Compact, low-noise, all-solid-state laser system for stimulated Raman scattering microscopy.

We present a highly stable and compact laser source for stimulated Raman scattering (SRS) microscopy. cw-seeding of an optical parametric amplifier pumped by a bulk femtosecond Yb-oscillator and self-phase modulation in a tapered fiber allow for broad tunability without any optical or electronic synchronization. The source features noise levels of the Stokes beam close to the shot-noise limit at MHz modulation frequencies. We demonstrate the superior performance of our system by SRS imaging of micrometer-sized polymer beads.

Core-Shell Architecture in Poly(3-hexylthiophene) Nanoparticles: Tuning of the Photophysical Properties for Enhanced Neuronal Photostimulation.

This study shows that entirely thiophene-based core@shell nanoparticles, in which the shell is made of the oxidized form of the core polymer (P3HT@PTDOx NPs), result in a type II interface at the particle surface. This enables the development of advanced photon nanotransducers with unique chemical-physical and biofunctional properties due to the core@shell nanoarchitecture. We demonstrate that P3HT@PTDOx NPs present a different spatial localization of the excitation energy with respect to the nonoxidized NPs, showing a prevalence of surface states as a result of a different alignment of the HOMO/LUMO energy levels between the core and shell. This allows for the efficient photostimulation of retinal neurons. Indeed, thanks to the stronger and longer-lived charge separation, P3HT@PTDOx NPs, administered subretinally in degenerate retinas from the blind Royal College of Surgeons rats, are more effective in photostimulation of inner retinal neurons than the gold standard P3HT NPs.

High-fidelity solvent-resistant replica molding of hydrophobic polymer surfaces produced by femtosecond laser nanofabrication.

We demonstrate that hydrophobic areas formed by femtosecond laser irradiation on poly(methyl methacrylate) (PMMA) and polystyrene (PS) polymer substrates can be faithfully replicated on samples of the same material via a solvent-resistant perfluoropolyether (PFPE) elastomer mold. The replicated PMMA and PS samples show nearly identical micro-nanoscale topography and hydrophobic wetting characteristics as the laser-patterned master substrates. This work combines the femtosecond laser capability of spatially tailoring the wettability with a high-resolution parallel replication method, offering the potential for the efficient production of microfluidic devices with selectively tailored flow behavior.

Fiber-format stimulated-Raman-scattering microscopy from a single laser oscillator.

A highly simplified architecture for stimulated-Raman-scattering microscopy is demonstrated, where multiple tunable narrowband picosecond pulses are generated by spectral compression of femtosecond pulses emitted by a single compact Er-fiber oscillator. The system provides high sensitivity (2x10(-7)) and spectral resolution (sub-15 cm(-1)), and it offers an unprecedented flexibility for multicolor imaging.

Multi-foci laser microfabrication of 3D polymeric scaffolds for stem cell expansion in regenerative medicine.

High quality large scale fabrication of cellular scaffolds, with three-dimensional resolution comparable to cell size, is an important task to enable regenerative medicine applications with stem cells. We are using two-photon polymerization to produce our stem cell culture substrate called Nichoid, which we already demonstrated capable of stimulating cell proliferation while maintaining their stemness, without the need of dangerous additives. Parallelization of this technique can be achieved with the use of a spatial light modulator: here we show the results obtained combining this device with fast linear stages to produce Nichoid-covered substrates by two-photon polymerization. The well-polymerized structures confirm that this approach is particularly convenient for porous structures, and allows a significant time saving by a factor of almost five, with minor design adjustments. A Live & Dead assay was performed on mesenchymal stem cells cultured into the Nichoid microstructures in order to verify that no difference in cell viability is present, compared to microstructures fabricated by a single focus. This parallel setup opens the possibility to obtain a much larger number of microstructured substrates, that are essential to test new stem cell-based therapies. This approach can be also used for the fast fabrication of other kinds of cell culture devices.

Interactions between structural and chemical biomimetism in synthetic stem cell niches.

Advancements in understanding stem cell functions and differentiation are of key importance for the clinical success of stem-cell-based therapies. 3D structural niches fabricated by two-photon polymerization are a powerful platform for controlling stem cell growth and differentiation. In this paper, we investigate the possibility of further controlling stem cell fate by tuning the mechanical properties of such niches through coating with thin layers of biomimetic hyaluronan-based and gelatin-based hydrogels. We first assess the biocompatibility of chemical coatings and then study the interactions between structural and chemical biomimetism on the response of MSCs in terms of proliferation and differentiation. We observed a clear effect of the hydrogel coating on otherwise identical 3D scaffolds. In particular, in gelatin-coated niches we observed a stronger metabolic activity and commitment toward the osteo-chondral lineage with respect to hyaluronan-coated niches. Conversely, a reduction in the homing effect was observed in all the coated niches, especially in gelatin-coated niches. This study demonstrates the feasibility of controlling independently different mechanical cues, in bioengineered stem cell niches, i.e. the 3D scaffold geometry and the surface stiffness. This will allow, on the one hand, understanding their specific role in stem cell proliferation and differentiation and, on the other hand, finely tuning their synergistic effect.

Three-dimensional structural niches engineered via two-photon laser polymerization promote stem cell homing.

A strategy to modulate the behavior of stem cells in culture is to mimic structural aspects of the native cell/extracellular matrix interaction. We applied femtosecond laser two-photon polymerization (2PP) to fabricate three-dimensional (3-D) microscaffolds, or "niches", using a hybrid organic-inorganic photoresist called SZ2080. The niches, of sizes fitting in a volume of 100×100×100 µm(3), were made by an external containment grid of horizontal parallel elements and by an internal 3-D lattice. We developed two niche heights, 20 and 80-100 µm, and four lattice pore dimensions (10, 20, 30 µm and graded). We used primary rat mesenchymal stem cells (MSCs) to study cell viability, migration and proliferation in the niches, up to 6 culture days. MSCs preferentially stayed on/in the structures once they ran into them through random migration from the surrounding flat surface, invaded those with a lattice pore dimension greater than 10 µm, and adhered to the internal lattice while the cell nuclei acquired a roundish morphology. In the niches, the highest MSC density was found in those areas where proliferation was observed, corresponding to the regions where the scaffold surface density available for cell adhesion was highest. The microgeometry inducing the highest cell density was 20 µm high with graded pores, in which cell invasion was favored in the central region of large porosity and cell adhesion was favored in the lateral regions of high scaffold surface density. Cell density in the niches, 17±6 cells/(100×100 µm(2)), did not significantly differ from that of the flat surface colonies. This implies that MSCs spontaneously homed and established colonies within the 3-D niches. This study brings to light the crucial role played by the niche 3-D geometry on MSC colonization in culture, with potential implications for the design of biomaterial scaffolds for synthetic niche engineering.

Femtosecond laser microstructuring for polymeric lab-on-chips.

This paper provides an overview of femtosecond laser microfabrication in polymeric materials, with emphasis on lab-on-chip applications. Due to the nonlinear interaction of femtosecond laser pulses with polymers, laser-induced modifications are localized to the focal volume, enabling high resolution patterning in 3D. Femtosecond laser microfabrication offers unmatched versatility in fabricating surface microchannels and diffractive optics by means of laser ablation, buried optical waveguides and micro-optics through refractive index modification and complex 3D microstructures in photoresists by two-photon polymerization. Femtosecond laser microfabrication technology opens the door to fabricating integrated lab-on-chip devices with a single tool.

Two-photon laser polymerization: from fundamentals to biomedical application in tissue engineering and regenerative medicine.

Three-dimensional material microstructuring by femtosecond laser-induced two-photon polymerization is emerging as an important tool in biomedicine. During two-photon polymerization, a tightly focused femtosecond laser pulse induces a crosslinking photoreaction in the polymer confined within the focal volume. As a rapid-prototyping technique, two-photon polymerization enables the fabrication of truly arbitrary three-dimensional micro- and nano-structures directly from computer models, with a spatial resolution down to 100 nm. In this review, we discuss the fundamentals, experimental methods, and materials used for two-photon polymerization; in addition, we present some applications of this technology related to microfluidics and to biomaterial scaffolds for tissue engineering and regenerative medicine.

Confocal ultrafast pump-probe spectroscopy: a new technique to explore nanoscale composites.

This article is devoted to the exploration of the benefits of a new ultrafast confocal pump-probe technique, able to study the photophysics of different structured materials with nanoscale resolution. This tool offers many advantages over standard stationary microscopy techniques because it directly interrogates excited state dynamics in molecules, providing access to both radiative and non-radiative deactivation processes at a local scale. In this paper we present a few different examples of its application to organic semiconductor systems. The first two are focussed on the study of the photophysics of phase-separated polymer blends: (i) a blue-emitting polyfluorene (PFO) in an inert matrix of PMMA and (ii) an electron donor polythiophene (P3HT) mixed with an electron acceptor fullerene derivative (PCBM). The experimental results on these samples demonstrate the capability of the technique to unveil peculiar interfacial dynamics at the border region between phase-segregated domains, which would be otherwise averaged out using conventional pump-probe spectroscopy. The third example is the study of the photophysics of isolated mesoscopic crystals of the PCBM molecule. Our ultrafast microscope could evidence the presence of two distinctive regions within the crystals. In particular, we could pinpoint for the first time areas within the crystals showing photobleaching/stimulated emission signals from a charge-transfer state.

Surface properties of femtosecond laser ablated PMMA.

The effects of femtosecond laser ablation on the physical and chemical properties at the surface of poly methylmethacrylate (PMMA) were studied. Femtosecond laser microfabrication caused the initially wetting behavior of PMMA to become nonwetting, mainly because of the laser-induced surface porosity at the submicroscale. Static and dynamic contact angle measurements along with morphological characterization revealed that after the laser irradiation, the system lies in an intermediate regime between those theorized by Wenzel and Cassie-Baxter. Spectroscopic analysis did not evidence any significant variation in the chemical properties of the processed polymeric surfaces.

Structural phase contrast in polycrystalline organic semiconductor films observed by broadband near-field optical spectroscopy.

We demonstrate a novel near-field absorption spectrometer with 100 nm spatial resolution based on an ultrabroadband Ti:sapphire oscillator coupled to an aperture NSOM, enabling the measurement of nanoscale absorption spectra. The instrument is particularly suited for structural phase-selective imaging of organic materials at the nanoscale. We demonstrate that variations in the local absorption spectrum allow us to distinguish between the crystalline and the amorphous phases in polycrystalline phtalocyanine films, thus providing previously unavailable information on their mesoscopic texture.