Signal back-collection Nonlinear optical endoscopy with Kagomé hollow-core fibers Vasyl MYTSKANIUK1, Alberto LOMBARDINI1, Siddharth SIVANKUTTY1, Jérôme WENGER1, Rémi HABERT2 Coralie FOURCADE-DUTIN2, Esben Ravn ANDRESEN1, Alexandre KUDLINSKI2, Hervé RIGNEAULT1 1 CNRS, Institut Fresnel UMR 7249, 13013 Marseille, France 2 Université Lille 1, IRCICA, Laboratoire PhLAM, 59655 Villeneuve d’Ascq, Franc Non-invasive in-vivo deep imaging of tissues and internal hollow organs has been a big challenge for the modern research community. Generally, in-vivo imaging itself is significantly affected by many factors related to excitation power and chemical labeling. We develop here non-linear fiber-based endoscope for high depth label free imaging with sub-micron resolution and >300 microns field of view. In order to reach this point, we have circumvented a few principle issues referring to femtosecond pulses delivery through an optical fiber and their non-linear interaction inside the fiber. A cutting-edge double clad Kagomé-lattice hollow core fiber has become the cardinal constituent for our endoscopic system. After propagating through such a fiber, femtosecond pulses preserve their temporal and spectral characteristics, since they travel basically in air. This also dramatically reduces all non-linear interactions of the ultrafast pulses inside of the fiber. The excitation light is delivered through the hollow core of the fiber, and the generated nonlinear signals are back-collected by the double cladding. Our prototype endoscope based on a four-quartered piezo-electric tube to scan the laser beam on the sample, is capable of performing nonlinear imaging like Second Harmonic Generation (SHG), two photon excited fluorescence (TPEF) and Coherent Anti-Stokes Raman Scattering (CARS) and shows great potential for in-vivo label-free imaging. Kagomé double clad fiber for NLO endoscopes (a), the fiber core (b) and transmission spectrum (c). Kagome double-clad fiber for nonlinear optical endoscopes We present here a novel hollow-core double-clad Kagomé fiber. This fiber is characterized by low Group Velocity Dispersion (GVD) over a broad transmission band, which allows for simple spectral tunability and multi-wavelength excitation. Propagation in an air core drastically reduces nonlinear interactions occurring along propagation in the fiber. The double clad in the fiber increases the numerical aperture of the fiber, enhancing the collection efficiency. Nonlinear Optical Endoscopy A nonlinear endoscope has to: Transmit excitation pulses Collect the generated signal Perform imaging on the sample Nonlinear Optical Endoscopy A nonlinear endoscope has to: Transmit excitation pulses Collect the generated signal Perform imaging on the sample Fiber-scanning endoscopy The excitation pulses are transmitted by the Kagomé fiber. The spiral pattern followed by the fiber is reproduced on the sample through the relay optics (achromatic lens assembly). The nonlinear signal is back-collected by the fiber and detected with a PMT. 40x NA=0.6 Kagome DC fiber OL PMT Piezoelectric tube DM Fs pulses Pulse duration Spectral tunability Signal back-collection Measured Literature [4] No temporal broadening is observed for 100-fs short pulses on the sample. The endoscope can perform imaging (or spectroscopy) over a broad spectral window. Coherent Raman imaging The double clad collects the signal emitted and diffused by the sample. The development of miniaturized CARS endoscopes has so far been limited by the unavoidable Four Wave Mixing (FWM) background generated in silica optical fibers. With the Kagome fiber, propagation through the hollow-core limits nonlinear interactions, allowing for high-contrast, high-resolution CARS endoscopy (shown here on 30micron latex beads and a human colon tissue ex-vivo, highlighting lipid structures). Photonic nanojet for higher image resolution The large mode field diameter of the Kagome is not ideal for a high-resolution, high-contrast nonlinear endoscope. We solve this problem with a dielectric micro-sphere attached to the distal end of the fiber and sealed by means of a CO2 laser. SEM image of the micro-bead after sealing to the fiber. TPEF and SHG imaging Coherent Raman imaging CARS images from polystyrene beads And fatty tissue from human colon biopsy TPEF images of a rat brain tissue ex-vivo (blue, bright spots are neurons) and SHG images of a rat tail tendon. The comparison of a CARS image generated by the vibrational CD-bonds (magenta) of deuterated glycerol dispersed into a sample of Stratum Corneum (SC), with an image originated from an autofluorescence signal yielded by the SC (blue). GPF-labeled neurons fluorescence emission at 584nm. SHG occurs when light is scattered by non-centrosymmetric materials. Collagen, naturally present in human tissues, is a strong SHG emitter.