1. Strategy of intratissue optical nanosurgery including flap-free non-invasive intrastromal ablation and multiphoton-mediated flap generation.

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1 Appendix 1. Strategy of intratissue optical nanosurgery including flap-free non-invasive intrastromal ablation and multiphoton-mediated flap generation. Fig.1 Schematic illustrating the flap-free non-invasive intrastromal ablation. Left: The two green quadrilaterals imply the laser-epithelial marking and the upper 4 squares indicate the intrastromal ablation located in the cornea depth of 120µm; Right: Showing the 4 lasermediated marks on the epithelial surface serving as relocalization of intrastromal surgery. The intrastromal ablation in the deep cornea located between the two rows could not be defined in visibility. Bar: 500µm. 2. Memory of the corneal lesions for the long-termed follow-up observations of the wound-healing process

Left: The two green quadrilaterals imply the laser-epithelial marking and the upper 4 squares indicate the intrastromal ablation located in the cornea depth of 120µm; Right: Showing the 4

2 Fig.2 Memorial skill called Clock-Memory for relocalization of intrastromal nanosurgery and ablation for the long-termed follow-up observations. The stromal lesion and epithelial marks can not be identified any more some days postoperation due to the wound healing. The limbus between cornea and sclera remains constant; therefore it is the most ideal orientation to relocalize the treated spot. Clock-Memory was designed to relocalize the treated spot in the cornea for intrastromal ablation and follow-up studies on the corneal flap

The limbus between cornea and sclera remains constant; therefore it is the most ideal orientation to relocalize the treated spot.

3 List of Figures and Diagrams Fig.1 Diagrams illustrating radial keratotomy (RK) P6 Fig.2 Diagrams illustrating PRK P6 Fig.3 Procedures of LASEK..P8 Fig.4 Procedures of Epi-LASIK.P9 Fig.4_1 Histological sections of Epi-LASIK.P9 Fig.5 Procedures of LASIK..P10 Fig.5_1 Photograph of microkeratome..p10 Fig.5_2 Flap-related complications P11 Fig.6 Procedures of Femto-LASIK.P12 Fig.6_1 Smooth flap bed generated with femtosecond high pulse energy lasers..p13 Fig.7 All-Femto-LASIK.P14 Fig.8_1 Imitated diagram of histological cross-section of rabbit cornea..p16 Fig.8_2 Three-dimensional illustration of corneal components.p16 Fig.8_3 TEM of corneal stroma..p18 Fig.9 Diagram depicting noncentrosymmetric collagen structure.p19 Fig.10 Nerve fibres in cornea displayed by gold chloride and optical imaging...p22 Fig.11 Schematic of intrastromal surgery induced by nanojoule ultrashort laser pulses..p25 Fig.12 Photographic illustration of animal laser eye surgery.p35 Fig.12_1 Modified photograph of laser operations on rabbit.p37 Fig.12_2 Schematic of experimental setup for multiphoton microscopy and intrastromal surgery.p38 Fig.12_3 Schematic illustrating intrastromal ablation..p38 Fig.13 Threshold power for intratissue surgery and optimized power for multiphoton imaging...p39 Fig.14 An indispensable instrument in this work: Zeiss slit lamp 120..P42 Fig.14_1 Epithelial marks taken by slit lamp P42 Fig.15 Comparison of average central cornea thickness of rabbits, human and porcine.p44 Fig.16_1 Nonlinear optical images of epithelial squamous cells and basal cells...p45 Fig.16_2 Nonlinear optical images of keratocytes and endothelial cells.p45 Fig.17_1 Optical nonlinear tomography of epithelium.p46 Fig.17_2 Optical nonlinear tomography of stromal keratocytes P47 Fig.17_3 In-vivo collagen lamellar nonlinear optical sectioning P48 Fig.18 Nonlinear optical imaging of stromal tissue with different wavelengths...p49 Fig.19 Real-time nonlinear optical observations of intrastromal femtosecond laser nanosurgery..p50 Fig.20 SHG image and λ-scanning of emission wavelength.p51 Fig.20_1 SHG observations on intrastromal nanosurgery 24 hours postoperation...p51 Fig.20_2 Reflexion imaging of intrastromal nanosurgery 24 hours postoperation.p52 Fig.21_1 On-line optical observations on intrastromal ablation.p53 Fig.21_2 Histological cross-sections displaying the immediate ablation outcomes...p

6_1 Smooth flap bed generated with femtosecond high pulse energy lasers..p13 Fig.7 All-Femto-LASIK.P14 Fig.8_1 Imitated diagram of histological cross-section of rabbit cornea..p16 Fig.

4 Fig.21_3 Histological observations on wound repair of intrastromal ablation at 4 th and 7 th days P55 Fig.21_4 Optical detecting on emergence of myofibroblasts.p56 Fig.22_1 Optical images during flap generation..p57 Fig.22_2 Photographs of corneal flaps taken immediately after the laser operation.p58 Fig.23 SEM of the lifted corneal flap..p58 Fig.24 Observations on wound repair at 1 st day..p59 Fig.24_1 Histological observations on the migrating inflammatory cells at 1 st day P59 Fig.25_1 Histological observations of a flap at 7 th day postoperation..p60 Fig.25_2 Flap at 16 th day.p61 Fig.25_3 Flap at 28 th day.p62 Fig.25_4 Flap at 66 th day.p63 Fig.25_5 Flap at 90 th day.p64 Fig.26 Photograph showing simultaneous generation of corneal flap and intrastromal lenticule P65 Fig.27 In-vivo cellular nonlinear optical tomography of myofibroblasts based on 2PF..P66 Fig.27_1 In-vivo optical imaging of the treated region 24 hours postoperation..p67 Fig.28 Histological observations on inflammatory cells and discrimination of eosinophils...p

.p59 Fig.24_1 Histological observations on the migrating inflammatory cells at 1 st day P59 Fig.25_1 Histological observations of a flap at 7 th day postoperation..p60 Fig.25_2 Flap at 16 th day.

5 Explanation of independence in writing this work Selbständigkeitserklärung hiermit erkläre ich, dass mir die Promotionsordnung der Medizinischen Fakultät der Friedrich-Schiller-Universität bekannt ist, ich die Dissertation selbst angefertigt habe und alle von mir benutzten Hilfsmittel, persönlichen Mitteilungen und Quellen in meiner Arbeit angegeben sind, mich folgende Personen bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskripts unterstützt haben: Professor Dr. med. habil. Karl-Jürgen Halbhuber und Professor Dr. rer. nat. habil. Karsten König, die Hilfe eines Promotionsberaters nicht in Anspruch genommen wurde und dass Dritte weder unmittelbar noch mittelbar geldwerte Leistungen von mir für Arbeiten erhalten haben, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen, dass ich die Dissertation noch nicht als Prüfungsarbeit für eine staatliche oder andere wissenschaftliche Prüfung eingereicht habe und dass ich die gleiche, eine in wesentlichen Teilen ähnliche oder eine andere Abhandlung nicht bei einer anderen Hochschule als Dissertation eingereicht habe. Jena, Juli 2006 Bao-Gui Wang

und Auswertung des Materials sowie bei der Herstellung des Manuskripts unterstützt haben: Professor Dr. med. habil.

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