Multiphoton-mediated corneal flap generation using the 80 MHz nanojoule femtosecond near-infrared laser.

PURPOSE: To evaluate whether corneal flaps can be generated by the 80 MHz near-infrared, intense nanojoule femtosecond laser based on multiphoton absorption. METHODS: A solid-state Ti:Sapphire femtosecond laser system was integrated in an inverted JenLab Femt-O-Cut laser scanning microscope. A diffraction-limited 40x objective was used to induce multiphoton ionization and plasma production. New Zealand albino rabbits and porcine eyes were used. Surgical outcomes were determined using frame and line scans with nanojoule pulses at a wavelength of 800 nm. RESULTS: Surgical performance was assessed by optical imaging, histology, and electron microscopy. No significant corneal turbidity was observed. Optical imaging and histological examinations detected virtually no perturbation in the surrounding tissue. Corneal flaps and stromal lenticules were generated. Wound repair of the unlifted flaps was observed up to 90 days postoperatively. CONCLUSIONS: Surgical results and follow-up studies confirm that this femtosecond laser at nanojoule pulse energy is able to generate corneal flaps precisely, without causing visible collateral damage to the surrounding tissue or overlying epithelium.

High-resolution two-photon excitation microscopy of ocular tissues in porcine eye.

BACKGROUND AND OBJECTIVE: Two-photon excitation laser scanning microscopy (TPM), based on nonlinear optical (NLO) response under high irradiance, is currently being extensively employed for diagnostic purposes in biomedical fields and becomes more and more an interesting imaging technique in the intact bulk tissue examination. In this study, this nonlinear-excitation imaging technique including two-photon-mediated autofluorescence (2PF) and second harmonic generation (SHG) was employed to investigate the microstructures in the whole-mount scleral, retinal, and corneal tissues of porcine eyes with intracellular spatial resolution and high signal-to-noise ratio. MATERIALS AND METHODS: Image acquisition was based on the intense 80 MHz femtosecond (fs) near-infrared (NIR) laser pulses, emitted from a mode-locked solid-state titanium:sapphire system. By integrating a high-numerical aperture diffraction-limited objective, the whole-mount ocular specimens could be viewed from the surface of eye globes further to a 200 microm depth. Under high light irradiance at the order of MW-GW/cm2, more than one photon was simultaneously absorbed by endogenous molecules in ocular tissues. RESULTS: The cellular and fibrous components of whole-mount scleral and corneal tissues were selectively displayed in situ by in-tandem detection of 2PF and SHG with high efficiency without the assistance of any exogenous dye. NLO images of fibroblasts and mature elastic fibers in sclerae as well as of the retina radial Müller glial cells, ganglion cells, bipolar cells, photoreceptors, and retina pigment epithelial (RPE) cells were acquired with subcellular spatial resolution. In particular, the microstructural topography of cells and extracellular components in the whole-mount ocular tissues was elucidated in situ. CONCLUSION AND OUTLOOK: The combination of the sensitive image acquisition technique allows to selectively studying of three-dimensional (3-D) architecture of cellular microstructures and extracellular matrix arrangement in situ at substantial depths in bulk tissues. The data obtained provided the primary knowledge for further studies of imaging entire eye globes based on two-photon excitation microscopy.

Multiphoton microscopy for monitoring intratissue femtosecond laser surgery effects.

BACKGROUND AND OBJECTIVE: Multiphoton microscopy/tomography has been used as a novel diagnostic method for corneal imaging with subcellular resolution. Here, we used this technique to monitor femtosecond laser intratissue surgery effects. MATERIALS AND METHODS: Multiphoton microscopy/tomography on rabbits based on intense 90 MHz femtosecond Ti: sapphire laser was realized at intensities of MW-GW/cm(2), whereas the surgical procedures were performed with the same system at a higher light intensity on the order of TW/cm(2). RESULTS: Multiphoton microscopy/tomography proved capable of determining the target of interest, and of visualizing and optically evaluating the in vivo intrastromal laser surgical outcomes with high efficiency. More interesting, using this technique, activated intrastromal keratocytes (myofibroblasts) were detected in vivo 24 hours after intrastromal surgery. CONCLUSION: Multiphoton microscopy/tomography is an efficient and convenient non-invasive imaging method which can be used not only to assess intrastromal microsurgical performance but also to perform in vivo follow-up observations on wound repair.

In-vivo intratissue ablation by nanojoule near-infrared femtosecond laser pulses.

Non-invasive intratissue ablation was performed in the cornea of living rabbits by using 80 MHz near-infrared intense nanojoule femtosecond laser pulses. The intratissue surgical effect was induced by multiphoton absorption at a wavelength of 800 nm and was ascertained by histological examination. Highly precise intratissue ablation was obtained with no detrimental effects to the overlying or underlying layers. Activated keratocytes in the laser-treated corneas were detected with two-photon imaging postoperatively. Intratissue femtosecond laser ablation thus has potential as a effective technique in refractive surgery for the treatment of visual disorders.

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers.

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 x 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman's layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.

Tissue engineering of aortic tissue: dire consequence of suboptimal elastic fiber synthesis in vivo.

OBJECTIVE: To study autologous tissue engineered blood vessels (TEBV) in the descending aorta of juvenile sheep. METHODS: Autologous vascular smooth muscle cells (vSMC) and endothelial cells were obtained from ovine carotid arteries. vSMC were seeded on bioresorbable scaffolds and dynamically cultured for 14 days. Following endothelialization an additional external ovine small intestinal submucosa wrapping was applied. Constructs were implanted in the descending aorta of juvenile sheep and removed after 1, 3, 6, 12 and 24 weeks for evaluation with histological, microscopical and biochemical techniques. RESULTS: Up to 3 months after implantation, grafts were fully patent, without any signs of dilatation, occlusion or intimal thickening. Scanning electron microscopy revealed a confluent luminal endothelial cell layer. In contrast, the 6 months graft displayed significant dilatation and partial thrombus formation. Histology displayed layered tissue formation resembling native aorta. Extracellular matrix (ECM) stains, immunostaining and Transmission Electron Microscopy (TEM) revealed alternating layers of vSMC and extracellular matrix consisting of collagen, elastin and glycosaminogycans. Compared to native aorta, the elastin content of the TE grafts was significantly reduced. CONCLUSION: In this study, we report for the first time, the implantation of a TEBV in the descending aorta in a large animal model. TEBV were fully functional for up to 3 months. At 6 months the graft remained functional but significantly dilated, most likely caused by an insufficient elastic fiber synthesis. Hence, future studies need to focus on the stimulation of elastin synthesis in TEBV.