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.

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.

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.

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.

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.

Recent studies of laser science in paintings conservation and research.

The removal of aged and deteriorated molecular overlayers from the surface of paintings is a delicate and critical intervention in Cultural Heritage (CH) conservation. This irreversible action gets particularly complicated given the multitude of materials that may be present within a painted work of art (often in ultrathin layers or traces), as well as the exceptional sensitivity of the original surfaces to environmental conditions such as heat, light, and so on. Lasers hold an important role among the available cleaning methodologies, as they enable high control and accuracy, material selectivity, and immediate feedback. Still, prior to their implementation, it is imperative to optimize the cleaning parameters, so to ensure that any potential implications to the remaining materials are minimal and well understood. Toward this aim, research at IESL-FORTH is focused on both refining and continuously updating the laser-cleaning protocols (by introducing novel laser technologies into the field, i.e., ultrashort laser pulses), as well as on investigating and studying the nature and extent of laser-induced physicochemical alterations to the involved materials. In this Account, extended work for the understanding of ultraviolet (UV) laser ablation of polymers is presented. Emphasis is placed on the use of model systems (polymers doped with chromophores of known photochemistry) to examine the in-depth laser-induced modifications at the processed surfaces and thus to illustrate the dependence of their nature and extent on laser parameters and material properties. Furthermore, studies for the potential use of femtosecond UV pulses to overcome certain limitations involved with the nanosecond ablation of molecular overlayers from CH surfaces are highlighted. In particular, it is demonstrated that in the femtosecond regime any chemical modifications are, qualitatively and quantitatively, highly defined, limited and nearly independent of the material properties, such as the absorptivity and the degree of polymerization/molecular weight. Thus, they can be highly potent in the treatment of molecular substrates, enabling new material processing schemes that have not been possible with nanosecond laser technology, as for example, processing of ultrathin varnish layers. Finally, a sensitive indicator is introduced to elucidate the extent of any photochemical or structural modification induced at the substrate on the process of the laser-assisted removal of overpaints. A realistic scenario of an overlayered modern painting is simulated by a sensitive polymer film covered with acrylic paint. The indicator is doped with photosensitizers of known photochemistry and strong fluorescence emission, which allow the employment of laser induced fluorescence (LIF) for the detection of any chemical modifications generated into the substrate during laser cleaning. In addition, nonlinear microscopy techniques are successfully employed to examine the extent of these modifications. The suggested methodology is proven to reliably and accurately detect potential changes, and thus, it can serve as a monitoring tool to fine-tune the cleaning protocol and safeguard the original painting.

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.

Electrowetting properties of micro/nanostructured black silicon.

This paper reports on the electrowetting on dielectric (EWOD) properties of dual rough black silicon (Si) surfaces, produced by pulsed laser structuring of Si wafers and subsequently coating with a thermally grown oxide and a chloroalkylsilane layer. By varying the laser fluence, it was possible to tune the black Si wettability, from hydrophobicity to water repellence, through a systematic and reproducible variation of the surface roughness at micro- and nanoscales. It is shown that a liquid droplet on these surfaces can be readily switched between superhydrophobicity and hydrophilicity by applying moderate external electric fields. This behavior is reversible or irreversible depending on the geometry of the patterned structures and the water repellence characteristics of the different surfaces. The fundamental role of structural and dynamic wettability characteristics on the switching behavior during the EWOD process is investigated and discussed. The results indicate the potential use of dual rough black Si for EWOD applications.

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.

Generation of Al nanoparticles via ablation of bulk Al in liquids with short laser pulses.

Highly stable aluminum nanoparticles (NPs) are generated via ablation of bulk Al in ethanol using either femtosecond (fs) or picosecond (ps) laser sources. The colloidal NPs solutions obtained with fs pulses exhibit a yellow coloration and show an increased optical absorption between 300 and 400 nm, tentatively assigned to the plasmon resonance of nanosized Al. The corresponding solutions after ps ablation are gray colored and opalescent. The average size of the NPs formed ranges from 20 nm for the fs case to 60 nm for the ps case, while a narrower distribution is obtained using the shorter pulses. High Resolution Transmission Electron Microscopy (HRTEM) studies indicate that the NPs are mostly amorphous with single crystalline inclusions. Al NPs generated with short laser pulses slowly react with air oxygen due to the presence of a native oxide cladding, which efficiently passivates their surface against further oxidation.

Shrinkage of microstructures produced by two-photon polymerization of Zr-based hybrid photosensitive materials.

An investigation of the shrinking behaviour of a zirconium-based sol-gel composite micro-structured by two-photon polymerization is presented and a simple, straightforward methodology allowing the evaluation of shrinkage is suggested. It is shown that volume reduction is directly related to the average laser power (irradiation dose) used for the microfabrication and becomes a critical issue near the polymerization threshold. It is demonstrated that this shrinkage can be employed beneficially to improve the structural resolution. This is demonstrated by the presence of stopbands in the photonic crystal nanostructures fabricated with controlled volume reduction. Well above the polymerization threshold, the studied material exhibits remarkably low shrinkage. Therefore, no additional effort for the pre-compensation of distortion and for the improvement of structural stability is required.

Third-harmonic generation and multi-photon excitation fluorescence imaging microscopy techniques for online art conservation diagnosis.

We present an appropriate methodology and results for using third-harmonic generation (THG) modality for nondestructive high resolution imaging measurements of varnished structures in model painted artifacts. Detection takes place in the reflection mode, demonstrating the ability of the technique to be applied to the evaluation of original artworks. Furthermore, multi-photon excitation fluorescence images were obtained, providing complementary information related to the identification of the chemical composition of the artifacts.

Fully-Non-Contact Masking-Based Holography Inspection on Dimensionally Responsive Artwork Materials.

Environmental control in galleries and museums is a necessity and is informed by the knowledge of ongoing processes of deterioration which can threaten the integrity and stability of artworks. Invisible dimensional changes in many works of art occur following environmental fluctuations as materials respond to the changes in humidity and temperature. The constant influence of dimensional changes usually remains invisible until displacement generates visible deterioration and irreversible damage. This paper exploits fully non contact coherent interferometry in a sequential masking procedure for visualising and studying surface deformation which is the direct effect of dimensional alterations induced by humidity changes. Surface deformation during dimensional displacements of constituent materials may occur on any artwork within an unstable environment. In this context, the presented research study explores the diagnostic potential of fully non contact sensors for the direct structural assessment of environmental effects as they occur in real time on works of art. The method is employed to characterise material responses, complementing and improving understanding of material behaviour in unstable environments.

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.

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.

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.

Optical gain from the open form of a photochromic molecule in the solid state.

This work presents evidence for line-narrowing from the UV photoexcited open form of the photochromic molecule, indolinospiropyran (1',3'-dihydro-1',3',3'-trimethyl-6-nitrospiro [2H-1-benzopyran-2,2'-(2H)-indole]) in the solid state. The line-narrowing is attributable to amplified spontaneous emission induced by optical gain and assisted by the waveguiding within the organic film. Optical gain throughout a band as large as 28 nm, with a maximum gain coefficient of 5.6 cm(-1), is observed in the merocyanine emission region (660-730 nm). These results open the way to the realization of hybrid devices based on the coupling between photochromic behavior and stimulated emission from conjugated molecules, such as lasing optical memories, and lasers gated by optical molecular switches.

Laser Doppler spectroscopy towards the detection of spontaneous muscle activity.

OBJECTIVE: We used laser Doppler spectroscopy to detect hyperactivity of muscle fibers caused by the presence of fibrillations, positive waves and fasciculations. METHODS: The proposed method relies upon the dynamic scattering of light by moving particles, which causes a Doppler shift of the original frequency. For resolution of small alterations in frequency the heterodyne detection was used. One hundred seventy-three normal and 109 EMG diagnosed denervated first dorsal interosseous muscles were examined. RESULTS: Denervated muscles with fasciculations showed significant differentiation from control muscles, whereas denervated muscles without fasciculations were not significantly differentiated. The smallest differentiation compared to controls was seen in chronic denervated muscles without spontaneous activity. CONCLUSIONS: Presence of fasciculations in denervated muscles allowed Doppler spectroscopy to differentiate them from normal muscles. Presence of atrophy reduced the diagnostic potential of the method. SIGNIFICANCE: Our findings can be used as a basis for further improving painless and noninvasive laser Doppler spectroscopy to be used clinically as an alternative to painful and invasive EMG.

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.