Force generation dynamics in native muscle tissues have been stringently optimized by evolution. Realizing similar contractile dynamics in a widely available biomaterial and subsequently fabricating macroscopic functional modules from them remains challenging. Herein, we tailor two-photon stereolithography to 3D print synthetic muscles made from bovine serum albumin to realize 1 mm long contractile fibers. We show that pH-dependent contractions in these synthetic muscles follow parabolic force-length relationships similar to biological muscles. Achieved stress outputs of 0.78 ± 0.13 N cm-2were within an order of magnitude of smooth and cardiac muscle. Stretch-shortening work loops performed under different strain rates in turn revealed a viscoelastic behavior and significant velocity dependence of work and net power, more similar to skeletal muscle. That an isotropic protein hydrogel can achieve such dynamics, reinforces the notion that these are not limited to sarcomere-level ordering and suggests a more general design space for non-canonical conformational dynamics to engineer performance improvements in artificial muscle materials.

Ultra-thin endoscopy requires miniaturized endoscope-tip imaging optics with sub-millimeter diameters. Emerging 3D-printed micro-optics by multiphoton lithography are suitably compact, and surpass the imaging quality of available alternatives such as GRIN optics. In Life Sciences, however, 3D-printed micro-optics have struggled with the necessity to operate in biological fluids. Further, their application for compact endoscopy using advanced microscopy methods such as confocal reflectance- and fluorescence imaging has remained unattained. Moving towards ultrathin, high performance microscopy at fiber-endoscope tips, we have developed compact monolithic 3D-printed micro-optics that successfully resolve micro-scale structures within an aberration-corrected field of view. Applied in liquid environments, they enable bright field-, confocal-, and fluorescence microscopy, promising to advance multimodal imaging for ultracompact, comprehensive tissue microscopy in biomedical endoscopy and Life Sciences.

Additive manufacturing has been invaluable for 3D microfluidic integration. We combine high-resolution two-photon printed microfluidic features with fast one-photon printing to reduce overall fabrication time by about 20-fold without compromising the accuracy of performance-critical features. Alignment strategies and interface design tolerances ensure fluidic sealing and the reproducible assembly between both 3D printing techniques. The resulting devices produce highly monodisperse GelMA-based foams with a polydispersity index of below 5% and tunable bubble radii between 235 and 400 μm. Combining multiple print modalities into a unified fabrication workflow will harness efficiency gains by utilizing high precision only where necessary.

In mammalian organisms, native tissue function depends on precise spatial organization down to the cellular level. Reconstituting tissue architectures in 2Din vitroplatforms can provide a means to study direct and indirect cell-cell interactions in a variety of tissue contexts while remaining compatible with high-throughput assays and high-resolution live imaging. We combine cost-effective stereolithography leveraging 3D printing with replica molding to stencil spatially defined, multicellular culture systems with sub-millimeter resolution onto planar substrates. The system is designed for ease of use, requires no complex fabrication setups and scales readily to 96-well plates. Sequential stencil application and removal under a biosafety cabinet enables controlled positioning of multiple cell types and supports the maturation of tissue assemblies. We demonstrate the utility of this stencil-based patterning strategy in three applications. First, we employ a combination of two circular stencils to recreate a structural feature characteristic of the tumor microenvironment of solid tumors: the encapsulation of colorectal cancer cells by cancer-associated fibroblast cells. Resulting cell patternings recapitulate key aspects of native tissue dynamics of the densely packed tumor tissues, in which cancer-associated fibroblast cells actively compress the cancer cells and confer targeted therapy resistance. Second, we probe an engineered synNotch-based signaling system in patterned cell patches that mimics morphogen gradient formation, where GFP-secreting sender cells generate a ligand-dependent gradient. Third, we recapitulate the characteristic crypt-villus architecture of the mammalian intestine by patterning intestinal organoids within a stencil-restricted crypt region and allowing differentiating cells to collectively migrate along a designed villus axis. The presented strategy allows for rebuilding multicellular tissue architecturesin vitrowith biologically relevant spatial precision for high-throughput drug screenings and dissection of tissue-specific cellular interactions.

Abstract Generative Lung Architecture Modeling (GLAM) is an integrated bioengineering framework that couples high-resolution three-dimensional tissue imaging with generative artificial intelligence to de novo design and 3D-bioprint anatomically detailed lung microtissue models. Native extracellular 3D matrix architectures of pulmonary parenchyma were extracted from healthy, fibrotic, and emphysematous in vivo mouse disease models and processed through a computational pipeline containing pre-trained image segmentation and 3D mesh generation. The resulting datasets were used to train a U-Net generative diffusion model with attention layers capable of synthesizing healthy and diseased lung tissue architectures. Microtissue cubes of about 200 - 300 µm edge length of native and synthetic datasets were fabricated through high-resolution two-photon stereolithography with gelatin-methacryloyl biomaterial ink and successfully seeded with cells, demonstrating biological compatibility. In closing the loop between biological imaging, generative modeling, and high-resolution biofabrication, this integrated framework establishes generative AI as a functional design layer for tissue engineering. The resulting lung microtissues retained architectural features of the native and original tissues, making them an application-ready platform for customizable and scalable fabrication of biological tissue surrogates for preclinical modeling, drug testing, and precision regenerative bioengineering.

Exothermic photopolymerization releases heat into the sample environment. Using NaYF4:Yb3+/Er3+ upconversion nanoparticle (UCNP) photoluminescence and a colinear lithography and thermometry laser configuration, we monitor thermal signatures in the focal spot during femtosecond direct laser writing in real time. A statistical short-pass filtering is introduced to reduce the standard error in temperature calibration compared to conventional Gaussian deconvolution. Thermometry performance of our set-up achieved a relative sensitivity of 0.89-1.58% K-1 and a measurement uncertainty of 0.2-0.4 K for 2 Hz sample rates. With this, the effect of scan speed, laser power, and photoinitiator concentration on accompanying local heating could be followed. Nonlinearities and thermal runaway effects with transient temperature spikes above 120-140°C demonstrate the need for a stringent reduction of the thermal burden when writing aqueous bioinks for biomedical applications. Physiological conditions were maintained only for fast 20 µm/s scan speeds, which limited temperature quenches to not exceed physiological temperatures. This paves the way to improve process control and to optimize for laser-assisted bioprinting and other related technologies.

Combining photonic integrated circuits (PICs) with microfluidic devices offers new possibilities for optofluidic lab-on-a-chip applications. Their accurate assembly and packaging, particularly with precise alignment, remain challenging. This work presents a versatile optofluidic integration of photonic and microfluidic chips, designed for seamless implementation in diverse laboratory environments. A 3D-printed holder for stable, cost-effective, and convenient fiber-to-chip bonding is used to configure optical in-and outputs. It can be readily adapted to accommodate various grating coupler designs. The microfluidic chip side is fabricated via soft-lithography in polydimethylsiloxane (PDMS), and positioned onto the photonic chip via cone-shaped alignment guides that were printed directly onto it. For this, the conventional PDMS plasma bonding process was adapted for passive self-alignment with the PIC chip between the 100 mu m-wide microfluidic channel and two grating couplers, each measuring 15 mu m in length and 50 mu m in width. In a proof-of-principle experiment, we detect the fluorescence of a fluorescein solution inside the microfluidic channels by delivering excitation and emission light through dedicated grating couplers. This configuration can address multiple sampling points along an optofluidic system to multiplex assays in a way that would be challenging to realize with conventional optical fibers.

Abstract In mammalian organisms, native tissue function depends on precise spatial organization down to the cellular level. Reconstituting tissue architectures in 2D in vitro platforms can provide a means to study direct and indirect cell-cell interactions in a variety of tissue contexts while remaining compatible with high-throughput assays and high-resolution live imaging. We combine cost-effective stereolithography leveraging 3D printing with replica molding to stencil spatially defined, multicellular culture systems with sub-millimeter resolution onto planar substrates. The system is designed for ease of use, requires no complex fabrication setups and scales readily to 96-well plates. Sequential stencil application and removal under a biosafety cabinet enables controlled positioning of multiple cell types and supported the maturation of tissue assemblies. We demonstrate the utility of this stencil-based patterning strategy in three applications. First, we employ a combination of two circular stencils to recreate a structural feature characteristic for the tumor microenvironment of solid tumors: the encapsulation of colorectal cancer cells by cancer-associated fibroblasts. Resulting cell patternings reproduce native tissue dynamics of the densely packed tumor tissues, in which cancer-associated fibroblast cells actively compress the cancer cells and confer targeted therapy resistance. Second, we probe the synthetic, diffusible morphogen system synNotch in patterned cell patches, where GFP-releasing cells generate a ligand-dependent gradient. Third, we recapitulate the characteristic crypt-villus architecture of the mammalian intestine by patterning intestinal organoids within a stencil-restricted crypt region and allowing differentiating cells to collectively migrate along a designed villus axis. The presented strategy allows for rebuilding multicellular tissue architectures in vitro with biologically relevant spatial precision for high-throughput drug screenings and dissection of tissue-specific cellular interactions.

Single-particle imaging at X-ray free-electron lasers relies on suitable sample injection of nanoscale macromolecules and particles into the gas phase at room temperature. A coaxial liquid-sheet strategy considerably extended the range of suitable samples to include conductivities from zero to 40000 µS cm−1 – a more than about an eightfold increase in range compared with conventional electrosprays. A helium chamber atmosphere in combination with an engineered gas-sheet protected aerosol formation against corona discharge and reduced background noise more than threefold. These results suggest new avenues to qualify ever more demanding biological and material science samples for single-particle imaging in the future.

Femtosecond 3D-printing offers tantalizing avenues for miniaturization and integration of micro optical systems. Available photoresists, however, restrain their utility in liquid immersion, especially in media with refractive indices larger than n = 1.33, such as glues or biomedical fluids. We present monolithic 3D-printed immersion optics, equipped with compact microfluidic sealing to protect the micro optical device from intrusion of liquid immersion media. We experimentally demonstrate diffraction limited performance in water, silicone-, and immersion oil, for a tailored aspherical-spherical doublet with a numerical aperture of NA = 0.625 and a footprint as small as a single mode optical fiber. Such compact monolithic immersion micro optics yield high potential to advance miniaturization for in situ biomedical sensing and robust coupling between fibers and photonic integrated circuits.

Imaging the structure and observing the dynamics of isolated proteins using single-particle X-ray diffractive imaging (SPI) is one of the potential applications of X-ray free-electron lasers (XFELs). Currently, SPI experiments on isolated proteins are limited by three factors: low signal strength, limited data and high background from gas scattering. The last two factors are largely due to the shortcomings of the aerosol sample delivery methods in use. Here we present our modified electrospray ionization (ESI) source, which we dubbed helium-ESI (He-ESI). With it, we increased particle delivery into the interaction region by a factor of 10, for 26 nm-sized biological particles, and decreased the gas load in the interaction chamber corresponding to an 80% reduction in gas scattering when compared to the original ESI. These improvements have the potential to significantly increase the quality and quantity of SPI diffraction patterns in future experiments using He-ESI, resulting in higher-resolution structures.

This work investigates the performance of the electrospray aerosol generator at the European X-ray Free Electron Laser (EuXFEL). This generator is, together with an aerodynamic lens stack that transports the particles into the X-ray interaction vacuum chamber, the method of choice to deliver particles for single-particle coherent diffractive imaging (SPI) experiments at the EuXFEL. For these experiments to be successful, it is necessary to achieve high transmission of particles from solution into the vacuum interaction region. Particle transmission is highly dependent on efficient neutralization of the charged aerosol generated by the electrospray mechanism as well as the geometry in the vicinity of the Taylor cone. We report absolute particle transmission values for different neutralizers and geometries while keeping the conditions suitable for SPI experiments. Our findings reveal that a vacuum ultraviolet ionizer demonstrates a transmission efficiency approximately seven times greater than the soft X-ray ionizer used previously. Combined with an optimized orifice size on the counter electrode, we achieve >40% particle transmission from solution into the X-ray interaction region. These findings offer valuable insights for optimizing electrospray aerosol generator configurations and data rates for SPI experiments.

Manufacturing of 3D-printed micro optics using two photon lithography (2PL) has been advancing rapidly over the last decade, enabling production of high-performance micro optics. Among many more, 3D-printed miniaturized sensors, imaging optics, OCT systems, spectrometers and optical tweezers appear to be promising for application in the biomedical field. Here, immersion of optical systems into aqueous solutions is required regularly, hence capsulation for protection of the optical system's interior is required. Yet, specific properties of the 2PL fabrication process render capsulation of fabricated optics a delicate task. In this talk, we outline a wholistic design strategy for 3D-printed immersion micro optics. The optical design and the mechanical manufacturing process are addressed, as well as approaches to combine metrology and simulation techniques for accurate assessment and performance optimization of manufactured systems. The feasibility of the proposed concept is experimentally validated. We discuss current limitations and evaluate the future potential of 3D-printed immersion micro optics.

One of the most challenging aspects of X-ray research is the delivery of liquid sample flows into the soft X-ray beam. Currently, cylindrical microjets are the most commonly used sample injection systems for soft X-ray liquid spectroscopy. However, they suffer from several drawbacks, such as complicated geometry due to their curved surface. In this study, we propose a novel 3D-printed nozzle design by introducing microscopic flat sheet jets that provide micrometre-thick liquid sheets with high stability, intending to make this technology more widely available to users. Our research is a collaboration between the EuXFEL and MAX IV research facilities. This collaboration aims to develop and refine a 3D-printed flat sheet nozzle design and a versatile jetting platform that is compatible with multiple endstations and measurement techniques. Our flat sheet jet platform improves the stability of the jet and increases its surface area, enabling more precise scanning and differential measurements in X-ray absorption, scattering, and imaging applications. Here, we demonstrate the performance of this new arrangement for a flat sheet jet setup with X-ray photoelectron spectroscopy, photoelectron angular distribution, and soft X-ray absorption spectroscopy experiments performed at the photoemission endstation of the FlexPES beamline at MAX IV Laboratory in Lund, Sweden.

Ammonium nitrate in aqueous solution was investigated with synchrotron radiation based photoelectron spectroscopy using two types of liquid jet nozzles. Electron emission from a cylindrical microjet of aqueous ammonium nitrate solution was measured at two different angles relative to the horizontal polarization of the incident synchrotron radiation, 90 degrees and 54.7 degrees (the "magic angle"), for a range of photon energies (470-530 eV). We obtained beta parameter values as a function of photon energy, based on a normalization procedure relying on simulations of background intensity with the SESSA (Simulation of Electron Spectra for Surface Analysis) package. The beta values are similar to literature data for O 1s ionization of liquid water, and the beta value of N 1s from NH4+ is higher than that for NO3-, by approximate to 0.1. The measurements also show that the photoelectron signal from NO3- exhibits a photon energy dependent cross section variation not observed in NH4+. Additional measurements using a flat jet nozzle found that the ammonium and nitrate peak area ratio was unaffected by changes in the takeoff angle, indicating a similar distribution of both ammonium and nitrate in the surface region.