Substrate microtopographies induce cellular alignment and affect nuclear force transduction.

Cellular physiology has been mainly studied by using two-dimensional cell culture substrates which lack in vivo-mimicking extracellular environment and interactions. Thus, there is a growing need for more complex model systems in life sciences. Micro-engineered scaffolds have been proven to be a promising tool in understanding the role of physical cues in the co-regulation of cellular functions. These tools allow, for example, probing cell morphology and migration in response to changes in chemo-physical properties of their microenvironment. In order to understand how microtopographical features, what cells encounter in vivo, affect cytoskeletal organization and nuclear mechanics, we used direct laser writing via two-photon polymerization (TPP) to fabricate substrates which contain different surface microtopographies. By combining with advanced high-resolution spectral imaging, we describe how the constructed grid and vertical line microtopographies influence cellular alignment, nuclear morphology and mechanics. Specifically, we found that growing cells on grids larger than 10 × 20 µm2 and on vertical lines increased 3D actin cytoskeleton orientation along the walls of microtopographies and abolished basal actin stress fibers. In concert, the nuclei of these cells were also more aligned, elongated, deformed and less flattened, indicating changes in nuclear force transduction. Importantly, by using fluorescence lifetime imaging microscopy for measuring Förster resonance energy transfer for a genetically encoded nesprin-2 molecular tension sensor, we show that growing cells on these microtopographic substrates induce lower mechanical tension at the nuclear envelope. To conclude, here used substrate microtopographies modulated the cellular mechanics, and affected actin organization and nuclear force transduction.

3D Polymer Architectures for the Identification of Optimal Dimensions for Cellular Growth of 3D Cellular Models.

Organ-on-chips and scaffolds for tissue engineering are vital assay tools for pre-clinical testing and prediction of human response to drugs and toxins, while providing an ethical sound replacement for animal testing. A success criterion for these models is the ability to have structural parameters for optimized performance. Here we show that two-photon polymerization fabrication can create 3D test platforms, where scaffold parameters can be directly analyzed by their effects on cell growth and movement. We design and fabricate a 3D grid structure, consisting of wall structures with niches of various dimensions for probing cell attachment and movement, while providing easy access for fluorescence imaging. The 3D structures are fabricated from bio-compatible polymer SZ2080 and subsequently seeded with A549 lung epithelia cells. The seeded structures are imaged with confocal microscopy, where spectral imaging with linear unmixing is used to separate auto-fluorescence scaffold contribution from the cell fluorescence. The volume of cellular material present in different sections of the structures is analyzed, to study the influence of structural parameters on cell distribution. Furthermore, time-lapse studies are performed to map the relation between scaffold parameters and cell movement. In the future, this kind of differentiated 3D growth platform, could be applied for optimized culture growth, cell differentiation, and advanced cell therapies.

Cellular Interaction of Bone Marrow Mesenchymal Stem Cells with Polymer and Hydrogel 3D Microscaffold Templates.

Biomimicking biological niches of healthy tissues or tumors can be achieved by means of artificial microenvironments, where structural and mechanical properties are crucial parameters to promote tissue formation and recreate natural conditions. In this work, three-dimensional (3D) scaffolds based on woodpile structures were fabricated by two-photon polymerization (2PP) of different photosensitive polymers (IP-S and SZ2080) and hydrogels (PEGDA 700) using two different 2PP setups, a commercial one and a customized one. The structures' properties were tuned to study the effect of scaffold dimensions (gap size) and their mechanical properties on the adhesion and proliferation of bone marrow mesenchymal stem cells (BM-MSCs), which can serve as a model for leukemic diseases, among other hematological applications. The woodpile structures feature gap sizes of 25, 50, and 100 µm and a fixed beam diameter of 25 µm, to systematically study the optimal cell colonization that promotes healthy cell growth and potential tissue formation. The characterization of the scaffolds involved scanning electron microscopy and mechanical nanoindenting, while their suitability for supporting cell growth was evaluated with live/dead cell assays and multistaining 3D confocal imaging. In the mechanical assays of the hydrogel material, we observed two different stiffness ranges depending on the indentation depth. Larger gap woodpile structures coated with fibronectin were identified as the most promising scaffolds for 3D BM-MSC cellular models, showing higher proliferation rates. The results indicate that both the design and the employed materials are suitable for further assays, where retaining the BM-MSC stemness and original features is crucial, including studies focused on BM disorders such as leukemia and others. Moreover, the combination of 3D scaffold geometry and materials holds great potential for the investigation of cellular behaviors in a co-culture setting, for example, mesenchymal and hematopoietic stem cells, to be further applied in medical research and pharmacological studies.

Two-photon polymerization simulation and fabrication of 3D microprinted suspended waveguides for on-chip optical interconnects.

Quantum and neuromorphic computational platforms in integrated photonic circuits require next-generation optical functionalities. Often, increasingly complex on-chip light-routing that allow superpositions not attainable by planar technologies are paramount e.g. for artificial neural networks. Versatile 3D waveguides are achievable via two-photon polymerization (TPP)-based microprinting. Here, a 3D morphology prediction tool which considers experimental TPP parameters, is presented, enabling on-chip 3D waveguide performance simulations. The simulations allow reducing the cost-intensive systematic experimental optimization process. Fabricated 3D waveguides show optical transmission properties in agreement with simulations, demonstrating that the developed morphology prediction methodology is beneficial for the development of versatile on-chip and potentially inter-chip photonic interconnect technology.

Photonic polymeric structures and electrodynamics simulation method based on a coupled oscillator finite-difference time-domain (O-FDTD) approach.

We use femtosecond laser-based two-photon polymerization (TPP) to fabricate a 2.5D micropillar array. Using an angular detection setup, we characterize the structure's scattering properties and compare the results against simulation results obtained from a novel electrodynamics simulation method. The algorithm employs a modified formulation of the Lorentz Oscillator Model and a leapfrog time differentiation to define a 2D coupled Oscillator Finite-Difference Time-Domain (O-FDTD). We validate the model by presenting several simulation examples that cover a wide range of photonic components, such as multi-mode interference splitters, photonic crystals, ring resonators, and Mach-Zehnder interferometers.

Multi-beam two-photon polymerization for fast large area 3D periodic structure fabrication for bioapplications.

Two-photon polymerization (TPP) is capable of fabricating 3D structures with dimensions from sub-µm to a few hundred µm. As a direct laser writing (DLW) process, fabrication time of 3D TPP structures scale with the third order, limiting its use in large volume fabrication. Here, we report on a scalable fabrication method that cuts fabrication time to a fraction. A parallelized 9 multi-beamlets DLW process, created by a fixed diffraction optical element (DOE) and subsequent stitching are used to fabricate large periodic high aspect ratio 3D microstructured arrays with sub-micron features spanning several hundred of µm2. The wall structure in the array is designed with a minimum of traced lines and is created by a low numerical aperture (NA) microscope objective, leading to self-supporting lines omitting the need for line-hatching. The fabricated periodic arrays are applied in a cell - 3D microstructure interaction study using living HeLa cells. First indications of increased cell proliferation in the presence of 3D microstructures compared to planar surfaces are obtained. Furthermore, the cells adopt an elongated morphology when attached to the 3D microstructured surfaces. Both results constitute promising findings rendering the 3D microstructures a suited tool for cell interaction experiments, e.g. for cell migration, separation or even tissue engineering studies.

SyncRGB-FLIM: synchronous fluorescence imaging of red, green and blue dyes enabled by ultra-broadband few-cycle laser excitation and fluorescence lifetime detection.

We demonstrate for the first time that an ultra-broadband 7 femtosecond (fs) few-cycle laser can be used for multicolor nonlinear imaging in a single channel detection geometry, when employing a time-resolved fluorescence detection scheme. On a multi-chromophore-labelled cell sample we show that the few-cycle laser can efficiently excite the multiple chromophores over a >400 nm two-photon absorption range. By combining the few-cycle laser excitation with time-correlated single-photon counting (TCSPC) detection to record two-photon fluorescence lifetime imaging microscopy (FLIM) images, the localization of different chromophores in the cell can be identified based on their fluorescence decay properties. The novel SyncRGB-FLIM multi-color bioimaging technique opens the possibility of real-time protein-protein interaction studies, where its single-scan operation translates into reduced laser exposure of the sample, resulting in more photoprotective conditions for biological specimens.

Differences in the optical properties of valve and girdle band in a centric diatom.

Diatoms are phototrophic single-celled microalgae encased in a cell wall (frustule) made of amorphous silicate. The frustule comprises two valves connected by a variable number of girdle bands, all exhibiting periodic micro/nanoporous structures. We studied the optical properties in water of girdle bands from the centric diatom Coscinodiscus granii, a frustule part that so far has received little attention by the scientific community. We show that valves and girdle bands exhibit different optical properties, as valves attenuate shorter wavelengths and girdle bands attenuate longer wavelengths of the visible light spectrum. Girdle bands show iridescent coloration in dependence of the light direction. Although the biological meaning of periodic nanoscale structures of frustules is still a matter of debate, the differences of valve and girdle band optical properties indicate that living diatoms are complex optical systems, where valves, girdles and pigments modulate light inside the cell.

Structure-based optics of centric diatom frustules: modulation of the in vivo light field for efficient diatom photosynthesis.

The optical properties of diatom silicate frustules inspire photonics and nanotechnology research. Whether light interaction with the nano-structure of the frustule also affects diatom photosynthesis has remained unclear due to lack of information on frustule optical properties under more natural conditions. Here we demonstrate that the optical properties of the frustule valves in water affect light harvesting and photosynthesis in live cells of centric diatoms (Coscinodiscus granii). Microscale cellular mapping of photosynthesis around localized spot illumination demonstrated optical coupling of chloroplasts to the valve wall. Photonic structures of the three-layered C. granii valve facilitated light redistribution and efficient photosynthesis in cell regions distant from the directly illuminated area. The different porous structure of the two sides of the valve exhibited photon trapping and forward scattering of blue light enhancing photosynthetic active radiation inside the cell. Photonic structures of diatom frustules thus alter the cellular light field with implications on diatom photobiology.

The UV filtering potential of drop-casted layers of frustules of three diatom species.

Diatoms are in focus as biological materials for a range of photonic applications. Many of these applications would require embedding a multitude of diatoms in a matrix (e.g. paint, crème or lacquer); however, most studies on the photonic and spectral properties of diatoms frustules (silica walls) have been carried out on single cells. In this study, for the first time, we test the spectral properties of layers of frustules of three diatom species (Coscinodiscus granii, Thalassiosira punctifera and Thalassiosira pseudonana), with special focus on transmission and reflectance in the UV range. The transmittance efficiency in the UV A and B range was: T. pseudonana (56-59%) >C. granii (53-54%) >T. punctifera (18-21%) for the rinsed frustules. To investigate the underlying cause of these differences, we performed X-ray scattering analysis, measurement of layer thickness and microscopic determination of frustule nanostructures. We further tested dried intact cells in the same experimental setup. Based on these data we discuss the relative importance of crystal structure properties, nanostructure and quantity of material on the spectral properties of diatom layers. Characterization of the UV protection performance of layers of diatom frustules is of central relevance for their potential use as innovative bio-based UV filters.

Tailoring the growth of p-6P nanofibres using ultrathin Au layers: an organic-metal-dielectric model system.

The influence of ultrathin Au cluster films on the growth of para-hexaphenyl (p-6P) fibres is investigated. Whereas p-6P at elevated temperatures forms long, mutually parallel fibres on plain mica, these fibres become shorter but taller on Au covered mica, up to a Au film thickness of approximately 8 nm. The degree to which fibres are mutually parallel decreases with increasing Au thickness. For thicker Au films the length of the fibres increases again, and their morphology changes from flat to faceted; for Au film thicknesses above 20 nm, fibre networks are formed. The spectroscopic properties of the fibres are not modified by the Au layer, enabling independent control of the fibre morphology by means of the intermediate metallic layer.