Flavie Braud

Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal

We present a two-photon microendoscope capable of in vivo label-free deep-tissue high-resolution fast imaging through a very long optical fiber. First, an advanced light-pulse spectro-temporal shaping device optimally precompensates for linear and nonlinear distortions occurring during propagation within the endoscopic fiber. This enables the delivery of sub-40-fs duration infrared excitation pulses at the output of 5 meters of fiber. Second, the endoscopic fiber is a custom-made double-clad polarization-maintaining photonic crystal fiber specifically designed to optimize the imaging resolution and the intrinsic luminescence backward collection. Third, a miniaturized fiber-scanner of 2.2 mm outer diameter allows simultaneous second harmonic generation (SHG) and two-photon excited autofluorescence (TPEF) imaging at 8 frames per second. This microendoscope’s transverse and axial resolutions amount respectively to 0.8 μm and 12 μm, with a field-of-view as large as 450 μm. This microendoscope’s unprecedented capabilities are validated during label-free imaging, ex vivo on various fixed human tissue samples, and in vivo on an anesthetized mouse kidney demonstrating an imaging penetration depth greater than 300 μm below the surface of the organ. The results reported in this manuscript confirm that nonlinear microendoscopy can become a valuable clinical tool for real-time in situ assessment of pathological states.

Emission of multiple dispersive waves from a single Raman-shifting soliton in an axially-varying optical fiber

We provide the experimental demonstration of the generation of multiple dispersive waves from a single soliton propagating in the vicinity of the first zero-dispersion wavelength of an axially-varying optical fiber. The fiber is designed such that the Raman-shifting soliton successively hits three times the longitudinally evolving zero-dispersion wavelength, which results in the emission of three distinct dispersive waves at different fiber lengths. These results illustrate how suitably controlled axially-varying fibers allow to tailor the soliton dynamics in a very accurate way.

Solitonization of a dispersive wave

We report the observation of a nonlinear propagation scenario in which a dispersive wave is transformed into a fundamental soliton in an axially varying optical fiber. The dispersive wave is initially emitted in the normal dispersion region and the fiber properties change longitudinally so that the dispersion becomes anomalous at the dispersive wave wavelength, which allows it to be transformed into a soliton. The solitonic nature of the field is demonstrated by solving the direct Zakharov-Shabat scattering problem. Experimental characterization performed in spectral and temporal domains show evidence of the solitonization process in an axially varying photonic crystal fiber.

Longitudinal soliton tunneling in optical fiber

We report the observation of the longitudinal soliton tunneling effect in axially varying optical fibers. A fundamental soliton, initially propagating in the anomalous dispersion region of a fiber, can pass through a normal dispersion barrier without being substantially affected. We perform experimental studies by means of spectral and temporal characterizations that show the evidence of the longitudinal soliton tunneling process. Our results are well supported by numerical simulations using the generalized nonlinear Schrödinger equation.

Development of a versatile multiphoton microendoscope for in vivo deep-tissue label-free biomedical imaging

We report the optimization of a miniaturized multiphoton endomicroscope enabling high resolution label-free in vivo imaging of deep cellular and tissular intrinsic constituents at the output of a very long and flexible custom-designed optical fiber.

Label-free in vivo in situ diagnostic imaging by cellular metabolism quantification with a flexible multiphoton endomicroscope (Conference Presentation)

Multiphoton microscopy is a cutting edge imaging modality leading to increasing advances in biology and also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there have been several developments of fiber-based multiphoton microendoscopes. The application for those probes is now limited by few major restrictions, such as the difficulty to collect autofluorescence signals from tissues and cells theses being inherently weak (e.g. the ones from intracellular NADH or FAD metabolites). This limitation reduces the usefulness of microendoscopy in general, effectively restraining it to morphological imaging modality requiring staining of the tissues. Our aim is to go beyond this limitation, showing for the first time label-free cellular metabolism monitoring, in vivo in situ in real time. The experimental setup is an upgrade of a recently published one (Ducourthial et.al, Scientific Reports, 2016) where femtosecond pulse fiber delivery is further optimized thank’s to a new transmissive-GRISM-based pulse stretcher permitting high energy throughput and wide bandwidth. This device allows fast sequential operation with two different excitation wavelengths for efficient two-photon excited NADH and FAD autofluorescence endoscopic detection (i.e. 860 nm for FAD and 760 nm for NADH), enabling cellular optical redox ratio quantification at 8 frames/s. The obtained results on cell models in vitro and also on animal models in vivo (e.g. neurons of a living mouse) prove that we accurately assess the level of NADH and FAD at subcellular resolution through a 3-meters-long fiber with our miniaturized probe (O.D. =2.2 mm).

Toward two-photon excited fluorescence lifetime endomicroscopy (Conference Presentation)

Fluorescence lifetime imaging microscopy (FLIM) represents a powerful tool for biological studies. Endoscopic FLIM applied to the intracellular native biomarker NADH and FAD represents a promising mean for in vivo in situ malignant tissue diagnosis in the medical field. Else, 2-photon-excited fluorescence (2PEF) provides increased 3D resolution and imaging depth. But very few demonstrations about 2PEF lifetime measurement through a fiber have been reported and none about endoscopic 2P-FLIM through a practical fiber length (< 3m). Our group has recently demonstrated the possibility to efficiently deliver through a very long optical fiber the short and intense excitation pulses required for 2P-FLIM. Our goal is now to check that collecting fluorescence through the same endoscopic fiber does not deteriorate the lifetime measurement. Relying on the basis previously published in case of 1PEF by P. French and co-workers (J. Biophotonics, 2015), we have experimentally quantitatively evaluated the influence on the lifetime measurement of the fiber chromatic and intermodal dispersions. The main result is that the fiber contribution to the system impulse response function, even in the case of a 3-meter long double-clad optical fiber, does not hinder the separation between free and bound NADH states using FLIM. Related calibrations and measurements will be detailed. Ongoing experiments about the development of a 2P-FLIM endomicroscope on the basis of an previously reported 2P-endomicroscope (Ducourthial et al., Sc. Reports, 2015), used under various configurations (i.e. point measurement in the center of the 2P-endomicroscope image, averaged lifetime, binned endoscopic 2P-FLIM image), will be also presented.

Topographic Optical Fibers : New Perspectives in Nonlinear Guided Optics

We investigate theoretically and experimentally basic nonlinear effects such as soliton propagation or modulation instability in what we called topographic optical fibers, i.e. fibers which parameters are longitudinally modulated.

Topographic Optical Fibers: A New Degree of Freedom in Nonlinear Optics

We investigate theoretically and experimentally basic nonlinear effects such as soliton propagation or modulation instability in what we called topographic optical fibers. We show that in these fibers which parameters are longitudinally modulated over a scale of a few meters, new dynamics are observed. As a consequence it adds a new degree of freedom in nonlinear optics and allows to experimentally explore original phenomena.