Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: the example of BV-2 microglia cells.

Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.

Controlling ripples' periodicity using temporally delayed femtosecond laser double pulses.

We demonstrate the capability to control the ripple periodicity on polycrystalline ZnO films by applying temporally delayed femtosecond double pulses. It is shown that there is a characteristic pulse separation time for which one can switch from low- to high- spatial-frequency ripple formation. Results are interpreted based on the relation of the characteristic delay time with the electron-phonon relaxation time of the material. Our results indicate that temporal pulse shaping can be advantageously used as a mean to control the periodic nanoripples' formation and thus the outcome of laser assisted nanofabrication process, which is desirable for the applications of nanopatterned transparent semiconductors.

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3.

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT(1)). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT(1) receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.

Tuning spectral properties of ultrafast laser ablation plasmas from brass using adaptive temporal pulse shaping.

Using automated laser pulse temporal shaping we report on enhancing spectral emission characteristics of ablation plasmas produced by laser irradiation of brass on ultrafast time scales. For different input irradiance levels, control of both atomic and ionic species becomes possible concerning the yield and the excitation state. The improved energy coupling determined by tailored pulses induces material ejection with lower mechanical load that translates into hot gas-phase regions with higher excitation degrees and reduced particulates.

Laser-induced topographies enable the spatial patterning of co-cultured peripheral nervous system cells.

The peripheral nervous system comprises glia and neurons that receive the necessary cues for their adhesion and proliferation from their extracellular milieu. In this study, a spatial platform of pseudoperiodic morphologies including patterns of nano- and micro- structures on Si were developed via direct ultrafast-laser structuring and were used as substrates for the patterning of co-cultured neuronal cells. The response of murine Schwann (SW10) and Neuro2a (N2a) cells were investigated both in monocultures and in a glia and neuronal co-culture system. Our results denoted that different types of neural tissue cells respond differently to the underlying topography, but furthermore, the presence of the glial cells alters the adhesion behavior of the neuronal cells in their co-culture. Therefore, we envisage that direct laser structuring that enables spatial patterning of the cells of the nervous system in a controllable manner according to the research needs, could in the future be a useful tool for understanding neural network interfaces and their electrical activity, synaptic processes and myelin formation.

Laser writing of nanostructures on bulk Al via its ablation in liquids.

Experimental results are presented on the formation of self-organized nanostructures (NSs) on a bulk Al target under its ablation in liquids--water and ethanol--with short laser pulses from 180 femtoseconds (fs) through 350 picoseconds (ps). NSs are characterized by atomic force microscopy, field emission scanning electron microscopy, optical absorption spectroscopy and x-ray diffraction. The period of NSs does not depend on the laser wavelength used from 248 through 800 nm and is approximately 200 nm. NSs on Al show the characteristic absorption peak in the near UV which has been attributed to plasmon oscillation of electrons. The wings of this peak, extending to the visible, lead to a distinct yellow coloration of the processed Al surface. Ultrafast laser structuring of bulk aluminum in liquids may be potentially a promising technique for efficient production of nanosized aluminum.

Second and third harmonic generation measurements of glues used for lining textile supports of painted artworks.

In this study, we present the implementation of non-linear spot measurements for obtaining specific and novel information related to various types of natural and synthetic glues used for lining of painted artworks. Third harmonic generation measurements were employed, in transmission mode, for the accurate and non-destructive thickness detection of lining glues. Furthermore, second harmonic generation signals were collected, in reflection mode, providing complementary information for the discrimination between different types of lining glues.

Laser induced forward transfer of metals by temporally shaped femtosecond laser pulses.

Temporally shaped, femtosecond laser pulses have been used for controlling the size and the morphology of micron-sized metallic structures obtained by using the Laser Induced Forward Transfer (LIFT) technique. We report the effect of pulse shaping on the size and morphology of the deposited structures of Au, Zn, Cr on a function of the pulse separation time ??t (from 0 to 10 ps) of double pulses of variable intensities generated by using a liquid crystal spatial light modulator (SLM). The observed differences in size and morphology are correlated with the outcome of pump-probe experiments for the study of electron-phonon scattering dynamics and subsequent energy transfer processes to the bulk in the different metals employed. We propose that in metals with weak electron-lattice coupling, the electron ballistic motion and the resulting fast electron scattering at the film surface, as well as the internal electron thermalization process are crucial to the morphology and size of the transferred material. Therefore, temporal shaping within the corresponding time scales of these processes may be used for tailoring the features of the metallic structures obtained by LIFT.

In vivo imaging of neurodegeneration in Caenorhabditis elegans by third harmonic generation microscopy.

In this study, neurodegeneration phenomena were investigated, by performing third harmonic generation imaging measurements on the nematode Caenorhabditis elegans, in vivo. The in vivo, precise identification of the contour of the degenerating neurons in the posterior part of the nematode and the monitoring, in real time, of the progression of degeneration in the worm, through third harmonic generation imaging measurements, were achieved. Femtosecond laser pulses (1028 nm) were utilized for excitation. Thus, the THG image contrast modality comprises a powerful diagnostic tool, providing valuable information and offering new insights into morphological changes and complex developmental processes in live biological specimens.

The laser-induced discoloration of stonework; a comparative study on its origins and remedies.

For understanding the phenomena associated with the discoloration observed in some cases of infrared laser cleaned stonework surfaces, a comparative study of three different types and morphologies of pollution encrustation and stone substrates was undertaken. Fragments originating from monuments with historic and/or artistic value, bearing homogeneous thin soiling on Pentelic marble (Athens, Greece), thick encrustation on Hontoria limestone (Burgos, Spain) and compact thin crust on gypsum decorations (Athens, Greece), have been studied on the basis of their composition and origin, together with the conditions that may induce yellowing effects upon their laser cleaning with IR wavelengths. While irradiation in the UV (i.e. at 355 nm) could not effectively remove the encrustations studied, irradiation at 1,064 nm was found efficient to remove all the studied pollution accumulations. Discoloration towards yellow was evident in all cases and at different levels, including the samples with intentional patination layer. To the limit of Raman detection no chemical alterations were detected on the irradiated areas while the presence of yellow polar compounds in all the pollution crusts studied supports the argument that the discoloration of the stone surfaces upon their IR irradiation may be due to the uncovering of existing yellow layers as result of the migration of these compounds inwards to the original stone surface. To correct and/or prevent such undesired coloration the use of IR and UV radiation both in sequential and synchronous mode was considered, with positive results.

Cell patterning via laser micro/nano structured silicon surfaces.

The surface topography of biomaterials can have an important impact on cellular adhesion, growth and proliferation. Apart from the overall roughness, the detailed morphological features, at all length scales, significantly affect the cell-biomaterial interactions in a plethora of applications including structural implants, tissue engineering scaffolds and biosensors. In this study, we present a simple, one-step direct laser patterning technique to fabricate nanoripples and dual-rough hierarchical micro/nano structures to control SW10 cell attachment and migration. It is shown that, depending on the laser processing conditions, distinct cell-philic or cell-repellant patterned areas can be attained with a desired motif. We envisage that our technique could enable spatial patterning of cells in a controllable manner, giving rise to advanced capabilities in cell biology research.

Metabolic profile of human coelomic fluid.

AIM: Till now there is very limited knowledge on the molecular content of coelomic fluid and cells. This study presents the first attempt to elucidate the metabolic profile of such samples. METHODOLOGY: Samples were collected via coelocentesis from 41 women during the first trimester of gestation. Metabolic content was assessed using four different analytical platforms. For targeted analysis a hydrophilic interaction chromatography ultra high performance LC-MS/MS method was applied. Holistic analysis performed by GC-MS, NMR spectroscopy and ion cyclotron ultra-high resolution MS (FT-ICR-MS) instrumentation. RESULTS & CONCLUSIONS: Our observations suggest coelomic fluid and cells as promising biosamples, rich in metabolites with potential use in mammalian system biology studies.

Making silicon hydrophobic: wettability control by two-lengthscale simultaneous patterning with femtosecond laser irradiation.

We report on the wettability properties of silicon surfaces, simultaneously structured on the micrometre-scale and the nanometre-scale by femtosecond (fs) laser irradiation to render silicon hydrophobic. By varying the laser fluence, it was possible to control the wetting properties of a silicon surface through a systematic and reproducible variation of the surface roughness. In particular, the silicon-water contact angle could be increased from 66° to more than 130°. Such behaviour is described by incomplete liquid penetration within the silicon features, still leaving partially trapped air inside. We also show how controllable design and tailoring of the surface microstructures by wettability gradients can drive the motion of the drop's centre of mass towards a desired direction (even upwards).

Spatial non-uniformity in exfoliated WS2 single layers.

Monolayers of transition metal dichalcogenides (TMDs) are atomically thin two-dimensional crystals with attractive optoelectronic properties, which are promising for emerging applications in nanophotonics. Here, we report on the extraordinary spatial non-uniformity of the photoluminescence (PL) and strain properties of exfoliated WS2 monolayers. Specifically, it is shown that the edges of such monolayers exhibit remarkably enhanced PL intensity compared to their respective central area. A comprehensive analysis of the recombination channels involved in the PL process demonstrates a spatial non-uniformity across the monolayer's surface and reflects on the non-uniformity of the intrinsic electron density across the monolayer. Auger electron imaging and spectroscopy studies complemented with PL measurements in different environments indicate that oxygen chemisorption and physisorption are the two fundamental mechanisms responsible for the observed non-uniformity. At the same time Raman spectroscopy analysis shows remarkable strain variations among the different locations of an individual monolayer, however such variations cannot be strictly correlated with the non-uniform PL emission. Our results shed light on the role of the chemical bonding in the competition between exciton complexes in monolayer WS2, providing a method of engineering new nanophotonic functions using WS2 monolayers. It is therefore envisaged that our findings could find diverse applications towards the development of TMD-based optoelectronic devices.

Microconical silicon structures influence NGF-induced PC12 cell morphology.

Micro-and nanofabrication techniques provide the opportunity to develop new types of cell culture platform, where the effect of various topographical cues on cellular functions such as proliferation and differentiation can be studied. In this study, PC12 cells were cultured on patterned silicon (Si) surfaces comprising arrays of microcones (MCs) exhibiting different geometrical characteristics and surface chemistries. It was illustrated that, in the absence of nerve growth factor (NGF), PC12 cells increased proliferation on all types of patterned surface, as compared to flat Si surfaces. However, in the presence of NGF, PC12 cells showed different responses, depending on the plating surface. Unlike low and intermediate rough MC surfaces, highly rough ones exhibiting large distances between MCs did not support PC12 cell differentiation, independently of the MCs' chemical coatings. These results suggest that the geometrical characteristics of MCs alone can influence specific cellular functions. Tailoring of the physical properties of arrays of Si MCs in order to identify which combinations of MC topologies and spatially defined chemistries are capable of driving specific cellular responses is envisaged.

Data in support on the shape of Schwann cells and sympathetic neurons onto microconically structured silicon surfaces.

This article contains data related to the research article entitled "Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth" in the Biomaterials journal [1]. Scanning electron microscopy (SEM) analysis is performed to investigate whether Schwann cells and sympathetic neurons alter their morphology according to the underlying topography, comprising arrays of silicon microcones with anisotropic geometrical characteristics [1]. It is observed that although soma of sympathetic neurons always preserves its round shape, this is not the case for Schwann cells that become highly polarized in high roughness microconical substrates.

Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth.

Patterning of neuronal outgrowth in vitro is important in tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. To date, this has been achieved with the aid of micro- and nanofabrication techniques giving rise to various anisotropic topographies, either in the form of continuous or discontinuous structures. In this study we propose a currently unexplored geometry of a 3D culture substrate for neuronal cell growth comprising discontinuous subcellular microstructures with anisotropic geometrical cross-section. Specifically, using laser precision 3D micro/nano fabrication techniques, silicon substrates comprising arrays of parallel oriented elliptical microcones (MCs) were fabricated to investigate whether a discontinuous geometry comprising anisotropic features at the subcellular level could influence the alignment of peripheral nervous system cell populations. It was shown that both Schwann cells and axons of sympathetic neurons were parallel oriented onto the MCs of elliptical shape, while they exhibited a random orientation onto the MCs of arbitrary shape. Notably, this topography-induced guidance effect was also observed in more complex cell culture systems, such as the organotypic culture whole dorsal root ganglia (DRG) explants. Our results suggest that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though the sizes of those range at the subcellular lengthscale. The laser-patterned arrays of MCs presented here could potentially be a useful platform for patterning neurons into artificial networks, allowing the study of neuronal cells interactions under 3D ex-vivo conditions.

Laser-induced fluorescence detection of cardiovascular atherosclerotic deposits via their natural emission and hypocrellin (HA) probing.

In order to develop a reliable laser-induced-fluorescence-guided angioplasty system, fluorescence spectra were recorded during exposure of normal and atherosclerotic cadaveric aortic tissue to He-Cd laser radiation (442 nm). A characteristic increase in the fluorescence signal at 600 nm for atheromatous tissue was observed after treatment of the samples with hypocrellin (HA). This, combined with the spectral distribution of tissue natural fluorescence, allowed the development of simple algorithms, based on the intensity difference and the full width at half-maximum (FWHM), and a subsequent index of discrimination between normal and various atheromatous tissues. Our results suggest that monitoring of this index through the catheter could enhance selective ablation, reducing the risk of normal vessel perforation.

Wavelength and pulse duration effects on laser induced changes on raw pigments used in paintings.

In this study, the reaction of widely used artist's pigments in raw form to pulsed laser radiation of different wavelengths and pulse duration was investigated. Vermilion, lead chromate and malachite (in the form of pellets) were irradiated using laser pulses of 500 fs at 248 nm, and pulses of 150 ps and 15 ns at 1064 and 213 nm. Optical microscopy, colorimetry, spectrofluorimetry, micro-Raman spectroscopy and X-ray photoelectron spectroscopy were employed to characterize the physicochemical changes induced to the pigments. Change of crystalline phase was identified for vermilion while reduction processes take place for lead chromate and malachite. It was found that these effects were minimized by application of ultraviolet ultrashort pulses (of femtosecond and picosecond duration) as compared with changes occurring by pulsed infrared irradiation (of both picosecond and nanosecond duration). The results presented are discussed in relation to previous research on painted mock-ups in order to elucidate the role and significance of the binding media in the laser induced discoloration of painted surfaces and thus to propose optimum laser cleaning practices.

Biomimetic micro∕nanostructured functional surfaces for microfluidic and tissue engineering applications.

This paper reviews our work on the application of ultrafast pulsed laser micro∕nanoprocessing for the three-dimensional (3D) biomimetic modification of materials surfaces. It is shown that the artificial surfaces obtained by femtosecond-laser processing of Si in reactive gas atmosphere exhibit roughness at both micro- and nanoscales that mimics the hierarchical morphology of natural surfaces. Along with the spatial control of the topology, defining surface chemistry provides materials exhibiting notable wetting characteristics which are potentially useful for open microfluidic applications. Depending on the functional coating deposited on the laser patterned 3D structures, we can achieve artificial surfaces that are (a) of extremely low surface energy, thus water-repellent and self-cleaned, and (b) responsive, i.e., showing the ability to change their surface energy in response to different external stimuli such as light, electric field, and pH. Moreover, the behavior of different kinds of cells cultured on laser engineered substrates of various wettabilities was investigated. Experiments showed that it is possible to preferentially tune cell adhesion and growth through choosing proper combinations of surface topography and chemistry. It is concluded that the laser textured 3D micro∕nano-Si surfaces with controllability of roughness ratio and surface chemistry can advantageously serve as a novel means to elucidate the 3D cell-scaffold interactions for tissue engineering applications.