Micro- and nanorobots (MNRs) are small autonomous devices capable of performing complex tasks and have been demonstrated for a variety of non-invasive biomedical applications, such as tissue engineering, drug delivery or assisted fertilization. However, translating such approaches to an in vivo environment is critical. Current imaging techniques do not allow localization and tracking of single or few micromotors at high spatiotemporal resolution in deep tissue. This thesis addresses some of these limitations, by exploring the use of two optical-based techniques (IR and photoacoustic imaging (PAI)) and a combination of both US and PAI. First, we employ an IR imaging setup to visualize mobile reflective micromotors under scattering phantoms and ex vivo mouse skull tissues, without using any labels. The reflective micromotor reflects more than tenfold the light intensity of a simple particle. However, the achieved penetration depth was ca. 100 μm (when using ex vivo tissues), limiting this technique to superficial biomedical applications. In this regard, PAI plays a role that combines the advantages of US such as penetration depth and real-time imaging with the molecular specificity of optics. For the first time, in this thesis, this method is evaluated for dynamic process monitoring, in particular for tracking single micromotor in real-time below ~1 cm deep phantom and ex vivo tissue. However, the precise function control of MNRs in living organisms, demand the combination of both anatomical and functional imaging methods. Therefore, in the end, we report the use of a hybrid US and PA system for the real-time tracking of magnetically driven micromotors (single and swarms) in phantoms, ex vivo, and in vivo (in mice bladder and uterus), envisioning their application for targeted drug-delivery. This achievement is of great importance and opens the possibilities to employ medical micromotors in a living organism and perform a medical task while being externally controlled and monitored.:ABSTRACT 1 1 INTRODUCTION 5 1.1 Motivation 5 1.2 Background 7 1.2.1 Microrobotics 7 1.2.2 Medical Imaging 9 1.3 Objectives and Structure of Thesis 12 2 FUNDAMENTALS 15 2.1 Optical Imaging 15 2.1.1. Reflection-based Imaging 17 2.1.2. Fluorescence-based Imaging 18 2.1.1 Light-Tissue Interaction 20 2.2 Photoacoustic Imaging 23 2.2.1 Theory 23 2.2.2 Implementation 25 2.3 Ultrasound Imaging 26 2.3.1 Theory 26 2.3.2 Implementation 28 3 MATERIALS AND METHODS 30 3.1 Fabrication of Magnetic Micropropellers 30 3.1.1 3D Laser Lithography of Polymeric Resin 30 3.1.2 Self-assembly of SiO2 Particles 31 3.1.3 Electron Beam Evaporation 32 3.1.4 Surface Functionalization 33 3.2 Fabrication of Phantom Tissue and Microfluidic Channels 34 3.2.1 Fabrication of PDMS-Glycerol Phantom 34 3.2.2 Fabrication of Agarose Phantom 35 3.2.3 Phantom based on Ex vivo Tissues (Chicken Breast and Mice Skull) 36 3.2.4 Microfluidic Channel Platform 37 3.3 Sample Characterization 38 3.3.1 Optical Microscopy 38 3.3.2 Scanning Electron Microscopy 38 3.4 Magnetic Actuation 39 3.4.1 Magnetic Force 39 3.4.2 Magnetic Torque 39 3.5 Ethic Statement for Mice Experiments 41 4 OPTICAL IMAGING OF MICROROBOTS 42 4.1 Concept of Reflective Micromotors 42 4.2 Fabrication of Reflective Micromotors 44 4.3 IR Imaging Actuation Setup 45 4.4 Actuation and Propulsion Performance below Phantom 47 4.5 Actuation and Propulsion Performance below Ex Vivo Skull Tissue 50 4.6 Actuation and Propulsion Performance in Blood 51 5 PHOTOACOUSTIC IMAGING OF MICROROBOTS 55 5.1 Absorbers for Deep Tissue Imaging 55 5.2 Absorber Micromotor Design and Fabrication 56 5.3 Photoacoustic Imaging Setup 58 5.4 Actuation Performance below Phantom Tissue 60 5.5 Actuation Performance below Ex Vivo Tissue 65 6 HYBRID ULTRASOUND AND PHOTOACOUSTIC IMAGING 67 6.1 Hybrid Ultrasound/Photoacoustic System 68 6.2 Fabrication and Characterization of Micromotors 69 6.3 Actuation and Propulsion Performance below Phantom 69 6.4 Actuation and Propulsion Performance below Ex Vivo Tissues 71 6.5 Actuation and Propulsion Performance in Mice 72 6.5.1 Swimming of Micromotors in Bladder 72 6.5.2 Actuation of Micromotors in Uterus 74 6.5.3 3D Multispectral Imaging 76 6.5.4 Towards Targeted Drug Delivery 77 7 SUMMARY AND PERSPECTIVES 80 7.1 Summary 80 7.2 Future Perspectives 83 7.2.1 Contrats Enhancing Labels 84 7.2.2 Novel Imaging Concepts 85 8 REFERENCES 88 9 APPENDIX 105 List of Figures 105 List of Tables 107 Abbreviations 108 List of Publications 109 Acknowledgements 110 Selbstständigkeitserklärung 111 Curriculum Vitae 112