Herein, we demonstrate the translation of Two-Photon Grayscale Lithography (2GL®), as well as Aligned 2-Photon Lithography (A2PL®), to biomedical applications. Specifically, we will present a novel workflow of aligned two-photon polymerization (2PP) microfabrication for 3D cell assays and perfusion inside microfluidic devices. For completeness, we also reveal how 2GL® can be applied to artificial intelligence (AI) generated topographies for enhanced and scalable 2.5D cell culturing. The versatility offered by both aligned and 2GL® printing holds great promise for various applications in biotechnology, tissue engineering, and microfluidics, creating new opportunities for innovation within established biomedical and pharmaceutical industries.

This paper presents advancements in aligned two-photon lithography (A2PL), where high resolution laser-based 3D fabrication technology is combined with high precision alignment routines. We demonstrate both camera- and confocal-based 3D detection algorithms for high-precision alignment, achieving accuracies below 100 nm. Two-photon lithography is utilized to fabricate photonic components such as micro-lenses for fiber-to-chip coupling, with losses as low as 1.7 dB and out-of-plane coupling structures. The workflow is readily adapted to other applications and material platforms, enabling scalable additive and multi-material fabrication.

Microneedles and, subsequently, microneedle arrays are emerging miniaturized medical devices for painless transdermal drug delivery. New and improved additive manufacturing methods enable novel microneedle designs to be realized for preclinical and clinical trial assessments. However, current literature reviews suggest that industrial manufacturers and researchers have focused their efforts on one-size-fits-all designs for transdermal drug delivery, regardless of patient demographic and injection site. In this perspective article, we briefly review current microneedle designs, microfabrication methods, and industrialization strategies. We also provide an outlook where microneedles may become personalized according to a patient’s demographic in order to increase drug delivery efficiency and reduce healing times for patient-centric care.

Two-photon polymerization (TPP) is widely used to create 3D micro- and nanoscale scaffolds for biological and mechanobiological studies, which often require the mechanical characterization of the TPP fabricated structures. To satisfy physiological requirements, most of the mechanical characterizations need to be conducted in liquid. However, previous characterizations of TPP fabricated structures are all conducted in air due to the limitation of conventional micro- and nanoscale mechanical testing methods. In this study, a new experimental method is reported for testing the mechanical properties of TPP-printed microfibers in liquid. The experiments show that the mechanical behaviors of the microfibers tested in liquid are significantly different from those tested in air. By controlling the TPP writing parameters, the mechanical properties of the microfibers can be tailored over a wide range to meet a variety of mechanobiology applications. In addition, it is found that, in water, the plasticly deformed microfibers can return to their predeformed shape after tensile strain is released. The shape recovery time is dependent on the size of microfibers. The experimental method represents a significant advancement in mechanical testing of TPP fabricated structures and may help release the full potential of TPP fabricated 3D tissue scaffolds for mechanobiological studies.

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell–cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between β-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors.

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) represent the best cell source for cardiac regenerative purposes but retain an immature phenotype after differentiation with significant limitations compared to adult cardiomyocytes. Apart from an incomplete cardiomyocyte-specific structure and micro-architecture, cells show at the level of Ca2+ signaling only slow Ca2+ release and reuptake properties. Here, we investigated the effect of restructuring single iPSC-CMs in specially designed 3D-micro-scaffolds on cell morphology and Ca2+ handling. Using direct laser writing, rectangular-shaped scaffolds were produced and single iPSC-CMs were seeded into these forms. Structural analyses revealed strong sarcolemmal remodeling processes and myofilament reorientation in 3D-shaped cells leading to enhanced clustered expression of L-type Ca2+ channels and ryanodine receptors and consequently, to faster Ca2+ transient kinetics. Spontaneous beating activity was enhanced and Ca2+ handling was more robust compared to non-patterned cells. Overall, our data demonstrate for the first time significant improvement of Ca2+ signaling properties in reshaped iPSC-CMs indicative of functional maturation by structural remodeling.

New promising therapeutic strategies for regeneration of the diseased heart focus on cardiomyocytes derived from pluripotent stem cells (iPSC-CM). Despite their cardiogenic properties, a well-known drawback of these cells is their immature structural and functional phenotype in contrast to adult cardiomyocytes. A characteristic feature of iPSC-CMs is the lack of a defined microarchitecture, which results in disorganized myofilament arrangement, altered electrophysiological characteristics and desynchronized calcium signals. As a consequence, iPSC-CMs exhibit spontaneous contractile activity and inefficient force production. The aim of this study was to test the hypothesis that a change in cell morphology influences the Ca2+-handling properties of iPSC-CMs leading to an improved functional phenotype comparable to adult cardiomyocytes. Using the method of direct laser writing, 3D-scaffolds of different geometries were produced from photolabile polyethylene glycol for single cell analysis. Cells were seeded in predesigned rectangular or hexagonal shapes and compared with control cells growing without any spatial limitations. Immunocytochemical investigations of the sarcomeric units revealed strong parallel alignment of myofilaments in rectangular scaffolds, while hexagonal-shaped cells exhibited diffuse myofilament orientation similar to control cells. Combining the techniques of whole-cell voltage clamp and confocal Ca2+ imaging using fluo-4, we discovered that despite similar L-type Ca2+ current densities (in pA/pF: control −12.2±0.9, rectangle −11.7±1.4, hexagon −10.6±1.4) the fast Ca2+-dependent current inactivation (t1) was significantly slower in rectangular cells due to structural cellular reorganization compared to hexagonal and control cells (in ms: control 11.3±1.1, rectangle 28.6±7.5, hexagon 16.7±1.9). Furthermore, spontaneous Ca2+ transients in rectangular-shaped cells showed a reduction in the peak-to-peak interval variation indicating enhanced signaling maturation (mean variance: control 0.21±0.07, rectangle 0.17±0.12; range: 1.61 vs. 1.05, respectively). In conclusion our data demonstrate that structural remodeling of iPSC-CMs by distinct cell shapes is paralleled by functional adaptations resulting in maturation of the Ca2+ handling machinery and contractile activity.

The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

Abstract3D‐Druck ist eine leistungsfähige Technik für die maßgeschneiderte Herstellung funktionaler Materialien. Dieser Aufsatz fasst den Stand der Technik im Hinblick auf 3D‐Laser‐Mikro‐ und ‐Nanodruck zusammen und erkundet die chemischen Herausforderungen, die derzeit die volle Etablierung dieser Technologie limitieren: von der Entwicklung fortgeschrittener Materialien für Anwendungen in der Zellbiologie und der Elektronik bis hin zu den bestehenden chemischen Grenzen des schnellen Schreibens mit Auflösungen unterhalb der Beugungsgrenze. Des Weiteren untersuchen wir Möglichkeiten zur Realisierung des direkten Laserschreibens mehrerer Materialien aus einem Photolack heraus, basierend auf wellenlängenselektiven photochemischen Prozessen (λ‐Orthogonalität). Schließlich betrachten wir chemische Prozesse, mit deren Hilfe adaptive 3D‐Strukturen geschrieben werden können, die auf externe Stimuli wie Licht, Wärme, pH‐Wert oder spezifische Moleküle reagieren, sowie fortgeschrittene Konzepte für abbaubare Stützstrukturen.

Recent developments in stimulated-emission depletion (STED) microscopy have led to a step change in the achievable resolution and allowed breaking the diffraction limit by large factors. The core principle is based on a reversible molecular switch, allowing for light-triggered activation and deactivation in combination with a laser focus that incorporates a point or line of zero intensity. In the past years, the concept has been transferred from microscopy to maskless laser lithography, namely direct laser writing (DLW), in order to overcome the diffraction limit for optical lithography. Herein, we propose and experimentally introduce a system that realizes such a molecular switch for lithography. Specifically, the population of intermediate-state photoenol isomers of α-methyl benzaldehydes generated by two-photon absorption at 700 nm fundamental wavelength can be reversibly depleted by simultaneous irradiation at 440 nm, suppressing the subsequent Diels-Alder cycloaddition reaction which constitutes the chemical core of the writing process. We demonstrate the potential of the proposed mechanism for STED-inspired DLW by covalently functionalizing the surface of glass substrates via the photoenol-driven STED-inspired process exploiting reversible photoenol activation with a polymerization initiator. Subsequently, macromolecules are grown from the functionalized areas and the spatially coded glass slides are characterized by atomic-force microscopy. Our approach allows lines with a full-width-at-half-maximum of down to 60 nm and line gratings with a lateral resolution of 100 nm to be written, both surpassing the diffraction limit.

3D printing is a powerful emerging technology for the tailored fabrication of advanced functional materials. This Review summarizes the state-of-the art with regard to 3D laser micro- and nanoprinting and explores the chemical challenges limiting its full exploitation: from the development of advanced functional materials for applications in cell biology and electronics to the chemical barriers that need to be overcome to enable fast writing velocities with resolution below the diffraction limit. We further explore chemical means to enable direct laser writing of multiple materials in one resist by highly wavelength selective (λ-orthogonal) photochemical processes. Finally, chemical processes to construct adaptive 3D written structures that are able to respond to external stimuli, such as light, heat, pH value, or specific molecules, are highlighted, and advanced concepts for degradable scaffolds are explored.

The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone.

We review fabrication and optics of polymeric high-Q whispering gallery mode resonators. They are employed as bio-sensors featuring selective parallel functionalization of arrays and integration into lab-on-a-chip systems. Liquid crystalline elastomers enable wide tunability of resonances. Elastomer substrates allow photonic molecules with tunable coupling gap.

Although much is known about chemotaxis- induced by gradients of soluble chemical cues - the molecular mechanisms involved in haptotaxis (migration induced by substrate-bound protein gradients) are largely unknown. We used micropatterning to produce discontinuous gradients consisting of μm-sized fibronectin-dots arranged at constant lateral but continuously decreasing axial spacing. Parameters like gradient slope, protein concentration and size or shape of the fibronectin dots were modified to determine optimal conditions for directional cell migration in gradient patterns. We demonstrate that fibroblasts predominantly migrate uphill towards a higher fibronectin density in gradients with a dot size of 2 × 2 μm, a 2% and 6% slope, and a low fibronectin concentration of 1 μg ml-1. Increasing dot size to 3.5 × 3.5 μm resulted in stationary cells, whereas rectangular dots (2 × 3 μm) orientated perpendicular to the gradient axis preferentially induce lateral migration. During haptotaxis, the Golgi apparatus reorients to a posterior position between the nucleus and the trailing edge. Using pharmacological inhibitors, we demonstrate that actomyosin contractility and microtubule dynamics are a prerequisite for gradient recognition indicating that asymmetric intracellular forces are necessary to read the axis of adhesive gradients. In the haptotaxis signalling cascade, RhoA and Cdc42, and the atypical protein kinase C zeta (aPKCζ), but not Rac, are located upstream of actomyosin contractility.

Light-harvesting micro-/nanohierarchical structures replicated from plants' epidermal cells are exploited for photovoltaic applications. Their broadband and omnidirectional antireflection properties, together with their light-trapping capability, are analyzed experimentally. Power conversion efficiency gains are reported after integrating those replicas onto optimized state-of-the-art organic solar cells. The proposed approach can be applied to different plant species and photovoltaic technologies. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Photonic molecules consisting of dye-doped polymeric WGM resonators exhibiting super-mode lasing are realized post fabrication by reducing the gap between the cavities. Flexible elastomer substrates allow a precise tuning of the coupling gap.

Measuring molecular orientation properties is very appealing for scientists in molecular and cell biology, as well as biomedical research. Orientational organization at the molecular scale is indeed an important brick to cells and tissues morphology, mechanics, functions and pathologies. Recent work has shown that polarized fluorescence imaging, based on excitation polarization tuning in the sample plane, is able to probe molecular orientational order in biological samples; however this applies only to information in 2D, projected in the sample plane. To surpass this limitation, we extended this approach to excitation polarization tuning in 3D. The principle is based on the decomposition of any arbitrary 3D linear excitation in a polarization along the longitudinal z-axis, and a polarization in the transverse xy–sample plane. We designed an interferometer with one arm generating radial polarization light (thus producing longitudinal polarization under high numerical aperture focusing), the other arm controlling a linear polarization in the transverse plane. The amplitude ratio between the two arms can vary so as to get any linear polarized excitation in 3D at the focus of a high NA objective. This technique has been characterized by polarimetry imaging at the back focal plane of the focusing objective, and modeled theoretically. 3D polarized fluorescence microscopy is demonstrated on actin stress fibers in non-flat cells suspended on synthetic polymer structures forming supporting pillars, for which heterogeneous actin orientational order could be identified. This technique shows a great potential in structural investigations in 3D biological systems, such as cell spheroids and tissues.