Laser-Structured Si and PLGA Inhibit the Neuro2a Differentiation in Mono- and Co-Culture with Glia.

BACKGROUND: The first step towards a successful neural tissue engineering therapy is the development of an appropriate scaffold and the in vitro study of the cellular response onto it. METHODS: Here, we fabricated nano- and micro- patterned Si surfaces via direct ultrafast laser irradiation, as well as their replicas in the biodegradable poly(lactide-co-glycolide), in order to use them as culture substrates for neuronal cells. The differentiation of neuro2a cells on the Si platforms and their replicas was studied both in a mono-culture and in a co-culture with glial cells (Schwann-SW10). RESULTS: It was found that the substrate's roughness inhibits the differentiation of the neuronal cells even in the presence of the differentiation medium, and the higher the roughness is, the more the differentiation gets limited. CONCLUSION: Our results highlight the importance of the substrate's topography for the controlled growth and differentiation of the neuronal cells and their further study via protein screening methods could shed light on the factors that lead to limited differentiation; thus, contributing to the long standing request for culture substrates that induce cells to differentiate.

Application of a Deep-Learning Technique to Non-Linear Images From Human Tissue Biopsies for Shedding New Light on Breast Cancer Diagnosis.

The development of label-free non-destructive techniques to be used as diagnostic tools in cancer research is of great importance for improving the quality of life for millions of patients. Previous studies have demonstrated that Third Harmonic Generation (THG) imaging could differentiate malignant from benign unlabeled human breast biopsies and distinguish the different grades of cancer. Towards the application of such technologies to clinic, in the present report, a deep learning technique was applied to THG images recorded from breast cancer tissues of grades 0, I, II, and III. By the implementation of a convolutional neural network (CNN) model, the differentiation of malignant from benign breast tissue samples and the discrimination of the different grades of cancer in a fast and accurate way were achieved. The obtained results provide a step ahead towards the use of optical diagnostic tools in conjunction with the CNN image classifier for the reliable and rapid malignancy diagnosis in clinic.

Monitoring aging-associated structural alterations in Caenorhabditis elegans striated muscles via polarization-dependent second-harmonic generation measurements.

The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging via non-invasive label free imaging techniques is an important issue with high biological significance. In this study, polarization-dependent second-harmonic generation (PSHG) was used to evaluate structural alterations in the striated muscles during Caenorhabditis elegans lifespan. Young and old wild-type animals were irradiated. The obtained results showed that it was not feasible to detect differences in the structure of myosin that could be correlated with the aging of the worms, via the implementation of the classical, widely used, cylindrical symmetry model and the calculation of the SHG anisotropy values. A trigonal symmetry model improved the extracted results; however, the best outcome was originated from the application of a general model. Myosin structural modifications were monitored via the estimation of the difference in spectral phases derived from discrete Fourier transform analysis. Age classification of the nematodes was achieved by employing both models, proving the usefulness of the usage of PSHG microscopy as a potential diagnostic tool for the investigation of muscle diseases.

Polarization-dependent second-harmonic generation for collagen-based differentiation of breast cancer samples.

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization-dependent second-harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second-harmonic generation (SHG) anisotropy and the "ratio parameter" values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.

Cells on hierarchically-structured platforms hosting functionalized nanoparticles.

In this work, we report on a novel approach to develop hierarchically-structured cell culture platforms incorporating functionalized gold nanoparticles (AuNPs). In particular, the hierarchical substrates comprise primary pseudo-periodic arrays of silicon microcones combined with a secondary nanoscale pattern of homogeneously deposited AuNPs terminated with bio-functional moieties. AuNPs with various functionalities (i.e. oligopeptides, small molecules and oligomers) were successfully attached onto the microstructures. Experiments with PC12 cells on hierarchical substrates incorporating AuNPs carrying the RGD peptide showed an impressive growth and NGF-induced differentiation of the PC12 cells, compared to that on the NP-free, bare, micropatterned substrates. The exploitation of the developed methodology for the binding of AuNPs as carriers of specific bio-functional moieties onto micropatterned culture substrates for cell biology studies is envisaged.

Photoacoustic imaging reveals hidden underdrawings in paintings.

A novel, non-invasive, imaging methodology, based on the photoacoustic effect, is introduced in the context of artwork diagnostics with emphasis on the uncovering of hidden features such as underdrawings or original sketch lines in paintings. Photoacoustic microscopy, a rapidly growing imaging method widely employed in biomedical research, exploits the ultrasonic acoustic waves, generated by light from a pulsed or intensity modulated source interacting with a medium, to map the spatial distribution of absorbing components. Having over three orders of magnitude higher transmission through strongly scattering media, compared to light in the visible and near infrared, the photoacoustic signal offers substantially improved detection sensitivity and achieves excellent optical absorption contrast at high spatial resolution. Photoacoustic images, collected from miniature oil paintings on canvas, illuminated with a nanosecond pulsed Nd:YAG laser at 1064 nm on their reverse side, reveal clearly the presence of pencil sketch lines coated over by several paint layers, exceeding 0.5 mm in thickness. By adjusting the detection bandwidth of the optically induced ultrasonic waves, photoacoustic imaging can be used for looking into a broad variety of artefacts having diverse optical properties and geometrical profiles, such as manuscripts, glass objects, plastic modern art or even stone sculpture.

Biomimetic surface structuring using cylindrical vector femtosecond laser beams.

We report on a new, single-step and scalable method to fabricate highly ordered, multi-directional and complex surface structures that mimic the unique morphological features of certain species found in nature. Biomimetic surface structuring was realized by exploiting the unique and versatile angular profile and the electric field symmetry of cylindrical vector (CV) femtosecond (fs) laser beams. It is shown that, highly controllable, periodic structures exhibiting sizes at nano-, micro- and dual- micro/nano scales can be directly written on Ni upon line and large area scanning with radial and azimuthal polarization beams. Depending on the irradiation conditions, new complex multi-directional nanostructures, inspired by the Shark's skin morphology, as well as superhydrophobic dual-scale structures mimicking the Lotus' leaf water repellent properties can be attained. It is concluded that the versatility and features variations of structures formed is by far superior to those obtained via laser processing with linearly polarized beams. More important, by exploiting the capabilities offered by fs CV fields, the present technique can be further extended to fabricate even more complex and unconventional structures. We believe that our approach provides a new concept in laser materials processing, which can be further exploited for expanding the breadth and novelty of applications.

3D plasmonic crystal metamaterials for ultra-sensitive biosensing.

We explore the excitation of plasmons in 3D plasmon crystal metamaterials and report the observation of a delocalized plasmon mode, which provides extremely high spectral sensitivity (>2600 nm per refractive index unit (RIU) change), outperforming all plasmonic counterparts excited in 2D nanoscale geometries, as well as a prominent phase-sensitive response (>3*10(4) deg. of phase per RIU). Combined with a large surface for bioimmobilization provided by the 3D matrix, the proposed sensor architecture promises a new important landmark in the advancement of plasmonic biosensing technology.

Application of multispectral imaging detects areas with neuronal myelin loss, without tissue labelling.

The application of multispectral imaging to discriminate myelinated and demyelinated areas of neural tissue is herein presented. The method is applied through a custom-made, multispectral imaging monochromator, coupled to a commercially available microscope. In the present work, a series of spinal cord sections were analysed derived from mice with experimental autoimmune encephalomyelitis (EAE), an experimental model widely used to study multiple sclerosis (MS). The multispectral microscope allows imaging of local areas with loss of myelin without the need of tissue labelling. Imaging with the aforementioned method and system is compared in a parallel way with conventional methods (wide-field and confocal fluorescence microscopies). The diagnostic sensitivity of our method is 90.4% relative to the 'gold standard' method of immunofluorescence microscopy. The presented method offers a new platform for the possible future development of an in vivo, real-time, non-invasive, rapid imaging diagnostic tool of spinal cord myelin loss-derived pathologies.

Implantable vaccine development using in vitro antigen-pulsed macrophages absorbed on laser micro-structured Si scaffolds.

To overcome the limiting antigenic repertoire of protein sub-units and the side effects of adjuvants applied in second generation vaccines, the present work combined in vitro and in vivo manipulations to develop biomaterials allowing natural antigen-loading and presentation in vitro and further activation of the immune response in vivo. 3-dimensional laser micro-textured implantable Si-scaffolds supported mouse macrophage adherence, allowed natural seeding with human serum albumin (antigen) and specific antibody and inflammatory cytokine production in vitro. Implantation of Si-scaffolds loaded with antigen-activated macrophages induced an inflammatory reaction along with antigen-specific antibody production in vivo, which could be detected even 30 days post implantation. Analysis of implant histology using scanning electron microscopy showed that Si-scaffolds could be stable for a 6-month period. Such technology leads to personalized implantable vaccines, opening novel areas of research and treatment.

Imaging ectopic fat deposition in Caenorhabditis elegans muscles using nonlinear microscopy.

The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a complex of pathological conditions including type-2 diabetes, hypertension, and cardiovascular diseases, is an important issue with high biological significance and requires accurate methods capable of monitoring lipid storage distribution and dynamics in vivo. In this study, the nonlinear phenomena of second and third harmonic generation (SHG, THG) have been employed simultaneously as label-free, nondestructive diagnostic techniques, for the monitoring and the complementary three-dimensional (3D) imaging and analysis of the muscular areas and the lipid content localization. THG microscopy was used as a quantitative tool in order to record the accumulation of lipids in nonadipose tissues in the pharyngeal muscles of 18 Caenorhabditis elegans (C. elegans) specimens, while the SHG imaging provided the detailed anatomical information about the structure of the muscles. The ectopic accumulation of fat on the pharyngeal muscles increases in wild-type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of ectopic fat accumulation with the process of aging. Our results can contribute to the unraveling of the link between the deposition of ectopic fat and aging, but mainly to the validation of SHG and THG microscopy modalities as new, noninvasive tools to localize and quantify selectively lipid formation and distribution.

Assessment of in-depth degradation of artificially aged triterpenoid paint varnishes using nonlinear microscopy techniques.

The present work investigates the applicability of nonlinear imaging microscopy for the precise assessment of degradation of the outer protective layers of painted artworks as a function of depth due to aging. Two fresh and artificially aged triterpenoid varnishes, dammar and mastic, were tested. Nonlinear imaging techniques have been employed as a new diagnostic tool for determination of the exact thickness of the affected region due to artificial aging of the natural varnishes. The measured thicknesses differ from the calculated mean penetration depths of the samples. These nondestructive, high resolution modalities are valuable analytical tools for aging studies and they have the potential to provide unique in-depth information. Single photon laser induced fluorescence measurements and Raman spectroscopy were used for the integrated investigation and analysis of aging effects in varnishes.

Label-free imaging of lipid depositions in C. elegans using third-harmonic generation microscopy.

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.

Pre-osteoblastic cell response on three-dimensional, organic-inorganic hybrid material scaffolds for bone tissue engineering.

Engineering artificial scaffolds that enhance cell adhesion and growth in three dimensions is essential to successful bone tissue engineering. However, the fabrication of three-dimensional (3D) tissue scaffolds exhibiting complex micro- and nano-features still remains a challenge. Few materials can be structured in three dimensions, and even those have not been characterized for their mechanical and biological properties. In this study, we investigate the suitability of three novel materials of different chemical compositions in bone tissue regeneration: a hybrid material consisting of methacryloxypropyl trimethoxysilane and zirconium propoxide, a hybrid organic-inorganic material of the above containing 50 mole% 2-(dimethylamino)ethyl methacrylate (DMAEMA) and a pure organic material based on polyDMAEMA. More specifically, we study the mechanical properties of the aforementioned materials and evaluate the biological response of pre-osteoblastic cells on them. We also highlight the use of a 3D scaffolding technology, Direct femtosecond Laser Writing (DLW), to fabricate complex structures. Our results show that, while all three investigated materials could potentially be used as biomaterials in tissue engineering, the 50% DMAEMA composite exhibits the best mechanical properties for structure fabrication with DLW and strong biological response.

Free-standing graphene on microstructured silicon vertices for enhanced field emission properties.

Controlled deposition of graphene in different orientations relative to the substrate is challenging and majority of existing deposition methods lead to sheets that lay flat on the substrate surface, limiting the potential applications in which the exposed sheet surface area and the atomically thin edges of graphene are exploited. Here we describe a simple and general solution-based methodology for the fabrication of random arrays of free-standing few-layer graphene (FLG) flakes on micro-spikes engraved on Si substrates. This should greatly benefit applications using free-standing graphene, such as in energy storage/conversion devices and bright electron sources. As a proof of concept, it is shown that the FLG sheets protruding on the top of micro-spikes are good electron emitters with turn-on fields as low as 2.3 V µm(-1) and field enhancement of few thousands. The emission performance and long-term stability achieved by this hierarchical deposition process are superior to that of planar graphene sheets and demonstrate promise for applications. Mechanisms leading to formation of free-standing FLG flakes are discussed.

Nonlinear microscopy techniques for assessing the UV laser polymer interactions.

A new diagnostic approach for assessing the in-depth laser induced modifications upon ultraviolet polymer irradiation is presented. The methodology relies on the observation of morphological alterations in the bulk material (Paraloid B72) by using third harmonic generation. This non destructive methodology allows the detailed and accurate imaging of the structurally laser modified zone extent in the vicinity of the irradiated area. Additionally, for the first time, the visualization and quantitative determination of the contour of the laser-induced swelling/bulk material interface is reported. The observed polymer surface swelling following single-pulse KrF laser irradiation at sub-ablation fluences is interpreted in the context of a model for laser-induced bubble formation due to droplet explosion mechanism.

Diffusion-assisted high-resolution direct femtosecond laser writing.

We present a new method for increasing the resolution of direct femtosecond laser writing by multiphoton polymerization, based on quencher diffusion. This method relies on the combination of a mobile quenching molecule with a slow laser scanning speed, allowing the diffusion of the quencher in the scanned area and the depletion of the multiphoton-generated radicals. The material we use is an organic-inorganic hybrid, while the quencher is a photopolymerizable amine-based monomer which is bound on the polymer backbone upon fabrication of the structures. We use this method to fabricate woodpile structures with a 400 nm intralayer period. This is comparable to the results produced by direct laser writing based on stimulated-emission-depletion microscopy, the method considered today as state-of-the-art in 3D structure fabrication. We optically characterize these woodpiles to show that they exhibit well-ordered diffraction patterns and stopgaps down to near-infrared wavelengths. Finally, we model the quencher diffusion, and we show that radical inhibition is responsible for the increased resolution.

Following the course of pre-implantation embryo patterning by non-linear microscopy.

Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.

Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers.

The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity.