Guillaume Ducourthial

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.

Development of a nonlinear fiber-optic spectrometer for human lung tissue exploration

Several major lung pathologies are characterized by early modifications of the extracellular matrix (ECM) fibrillar collagen and elastin network. We report here the development of a nonlinear fiber-optic spectrometer, compatible with an endoscopic use, primarily intended for the recording of second-harmonic generation (SHG) signal of collagen and two-photon excited fluorescence (2PEF) of both collagen and elastin. Fiber dispersion is accurately compensated by the use of a specific grism-pair stretcher, allowing laser pulse temporal width around 70 fs and excitation wavelength tunability from 790 to 900 nm. This spectrometer was used to investigate the excitation wavelength dependence (from 800 to 870 nm) of SHG and 2PEF spectra originating from ex vivo human lung tissue samples. The results were compared with spectral responses of collagen gel and elastin powder reference samples and also with data obtained using standard nonlinear microspectroscopy. The excitation-wavelength-tunable nonlinear fiber-optic spectrometer presented in this study allows performing nonlinear spectroscopy of human lung tissue ECM through the elastin 2PEF and the collagen SHG signals. This work opens the way to tunable excitation nonlinear endomicroscopy based on both distal scanning of a single optical fiber and proximal scanning of a fiber-optic bundle.

Label free multiphoton imaging of human pulmonary tissues through two-meter-long microstructured fiber and multicore image-guide

This work deals with label free multiphoton imaging of the human lung tissue extra-cellular matrix (ECM) through optical fibers. Two devices were developed, the first one using distal scanning associated to a double clad large mode area (LMA) air-silica microstructured fiber, the second one using proximal scanning of a miniature multicore image guide (30000 cores inside a 0.8 mm diameter). In both cases, the main issue has been efficient linear and nonlinear distortion pre-compensation of excitation pulses. By inserting before the delivery fiber a compact (10 cm × 10 cm footprint) grisms-based stretcher (a grating in close contact with a prism) made of readily available commercial components, we achieved as short as 35-femtosecond-duration pulses that were temporally compressed at the direct exit of a 2-meter-long fiber. Interestingly, this femtosecond pulse fiber delivery device is also wavelength tunable over more than 100 nm inside the Ti: Sapphire emission band. With the help of distal scan system, those unique features allowed us to record elastin (through two-photon fluorescence) and collagen (through second harmonic generation) fibered network images. These images were obtained ex-vivo with only 15 mW @ 80 MHz of IR radiation delivered to the alveoli or bronchus tissues. 3D imaging with 400-μm-penetration depth inside the tissue was possible working with a 2-meter-long LMA fiber. With the help of proximal scanning, the miniature image guide allowed us to perform endoscopic real time microimaging of the ECM ex vivo.

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.