@article{21547, abstract = {Flatbands have become a cornerstone of contemporary condensed-matter physics and photonics. In electronics, flatbands entail comparable energy bandwidth and Coulomb interaction, leading to correlated phenomena such as the fractional quantum Hall effect and recently those in magic-angle systems. In photonics, they enable properties including slow light1 and lasing2. Notably, flatbands support supercollimation—diffractionless wavepacket propagation—in both systems3,4. Despite these intense parallel efforts, flatbands have never been shown to affect the core interaction between free electrons and photons. Their interaction, pivotal for free-electron lasers5, microscopy and spectroscopy6,7, and particle accelerators8,9, is, in fact, limited by a dimensionality mismatch between localized electrons and extended photons. Here we reveal theoretically that photonic flatbands can overcome this mismatch and thus remarkably boost their interaction. We design flatband resonances in a silicon-on-insulator photonic crystal slab to control and enhance the associated free-electron radiation by tuning their trajectory and velocity. We observe signatures of flatband enhancement, recording a two-order increase from the conventional diffraction-enabled Smith–Purcell radiation. The enhancement enables polarization shaping of free-electron radiation and characterization of photonic bands through electron-beam measurements. Our results support the use of flatbands as test beds for strong light–electron interaction, particularly relevant for efficient and compact free-electron light sources and accelerators.}, author = {Yang, Yi and Roques-Carmes, Charles and Kooi, Steven E. and Tang, Haoning and Beroz, Justin and Mazur, Eric and Kaminer, Ido and Joannopoulos, John D. and Soljačić, Marin}, issn = {1476-4687}, journal = {Nature}, pages = {42--47}, publisher = {Springer Nature}, title = {{Photonic flatband resonances for free-electron radiation}}, doi = {10.1038/s41586-022-05387-5}, volume = {613}, year = {2023}, } @article{21585, abstract = {Efficient learning algorithms are implemented in a silicon photonic neural network chip}, author = {Roques-Carmes, Charles}, issn = {1095-9203}, journal = {Science}, number = {6643}, pages = {341--342}, publisher = {American Association for the Advancement of Science}, title = {{Learning photons go backward}}, doi = {10.1126/science.adh0724}, volume = {380}, year = {2023}, } @article{21553, abstract = {When impinging on optical structures or passing in their vicinity, free electrons can spontaneously emit electromagnetic radiation, a phenomenon generally known as cathodoluminescence. Free-electron radiation comes in many guises: Cherenkov, transition, and Smith–Purcell radiation, but also electron scintillation, commonly referred to as incoherent cathodoluminescence. While those effects have been at the heart of many fundamental discoveries and technological developments in high-energy physics in the past century, their recent demonstration in photonic and nanophotonic systems has attracted a great deal of attention. Those developments arose from predictions that exploit nanophotonics for novel radiation regimes, now becoming accessible thanks to advances in nanofabrication. In general, the proper design of nanophotonic structures can enable shaping, control, and enhancement of free-electron radiation, for any of the above-mentioned effects. Free-electron radiation in nanophotonics opens the way to promising applications, such as widely tunable integrated light sources from x-ray to THz frequencies, miniaturized particle accelerators, and highly sensitive high-energy particle detectors. Here, we review the emerging field of free-electron radiation in nanophotonics. We first present a general, unified framework to describe free-electron light–matter interaction in arbitrary nanophotonic systems. We then show how this framework sheds light on the physical underpinnings of many methods in the field used to control and enhance free-electron radiation. Namely, the framework points to the central role played by the photonic eigenmodes in controlling the output properties of free-electron radiation (e.g., frequency, directionality, and polarization). We then review experimental techniques to characterize free-electron radiation in scanning and transmission electron microscopes, which have emerged as the central platforms for experimental realization of the phenomena described in this review. We further discuss various experimental methods to control and extract spectral, angular, and polarization-resolved information on free-electron radiation. We conclude this review by outlining novel directions for this field, including ultrafast and quantum effects in free-electron radiation, tunable short-wavelength emitters in the ultraviolet and soft x-ray regimes, and free-electron radiation from topological states in photonic crystals.}, author = {Roques-Carmes, Charles and Kooi, Steven E. and Yang, Yi and Rivera, Nicholas and Keathley, Phillip D. and Joannopoulos, John D. and Johnson, Steven G. and Kaminer, Ido and Berggren, Karl K. and Soljačić, Marin}, issn = {1931-9401}, journal = {Applied Physics Reviews}, number = {1}, publisher = {AIP Publishing}, title = {{Free-electron–light interactions in nanophotonics}}, doi = {10.1063/5.0118096}, volume = {10}, year = {2023}, } @article{21586, abstract = {Quantum field theory suggests that electromagnetic fields naturally fluctuate, and these fluctuations can be harnessed as a source of perfect randomness. Many potential applications of randomness rely on controllable probability distributions. We show that vacuum-level bias fields injected into multistable optical systems enable a controllable source of quantum randomness, and we demonstrated this concept in an optical parametric oscillator (OPO). By injecting bias pulses with less than one photon on average, we controlled the probabilities of the two possible OPO output states. The potential of our approach for sensing sub–photon-level fields was demonstrated by reconstructing the temporal shape of fields below the single-photon level. Our results provide a platform to study quantum dynamics in nonlinear driven-dissipative systems and point toward applications in probabilistic computing and weak field sensing.}, author = {Roques-Carmes, Charles and Salamin, Yannick and Sloan, Jamison and Choi, Seou and Velez, Gustavo and Koskas, Ethan and Rivera, Nicholas and Kooi, Steven E. and Joannopoulos, John D. and Soljačić, Marin}, issn = {1095-9203}, journal = {Science}, number = {6654}, pages = {205--209}, publisher = {American Association for the Advancement of Science}, title = {{Biasing the quantum vacuum to control macroscopic probability distributions}}, doi = {10.1126/science.adh4920}, volume = {381}, year = {2023}, } @article{21511, abstract = {Converting ionizing radiation into visible light is essential in a wide range of fundamental and industrial applications, such as electromagnetic calorimeters in high-energy particle detectors, electron detectors, image intensifiers, and X-ray imaging. These different areas of technology all rely on scintillators or phosphors, i.e., materials that emit light upon bombardment by high-energy particles. In all cases, the emission is through spontaneous emission. The fundamental nature of spontaneous emission poses limitations on all these technologies, imposing an intrinsic trade-off between efficiency and resolution in all imaging applications: thicker phosphors are more efficient due to their greater stopping power, which however comes at the expense of image blurring due to light spread inside the thicker phosphors. Here, the concept of inverse-designed nanophotonic scintillators is proposed, which can overcome the trade-off between resolution and efficiency by reshaping the intrinsic spontaneous emission. To exemplify the concept, multilayer phosphor nanostructures are designed and these nanostructures are compared to state-of-the-art phosphor screens in image intensifiers, showing a threefold resolution enhancement simultaneous with a threefold efficiency enhancement. The enabling concept is applying the ubiquitous Purcell effect for the first time in a new context—for improving image resolution. Looking forward, this approach directly applies to a wide range of technologies, including X-ray imaging applications.}, author = {Shultzman, Avner and Segal, Ohad and Kurman, Yaniv and Roques-Carmes, Charles and Kaminer, Ido}, issn = {2195-1071}, journal = {Advanced Optical Materials}, number = {8}, publisher = {Wiley}, title = {{Enhanced imaging using inverse design of nanophotonic scintillators}}, doi = {10.1002/adom.202202318}, volume = {11}, year = {2023}, } @inbook{21739, abstract = {We revisit the derivation of the time-dependent Hartree–Fock equation for interacting fermions in a regime coupling a mean-field and a semiclassical scaling, contributing two comments to the result obtained in 2014 by Benedikter, Porta, and Schlein. First, the derivation holds in arbitrary space dimension. Second, by using an explicit formula for the unitary implementation of particle-hole transformations, we cast the proof in a form similar to the coherent state method of Rodnianski and Schlein for bosons.}, author = {Benedikter, Niels P and Desio, Davide}, booktitle = {Quantum Mathematics I}, editor = {Correggi, Michele and Falconi, Marco}, isbn = {9789819958931}, issn = {2281-5198}, pages = {319--333}, publisher = {Springer Nature}, title = {{Two Comments on the Derivation of the Time-Dependent Hartree–Fock Equation}}, doi = {10.1007/978-981-99-5894-8_13}, volume = {57}, year = {2023}, } @article{13049, abstract = {We propose a computational design approach for covering a surface with individually addressable RGB LEDs, effectively forming a low-resolution surface screen. To achieve a low-cost and scalable approach, we propose creating designs from flat PCB panels bent in-place along the surface of a 3D printed core. Working with standard rigid PCBs enables the use of established PCB manufacturing services, allowing the fabrication of designs with several hundred LEDs. Our approach optimizes the PCB geometry for folding, and then jointly optimizes the LED packing, circuit and routing, solving a challenging layout problem under strict manufacturing requirements. Unlike paper, PCBs cannot bend beyond a certain point without breaking. Therefore, we introduce parametric cut patterns acting as hinges, designed to allow bending while remaining compact. To tackle the joint optimization of placement, circuit and routing, we propose a specialized algorithm that splits the global problem into one sub-problem per triangle, which is then individually solved. Our technique generates PCB blueprints in a completely automated way. After being fabricated by a PCB manufacturing service, the boards are bent and glued by the user onto the 3D printed support. We demonstrate our technique on a range of physical models and virtual examples, creating intricate surface light patterns from hundreds of LEDs.}, author = {Freire, Marco and Bhargava, Manas and Schreck, Camille and Hugron, Pierre-Alexandre and Bickel, Bernd and Lefebvre, Sylvain}, issn = {1557-7368}, journal = {Transactions on Graphics}, keywords = {PCB design and layout, Mesh geometry models}, location = {Los Angeles, CA, United States}, number = {4}, publisher = {Association for Computing Machinery}, title = {{PCBend: Light up your 3D shapes with foldable circuit boards}}, doi = {10.1145/3592411}, volume = {42}, year = {2023}, } @inproceedings{21592, abstract = {We demonstrate improved X-ray imaging using nanophotonic scintillators. Our scintillators rely on Purcell enhancement for brighter and faster emission. Applying this concept in radiology and nuclear medicine could enable a significant reduction of X-ray dose.}, author = {Schuetz, Roman and Kurman, Yaniv and Lahav, Neta and Shultzman, Avner and Roques-Carmes, Charles and Lifshits, Alon and Zaken, Segev and Strassberg, Rotem and Be’er, Orr and Bekenstein, Yehonadav and Kaminer, Ido}, booktitle = {Conference on Lasers and Electro-Optics}, location = {San Jose, CA, United States}, publisher = {Optica Publishing Group}, title = {{Purcell-enhanced X-ray imaging in ultra-thin scintillators}}, doi = {10.1364/cleo_at.2023.aw3q.7}, year = {2023}, } @inproceedings{21630, abstract = {We demonstrate the generation of random bits with tunable probability distribution in an optical parametric oscillator. Bits are encoded into the phase statistics of the signal field, which are tuned by a small bias field.}, author = {Roques-Carmes, Charles and Salamin, Yannick and Sloan, Jamison and Velez, Gustavo and Koskas, Ethan and Choi, Seou and Rivera, Nicholas and Kooi, Steven E. and Joannopoulos, John and Soljačić, Marin}, booktitle = {Conference on Lasers and Electro-Optics}, location = {San Jose, CA, United States}, publisher = {Optica Publishing Group}, title = {{Tunable probabilities from the quantum vacuum}}, doi = {10.1364/cleo_si.2023.sth3f.3}, year = {2023}, } @inproceedings{21631, abstract = {We present inverse-designed multilayer nanophotonic scintillators with optimal efficiency, directionality, and point-spread function, for applications in x-ray imaging.}, author = {Shultzman, Avner and Segal, Ohad and Kurman, Yaniv and Roques-Carmes, Charles and Kaminer, Ido}, booktitle = {Conference on Lasers and Electro-Optics}, location = {San Jose, CA, United States}, publisher = {Optica Publishing Group}, title = {{Overcoming the imaging limits of high-energy particle detection via nanophotonic inverse-design}}, doi = {10.1364/cleo_si.2023.sth4g.8}, year = {2023}, } @inproceedings{21629, abstract = {We measure the second-order coherence function g(2) for X-ray-driven light emission (scintillation), observing that it is bunched (g(2) > 1), and can achieve extreme bunching values (g(2)~97) in perovskite nano-crystals.}, author = {Katznelson, Shaul and Tziperman, Offek and Bucher, Tomer and Abudi, Tom Lenkiewicz and Schuetz, Roman and Be'er, Orr and Levy, Shai and Bekenstein, Yehonadav and Roques-Carmes, Charles and Kaminer, Ido}, booktitle = {Conference on Lasers and Electro-Optics}, location = {San Jose, CA, United States}, publisher = {Optica Publishing Group}, title = {{X-ray-driven photon bunching}}, doi = {10.1364/cleo_si.2023.sm1h.6}, year = {2023}, } @inproceedings{21595, abstract = {We present a method for x-ray spectroscopy, combining nanophotonic scintillator inverse design with an image reconstruction algorithm. We demonstrate our pipeline on 3-energy x-ray spectroscopy, achieving 8% reconstruction error under 1% Gaussian noise}, author = {Li, William F. and Roques-Carmes, Charles and Lin, Zin and Johnson, Steven G. and Soljačić, Marin}, booktitle = {Conference on Lasers and Electro-Optics}, location = {San Jose, CA, United States}, publisher = {Optica Publishing Group}, title = {{X-ray spectroscopy with end-to-end optimized nanophotonic scintillators}}, doi = {10.1364/cleo_fs.2023.fw4c.4}, year = {2023}, } @article{21810, abstract = {The next-generation semiconductors and devices, such as halide perovskites and flexible electronics, are extremely sensitive to water, thus demanding highly effective protection that not only seals out water in all forms (vapor, droplet, and ice), but simultaneously provides mechanical flexibility, durability, transparency, and self-cleaning. Although various solid-state encapsulation methods have been developed, no strategy is available that can fully meet all the above requirements. Here, we report a bioinspired liquid-based encapsulation strategy that offers protection from water without sacrificing the operational properties of the encapsulated materials. Using halide perovskite as a model system, we show that damage to the perovskite from exposure to water is drastically reduced when it is coated by a polymer matrix with infused hydrophobic oil. With a combination of experimental and simulation studies, we elucidated the fundamental transport mechanisms of ultralow water transmission rate that stem from the ability of the infused liquid to fill-in and reduce defects in the coating layer, thus eliminating the low-energy diffusion pathways, and to cause water molecules to diffuse as clusters, which act together as an excellent water permeation barrier. Importantly, the presence of the liquid, as the central component in this encapsulation method provides a unique possibility of reversing the water transport direction; therefore, the lifetime of enclosed water-sensitive materials could be significantly extended via replenishing the hydrophobic oils regularly. We show that the liquid encapsulation platform presented here has high potential in providing not only water protection of the functional device but also flexibility, optical transparency, and self-healing of the coating layer, which are critical for a variety of applications, such as in perovskite solar cells and bioelectronics.}, author = {Lemaire, Baptiste and Yu, Yanhao and Molinari, Nicola and Wu, Haichao and Goodwin, Zachary A. H. and Stricker, Friedrich J and Kozinsky, Boris and Aizenberg, Joanna}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {water permeability, photoelectronic materials, device encapsulation, liquid-infused polymers}, number = {34}, publisher = {National Academy of Sciences}, title = {{Flexible fluid-based encapsulation platform for water-sensitive materials}}, doi = {10.1073/pnas.2308804120}, volume = {120}, year = {2023}, } @article{21813, abstract = {Aligned liquid crystal polymers are materials of interest for electronic, optic, biological and soft robotic applications. The manufacturing and processing of these materials have been widely explored with mechanical alignment establishing itself as a preferred method due to its ease of use and widespread applicability. However, the fundamental chemistry behind the required two‐step polymerization for mechanical alignment has limitations in both fabrication and substrate compatibility. In this work we introduce a new protection‐deprotection approach utilizing a two‐stage Diels–Alder cyclopentadiene‐maleimide step‐growth polymerization to enable mild yet efficient, fast, controlled, reproducible and user‐friendly polymerizations, broadening the scope of liquid crystal systems. Thorough characterization of the films by DSC, DMA, POM and WAXD show the successful synthesis of a uniaxially aligned liquid crystal network with thermomechanical actuation abilities.}, author = {Guillen Campos, Jesus and Stricker, Friedrich J and Clark, Kyle D. and Park, Minwook and Bailey, Sophia J. and Kuenstler, Alexa S. and Hayward, Ryan C. and Read de Alaniz, Javier}, issn = {1521-3773}, journal = {Angewandte Chemie International Edition}, number = {1}, publisher = {Wiley}, title = {{Controlled Diels–Alder “Click” strategy to access mechanically aligned main‐chain liquid crystal networks}}, doi = {10.1002/anie.202214339}, volume = {62}, year = {2023}, } @article{21807, abstract = {Multifaceted material responses upon exposure to stimuli are key for developing life-like materials. Developing such synthetic systems, though not trivial, typically relies on orthogonal stimuli to enable control of molecular systems that enable multi-responsive behavior. Access to complex tunable reaction mechanisms with diverse energy landscapes offers an alternative strategy for controlling out-of-equilibrium processes without requiring orthogonal stimuli for each responsive unit. Donor-acceptor Stenhouse adducts (DASAs) are a class of photoswitches that have complex, tunable, and environmentally sensitive reaction pathways. We present the control of donor-acceptor Stenhouse adduct equilibrium and photoswitching kinetics through changes in the polarity of their environment. Polarity and light can be used to selectively control the pathway outcomes of three DASA derivatives where the orthogonal response comes from changes in the energy landscape and is not driven by their orthogonal response to the given stimuli. This work paves the way to designing multi-responsive and self-regulating life-like materials.}, author = {Stricker, Friedrich J and Peterson, Julie and Sandlass, Sara K. and de Tagyos, Aurora and Sroda, Miranda and Seshadri, Serena and Gordon, Michael J. and Read de Alaniz, Javier}, issn = {2451-9294}, journal = {Chem}, number = {7}, pages = {1994--2005}, publisher = {Elsevier}, title = {{Selective control of donor-acceptor Stenhouse adduct populations with non-selective stimuli}}, doi = {10.1016/j.chempr.2023.05.011}, volume = {9}, year = {2023}, } @article{21818, abstract = {Surface-aligned liquid-crystal networks (LCNs) offer a solution for developing functional materials capable of performing a range of tasks, including actuation, shape memory, and surfaces patterning. Here we show that Diels–Alder cycloaddition can be used to prepare the backbone of planar aligned LCNs under mild ambient conditions without the addition of additives or UV irradiation. The mechanical properties of the networks have robust viscoelastic modulus and stiffness with a reversible local free volume change upon physical aging. This study shows new opportunities to design surface-aligned LCNs based on additive free step-growth Diels–Alder polymerization and enables the potential to incorporate a wider range of photochromic materials into LCNs.}, author = {Park, Minwook and Stricker, Friedrich J and Campos, Jesus Guillen and Clark, Kyle D. and Lee, Jaejun and Kwon, Younghoon and Valentine, Megan T. and Read de Alaniz, Javier}, issn = {2161-1653}, journal = {ACS Macro Letters}, number = {1}, pages = {33--39}, publisher = {American Chemical Society}, title = {{Design of surface-aligned main-chain liquid-crystal networks prepared under ambient, light-free conditions using the Diels–Alder cycloaddition}}, doi = {10.1021/acsmacrolett.2c00616}, volume = {12}, year = {2023}, } @inproceedings{14459, abstract = {Autoencoders are a popular model in many branches of machine learning and lossy data compression. However, their fundamental limits, the performance of gradient methods and the features learnt during optimization remain poorly understood, even in the two-layer setting. In fact, earlier work has considered either linear autoencoders or specific training regimes (leading to vanishing or diverging compression rates). Our paper addresses this gap by focusing on non-linear two-layer autoencoders trained in the challenging proportional regime in which the input dimension scales linearly with the size of the representation. Our results characterize the minimizers of the population risk, and show that such minimizers are achieved by gradient methods; their structure is also unveiled, thus leading to a concise description of the features obtained via training. For the special case of a sign activation function, our analysis establishes the fundamental limits for the lossy compression of Gaussian sources via (shallow) autoencoders. Finally, while the results are proved for Gaussian data, numerical simulations on standard datasets display the universality of the theoretical predictions.}, author = {Shevchenko, Aleksandr and Kögler, Kevin and Hassani, Hamed and Mondelli, Marco}, booktitle = {Proceedings of the 40th International Conference on Machine Learning}, issn = {2640-3498}, location = {Honolulu, Hawaii, HI, United States}, pages = {31151--31209}, publisher = {ML Research Press}, title = {{Fundamental limits of two-layer autoencoders, and achieving them with gradient methods}}, volume = {202}, year = {2023}, } @phdthesis{12897, abstract = {Inverse design problems in fabrication-aware shape optimization are typically solved on discrete representations such as polygonal meshes. This thesis argues that there are benefits to treating these problems in the same domain as human designers, namely, the parametric one. One reason is that discretizing a parametric model usually removes the capability of making further manual changes to the design, because the human intent is captured by the shape parameters. Beyond this, knowledge about a design problem can sometimes reveal a structure that is present in a smooth representation, but is fundamentally altered by discretizing. In this case, working in the parametric domain may even simplify the optimization task. We present two lines of research that explore both of these aspects of fabrication-aware shape optimization on parametric representations. The first project studies the design of plane elastic curves and Kirchhoff rods, which are common mathematical models for describing the deformation of thin elastic rods such as beams, ribbons, cables, and hair. Our main contribution is a characterization of all curved shapes that can be attained by bending and twisting elastic rods having a stiffness that is allowed to vary across the length. Elements like these can be manufactured using digital fabrication devices such as 3d printers and digital cutters, and have applications in free-form architecture and soft robotics. We show that the family of curved shapes that can be produced this way admits geometric description that is concise and computationally convenient. In the case of plane curves, the geometric description is intuitive enough to allow a designer to determine whether a curved shape is physically achievable by visual inspection alone. We also present shape optimization algorithms that convert a user-defined curve in the plane or in three dimensions into the geometry of an elastic rod that will naturally deform to follow this curve when its endpoints are attached to a support structure. Implemented in an interactive software design tool, the rod geometry is generated in real time as the user edits a curve and enables fast prototyping. The second project tackles the problem of general-purpose shape optimization on CAD models using a novel variant of the extended finite element method (XFEM). Our goal is the decoupling between the simulation mesh and the CAD model, so no geometry-dependent meshing or remeshing needs to be performed when the CAD parameters change during optimization. This is achieved by discretizing the embedding space of the CAD model, and using a new high-accuracy numerical integration method to enable XFEM on free-form elements bounded by the parametric surface patches of the model. Our simulation is differentiable from the CAD parameters to the simulation output, which enables us to use off-the-shelf gradient-based optimization procedures. The result is a method that fits seamlessly into the CAD workflow because it works on the same representation as the designer, enabling the alternation of manual editing and fabrication-aware optimization at will.}, author = {Hafner, Christian}, isbn = {978-3-99078-031-2}, issn = {2663-337X}, pages = {180}, publisher = {Institute of Science and Technology Austria}, title = {{Inverse shape design with parametric representations: Kirchhoff Rods and parametric surface models}}, doi = {10.15479/at:ista:12897}, year = {2023}, } @article{13188, abstract = {The Kirchhoff rod model describes the bending and twisting of slender elastic rods in three dimensions, and has been widely studied to enable the prediction of how a rod will deform, given its geometry and boundary conditions. In this work, we study a number of inverse problems with the goal of computing the geometry of a straight rod that will automatically deform to match a curved target shape after attaching its endpoints to a support structure. Our solution lets us finely control the static equilibrium state of a rod by varying the cross-sectional profiles along its length. We also show that the set of physically realizable equilibrium states admits a concise geometric description in terms of linear line complexes, which leads to very efficient computational design algorithms. Implemented in an interactive software tool, they allow us to convert three-dimensional hand-drawn spline curves to elastic rods, and give feedback about the feasibility and practicality of a design in real time. We demonstrate the efficacy of our method by designing and manufacturing several physical prototypes with applications to interior design and soft robotics.}, author = {Hafner, Christian and Bickel, Bernd}, issn = {1557-7368}, journal = {ACM Transactions on Graphics}, keywords = {Computer Graphics, Computational Design, Computational Geometry, Shape Modeling}, number = {5}, publisher = {Association for Computing Machinery}, title = {{The design space of Kirchhoff rods}}, doi = {10.1145/3606033}, volume = {42}, year = {2023}, } @phdthesis{12491, abstract = {The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. Despite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. In this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). To this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. In order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. High-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. In summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.}, author = {Zens, Bettina}, isbn = {978-3-99078-027-5}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, FIB milling, method development, FIBSEM, extracellular matrix, ECM, cell-derived matrices, CDMs, cell culture, high pressure freezing, HPF, structural biology, tomography, collagen}, pages = {187}, publisher = {Institute of Science and Technology Austria}, title = {{Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography}}, doi = {10.15479/at:ista:12491}, year = {2023}, }