All fiber nonlinear microscopy at 1550 nm using a double-clad fiber coupler

Nonlinear microscopy has already shown its impact in biological research, namely in the fields of neurobiology, immunology, cancer research and embryology. Typically, these microscopes operate under free space propagation, using a dichroic mirror to separate the nonlinear signals from the excitation laser. While powerful such implementations are difficult to translate from the laboratory to a clinical setting where the environment is less controlled. Therefore, we propose an alignment-free all-fiber nonlinear microscopy system at 1550 nm based on double-clad fibers (DCF). As sectioning is performed through nonlinear effects, nonlinear microscopy does not require a detection pinhole, and. the DCF inner cladding can be used for efficient collection of nonlinear signals. The built system allows for multiplexing second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF), collected from the inner cladding; and reflectance confocal microscopy (RCM), detected from the core acting as the confocal pinhole. Finally, an asymmetric double-clad fiber coupler (DCFC) is used to address efficiently both DCF channels. This all-fiber system is more compact and less sensitive to alignment, but requires carefully managing the transmission of the femtosecond pulse in the fiber. This is addressed using dispersion compensation fiber, pulse compression and solitonic propagation. Additionally, with a source centered at 1550 nm, we benefit from reduced sample scattering thus increasing the depth of field in comparison with systems operating at 800 nm. Overall we believe that the developed system could be transferred in clinics to enable in-vivo and in-situ imaging of human patient.