Two-photon microscopy with a frequency-doubled fully fusion-spliced fiber laser at 1840 nm.

We introduce a fiber-based laser system providing 130 fs pulses with 3.5 nJ energy at 920 nm at a 43 MHz repetition rate and illustrate the potential of the source for two-photon excited fluorescence microscopy of living mouse brain. The laser source is based on frequency-doubling high-energy solitons generated and frequency-shifted to 1840 nm in large mode area fibers. This simple laser system could unleash the potential of two-photon microscopy techniques in the biology laboratory where green fluorescent proteins with two-photon absorption spectrum peaking around 920 nm are routinely used.

Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy.

In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements.