1. Deep Stromal Dissection for Endothelial Keratoplasty Obtained with a Femtosecond Laser and a Microkeratome with Different Head Advancement Speeds. A Scanning Electron Microscopy Study Deep Stromal Dissection for Endothelial Keratoplasty Obtained with a Femtosecond Laser and a Microkeratome with Different Head Advancement Speeds. A Scanning Electron Microscopy Study P. Scaroni, R. Leaci, D. Dallatana, A. Neri, L. Fontana°, C. Macaluso. Ophthalmology, University of Parma, Parma, Italy. °Eye Bank Institute of Emilia Romagna, Bologna, Italy. FINANCIAL DISCLOSURE FOR ALL AUTHORS :NONE

2. Purpose: To explore the surface quality of deep stromal dissections in bank eyes obtained with either a femtosecond laser or a microkeratome used with three different head advancement speeds.Purpose:

3. Methods: 12 sclerocorneal donor buttons, not suitable for human transplantation, mounted on an artificial chamber. 1000 KHz Femtosecond laser (Femto LDV, Ziemer, Switzerland) at 400µm depth : –FEMT group: 3 Corneas Microkeratome Carriazo Barraquer, Moria, France. Mounting a 300µm head. Different speeds to perform the dissection: –Microkeratome FAST 2-4 sec –Microkeratome MEDIUM 8-10 sec –Microkeratome SLOW 18-20 sec Methods: 12 sclerocorneal donor buttons, not suitable for human transplantation, mounted on an artificial chamber. 1000 KHz Femtosecond laser (Femto LDV, Ziemer, Switzerland) at 400µm depth : –FEMT group: 3 Corneas Microkeratome Carriazo Barraquer, Moria, France. Mounting a 300µm head. Different speeds to perform the dissection: –Microkeratome FAST 2-4 sec –Microkeratome MEDIUM 8-10 sec –Microkeratome SLOW 18-20 sec Following routine preparation, all endothelial buttons were analyzed with a scanning electron microscope (SEM 501, Philips, Germany) at 20X (allowing visualization of the whole corneal specimen) and 160X magnifications. Qualitative evaluation: Overall homogeneity of the cut at low (20x) magnification Overall homogeneity of the cut at low (20x) magnification Detection of surface irregularities of the central cornea at high (160x) magnification. A masked technician examined the rest of the corneal surface for areas showing any difference in surface quality when compared to the center. Detection of surface irregularities of the central cornea at high (160x) magnification. A masked technician examined the rest of the corneal surface for areas showing any difference in surface quality when compared to the center. As in the femtolaser group the technician found significant differences in areas quality, both best and worst areas were selected As in the femtolaser group the technician found significant differences in areas quality, both best and worst areas were selected Following routine preparation, all endothelial buttons were analyzed with a scanning electron microscope (SEM 501, Philips, Germany) at 20X (allowing visualization of the whole corneal specimen) and 160X magnifications. Qualitative evaluation: Overall homogeneity of the cut at low (20x) magnification Overall homogeneity of the cut at low (20x) magnification Detection of surface irregularities of the central cornea at high (160x) magnification. A masked technician examined the rest of the corneal surface for areas showing any difference in surface quality when compared to the center. Detection of surface irregularities of the central cornea at high (160x) magnification. A masked technician examined the rest of the corneal surface for areas showing any difference in surface quality when compared to the center. As in the femtolaser group the technician found significant differences in areas quality, both best and worst areas were selected As in the femtolaser group the technician found significant differences in areas quality, both best and worst areas were selected

4. 20X magnifications The stromal surface of the endothelial buttons of the FEMTOLASER group showed a generally excellent smoothness, but with some small rough areas (red arrow). FEMTOLASERFEMTOLASER

5. BEST QUALITY AREAS BEST QUALITY AREAS (160X magnifications) Best areas obtained with the femtolaser: the surface is regular and the stromal fibers have been cut and not ripped off FEMTOLASERFEMTOLASER

6. WORSE QUALITY AREAS (160X magnifications) WORSE QUALITY AREAS (160X magnifications) In the images above it’is clear that many stromal fibers have been torn and not cut and the quality of the surfaces is poor FEMTOLASERFEMTOLASER

7. FAST GROUP performing the cut to obtain the lamellas in 2-4 sec (160X magnifications) FAST GROUP performing the cut to obtain the lamellas in 2-4 sec (160X magnifications) Differently from the FEMTOLASER group, all microkeratome dissected endothelial buttons failed to show significant variations in smoothness across the cut surface. MICROKERATOMEMICROKERATOME

8. MEDIUM GROUP performing the cut to obtain the lamellas in 8-10 sec (160X magnifications) MEDIUM GROUP performing the cut to obtain the lamellas in 8-10 sec (160X magnifications) MICROKERATOMEMICROKERATOME

9. SLOW GROUP performing the cut to obtain the lamellas in 18-20 sec (160X magnifications) SLOW GROUP performing the cut to obtain the lamellas in 18-20 sec (160X magnifications) While the surface of the MEDIUM and of the SLOW groups were regular, the FAST group showed a rougher surface. The smoothness obtained in the best areas of the FEMTOLASER group was unmatched by the surfaces obtained in any of the microkeratome groups. While the surface of the MEDIUM and of the SLOW groups were regular, the FAST group showed a rougher surface. The smoothness obtained in the best areas of the FEMTOLASER group was unmatched by the surfaces obtained in any of the microkeratome groups. MICROKERATOMEMICROKERATOME

10. MicrokeratomeMicrokeratome FemtolaserFemtolaser Stroma after Descemet stripping The quality of the surfaces obtained with microkeratome and femtolaser can be compared. And both can be compared with the smoothness of the receiving bed for endothelial transplantation, i.e. the stroma after Descemet stripping

11. Conclusions: 1.MICROKERATOME dissection of endothelial corneal buttons resulted in consistent regular cut surfaces, but it is advisable to complete the head movement rather slowly, in at least 8-10 seconds. Conclusions: 1.MICROKERATOME dissection of endothelial corneal buttons resulted in consistent regular cut surfaces, but it is advisable to complete the head movement rather slowly, in at least 8-10 seconds.

12. 2.FEMTOSECOND laser technology has the potential for generating endothelial corneal buttons with extremely smooth surfaces, even better than those that can be obtained with a microkeratome. Nevertheless, the deep stromal location of the cut may limit the overall regularity of the dissected surface. It is conceivable that the suboptimal optical properties of rejected eye bank tissues could have heavily contributed to this problem, limiting proper laser focusing in the deep stroma. Thank you Thank you…