Corneal lamellar cutting with a blade or femtosecond laser (FSL) is commonly used during refractive surgery and corneal grafts. Surface roughness of the cutting plane influences postoperative visual acuity but is difficult to assess reliably. For the first time, we compared chromatic confocal microscopy (CCM) with scanning electron microscopy, atomic force microscopy (AFM) and focus-variation microscopy (FVM) to characterize surfaces of variable roughness after FSL cutting. The small area allowed by AFM hinders conclusive roughness analysis, especially with irregular cuts. FVM does not always differentiate between smooth and rough surfaces. Finally, CCM allows analysis of large surfaces and differentiates between surface states.

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International audienceAbstractPurpose To compare femtosecond laser endothelial graft precut from epithelial and endothelial side, on corneas stored in a new corneal bioreactorMethods Human corneas unsuitable for transplantation for serological reasons and procured by the Auvergne-Loire eye bank (Saint Etienne, France) were used after informed consent of the relatives, as authorized by French bioethics laws. Femtosecond laser lamellar cuts were realized on corneas stored in a new corneal bioreactor, patented by our laboratory, designed to maintain a constant endothelial pressure and to permit an epithelial aplanation without opening the device. Lamellar cuts of several thickness were realized with corneal epithelium or corneal endothelium side up, but always with epithelial aplanation. OCT images (Casia SS1000, Tome) were realized in the bioreactor before and after each cut. Endothelial viability was determined by using a triple Hoechst/Ethidium/Calcein labeling (HEC, IOVS Pipparelli 2011) coupled with image analysis of the whole endothelial surface immediately after the cut. Surface roughness was assessed by scanning electron microscopyResults This study demonstrates the possibility to realise femtoseconde laser endothelial graft precut directly in a dedicated storage device. First results of endothelial viability in function of lamellar graft thickness and lamellar cut side will be presented as well as surface roughnessConclusion Capacity to realize femtosecond laser endothelial graft precut directly in a storage device could improve the quality and security of delivered lamellar graft

International audiencePurpose NanoFemtoTransfection (NFT) is an innovative and promising technique of gene transfer (Nature nanotechnology 2011). It consists in permeabilizing cell membrane with nanoparticles (NP) of black carbon activated by Ti-Sapphire femtosecond laser (FsL) in order to safely introduce molecule of interest. Initially described for cell suspension, we adapted during last year NFT to human corneal endothelial cells (EC) Aim: to further optimize NFT of EC in vitroMethods The model of NFT of calcein (a small molecule) in an EC line (B4G12, DMSZ, Germany) was used to optimize exposition times, fluence and Ti-Saphir FsL beam movements in order to determine the best efficiency/toxicity ratio. Calcein transfer was observed with fluorescent microscopy (IX81, Olympus) and quantified by flow cytometry (FACSCalibur, BD, CA). Viability of EC was assessed by propidium iodide staining. Staining of actine with phalloidin and immunostaining of ZO-1 was performed to study cell alteration caused by NFTResults With a static laser beam increasing FsL fluence and exposure time increased transfection efficiency but also EC mortality. The use of mobile FsL beam (with a XY micrometric stage, cheaper than a scanner), permitted an irradiation of the entire area of EC culture resulting in a noticeably increased efficiency that reached 38% of transfected EC and a limited toxicity (3%). Calcein positive EC presented the same phalloidin and ZO-1 patterns as control ECConclusion Optimized NFT parameters are currently being tested in our laboratory to transfect plasmids. Grants: Fondation pour la Recherche Médicale, Fondation des Aveugles de Franc

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