We describe a dual, second harmonic generation (SHG) and third harmonic generation (THG) microscope, with the aim to obtain large-scale images of the cornea that can simultaneously resolve the micron-thick thin layers. We use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering and allows simultaneous SHG and THG imaging. We measure one-dimensional SHG and THG profiles across the entire thickness of pig cornea, detected in both the forward and backward directions. These profiles allow us to clearly distinguish all the porcine corneal layers (epithelium, stroma, Descemet's membrane and endothelium). From these profiles, longitudinal cross sectional images of the corneal layers are generated, providing large scale topographic information with high-spatial resolution. The ability to obtain both SHG and THG signals in epi-detection on fresh eyes gives promising hopes for in vivo applications.
"Moreover, another asset of nonlinear optics is the use of near-infrared laser light which propagates deeper in the tissue thanks to lower absorption and scattering . Studies have been reported in a variety of ex vivo and in vitro tissues (animal models  and human tissue samples [7–9]); multiphoton microendoscopy was also used in vivo for dermatologic applications . "
[Show abstract][Hide abstract] ABSTRACT: 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.
"The corneal epithelium (for the central cornea) was imaged through TPEF microscopy. This is composed of several layers of different cells, in which superficial, wing and basal cells are lined up from surface to bottom with increasing nucleus-to-cytoplasm ratio as depth increases, except basal cells that appear like small disks [33,34]. Figures 1(b-f)
[Show abstract][Hide abstract] ABSTRACT: The performance of femtosecond (fs) laser intrastromal ablation was evaluated with backscattering-mode adaptive-optics multiphoton microscopy in ex vivo chicken corneas. The pulse energy of the fs source used for ablation was set to generate two different ablation patterns within the corneal stroma at a certain depth. Intrastromal patterns were imaged with a custom adaptive-optics multiphoton microscope to determine the accuracy of the procedure and verify the outcomes. This study demonstrates the potential of using fs pulses as surgical and monitoring techniques to systematically investigate intratissue ablation. Further refinement of the experimental system by combining both functions into a single fs laser system would be the basis to establish new techniques capable of monitoring corneal surgery without labeling in real-time. Since the backscattering configuration has also been optimized, future in vivo implementations would also be of interest in clinical environments involving corneal ablation procedures.
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