonds of lipids was successfully used to perform a quantitative analysis of the myelin loss in a cuprizone model of sclerosis multiplex (SM) depending on the drug treatment. This year, we have aimed at label-free imaging of proteins as well as NO in living tissues, such as in murine skin or in the brain. To this end, we optimized our CARS imaging system for protein and NO detection, which required several modifications and improvement in our optics and the laser system. Note that the concentration (and hence the CARS signal) of NO is orders of magnitude smaller than that of the lipids in myelin, that is why NO imaging is a challenging task. For demonstrative purposes, we show two CARS images of murine skin recorded for “lipid” and “protein” settings (Fig. 2, left) and one CARS image of an artificial sample containing NO in a gaseous state (Fig. 2, right). In the latter experiment, NO was generated from an aqueous solvent of sodium nitroprusside (SNP) after exposing it to white light for a few tens of seconds before the imaging.

Figure 2. Left: CARS images of the stratum corneum of murine skin for “lipid” (green) and “protein” (blue) settings. Right: CARS image of NO distribution in a SNP solvent containing artificial tissue.

Currently, we are working on the development of more sophisticated, non-resonant background-free imaging techniques for 3D distribution detection of low-concentration molecules (e.g. NO), such as FM-CARS or our newly developed TD-CARS method.

Broadband beam steering mirrors in nonlinear microscopy. — Broadband dielectric beam steering mirrors are key components for nonlinear microscopes comprising tunable Ti:sapphire lasers. This year we characterized dispersion of a few mirror samples by spectral interferometry and showed how they affected our imaging quality. As an example, we show the measured group delay of a BB1-E03 mirror (product Thorlabs Inc.) for s- and p-polarized light (Fig. 3, up), and how it affects the pulse shape after four reflections at around a resonance wavelength (Fig. 3, down). In practice, one obtains considerably lower signal in a nonlinear microscope when the peak intensity of the optical pulses is reduced by such unexpected dispersive effects (see Fig. 3, down).

Figure 3. Up: Measured group-delay vs. wavelength functions of an ultra-broadband dielectric mirror (two reflection, AOI: 45 degrees, Thorlabs Inc. BB1-E03). Down: Calculated intensity vs. time functions of transform limited 130 fs pulses being reflected on the same dielectric mirrors for s-polarized light at 45 degrees of AOI at different central wavelenghts.