Laura Andolfi

Pushing the High-energy limit of plasmonics

The localized surface plasmon resonance of metal nanoparticles allows confining the eletromagnetic field in nanosized volumes, creating high-field hot spots, most useful for enhanced nonlinear optical spectroscopies. The commonly employed metals, Au and Ag, yield plasmon resonances only spanning the visible/near-infrared range. Stretching upward, the useful energy range of plasmonics requires exploiting different materials. Deep-ultraviolet plasmon resonances happen to be achievable with one of the cheapest and most abundant materials available: aluminum indeed holds the promise of a broadly tunable plasmonic response, theoretically extending far into the deep-ultraviolet. Complex nanofabrication and the unavoidable Al oxidation have so far prevented the achievement of this ultimate high-energy response. A nanofabrication technique producing purely metallic Al nanoparticles has at last allowed to overcome these limits, pushing the plasmon resonance to 6.8 eV photon energy (≈180 nm) and thus significantly broadening the spectral range of plasmonics numerous applications.

Membrane stiffening by STOML3 facilitates mechanosensation in sensory neurons

Sensing force is crucial to maintain the viability of all living cells. Despite its fundamental importance, how force is sensed at the molecular level remains largely unknown. Here we show that stomatin-like protein-3 (STOML3) controls membrane mechanics by binding cholesterol and thus facilitates force transfer and tunes the sensitivity of mechano-gated channels, including Piezo channels. STOML3 is detected in cholesterol-rich lipid rafts. In mouse sensory neurons, depletion of cholesterol and deficiency of STOML3 similarly and interdependently attenuate mechanosensitivity while modulating membrane mechanics. In heterologous systems, intact STOML3 is required to maintain membrane mechanics to sensitize Piezo1 and Piezo2 channels. In C57BL/6N, but not STOML3−/− mice, tactile allodynia is attenuated by cholesterol depletion, suggesting that membrane stiffening by STOML3 is essential for mechanical sensitivity. Targeting the STOML3–cholesterol association might offer an alternative strategy for control of chronic pain.

Nanomechanics controls neuronal precursors adhesion and differentiation

The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell–substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine. Biotechnol. Bioeng. 2013; 110: 2301–2310.

Acetylated tubulin is essential for touch sensation in mice.

At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch. DOI: http://dx.doi.org/10.7554/eLife.20813.001

Cubosomes for in vivo fluorescence lifetime imaging

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Multiple dye-doped NIR-emitting silica nanoparticles for both flow cytometry and in vivo imaging

Dye-doped near infrared-emitting silica nanoparticles (DD-NIRsiNPs) represent a valuable tool in bioimaging, because they provide sufficient brightness, resistance to photobleaching and consist of hydrophilic non-toxic materials. Here, we report the development of multiple dye-doped NIR emitting siNPs (mDD-NIRsiNPs), based on silica–PEG core–shell nanostructures doped with a donor–acceptor couple, exhibiting a tunable intensity profile across the NIR spectrum and suitable for both multiparametric flow cytometry analyses and time-domain optical imaging. In order to characterize the optical properties and fluorescence applications of the mDD-NIRsiNPs, we have characterized their performance by analyzing their in vivo biodistribution in healthy mice as well as in lymphoma bearing xenografts, and their suitability as contrast imaging agents for cell labeling and tracking. The mDD-NIRsiNPs features will be useful in designing new applications for imaging agents based on silica nanoparticles for different experimental disease models.

The application of scanning near field optical imaging to the study of human sperm morphology

BackgroundThe morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures.ResultsIn this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols.ConclusionsSuch findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together.

Multimodal near-infrared-emitting PluS Silica nanoparticles with fluorescent, photoacoustic, and photothermal capabilities.

Purpose The aim of the present study was to develop nanoprobes with theranostic features, including – at the same time – photoacoustic, near-infrared (NIR) optical imaging, and photothermal properties, in a versatile and stable core–shell silica-polyethylene glycol (PEG) nanoparticle architecture. Materials and methods We synthesized core–shell silica-PEG nanoparticles by a one-pot direct micelles approach. Fluorescence emission and photoacoustic and photothermal properties were obtained at the same time by appropriate doping with triethoxysilane-derivatized cyanine 5.5 (Cy5.5) and cyanine 7 (Cy7) dyes. The performances of these nanoprobes were measured in vitro, using nanoparticle suspensions in phosphate-buffered saline and blood, dedicated phantoms, and after incubation with MDA-MB-231 cells. Results We obtained core–shell silica-PEG nanoparticles endowed with very high colloidal stability in water and in biological environment, with absorption and fluorescence emission in the NIR field. The presence of Cy5.5 and Cy7 dyes made it possible to reach a more reproducible and higher doping regime, producing fluorescence emission at a single excitation wavelength in two different channels, owing to the energy transfer processes within the nanoparticle. The nanoarchitecture and the presence of both Cy5.5 and Cy7 dyes provided a favorable agreement between fluorescence emission and quenching, to achieve optical imaging and photoacoustic and photothermal properties. Conclusion We obtained rationally designed nanoparticles with outstanding stability in biological environment. At appropriate doping regimes, the presence of Cy5.5 and Cy7 dyes allowed us to tune fluorescence emission in the NIR for optical imaging and to exploit quenching processes for photoacoustic and photothermal capabilities. These nanostructures are promising in vivo theranostic tools for the near future.

The phototransduction machinery in the rod outer segment has a strong efficacy gradient

Significance Phototransduction is now considered to be a quite thoroughly understood phenomenon. It is well known that new discs are continuously generated at the base of the outer segments (OSs) and old discs are shed at their tip, but the rod OSs are considered a well-stirred compartment with minor inhomogeneities. To verify this assumption and to better understand the machinery within the OS, we developed a new methodology to deliver highly localized lights. We found that, as the light stimulus is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreased by 5–10 times. This gradient of efficacy is attributed to a progressive loss of phosphodiesterase. Therefore, OSs are highly inhomogeneous compartments. Rod photoreceptors consist of an outer segment (OS) and an inner segment. Inside the OS a biochemical machinery transforms the rhodopsin photoisomerization into electrical signal. This machinery has been treated as and is thought to be homogenous with marginal inhomogeneities. To verify this assumption, we developed a methodology based on special tapered optical fibers (TOFs) to deliver highly localized light stimulations. By using these TOFs, specific regions of the rod OS could be stimulated with spots of light highly confined in space. As the TOF is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreases, demonstrating that the efficacy of the transduction machinery is not uniform and is 5–10 times higher at the base than at the tip. This gradient of efficacy of the transduction machinery is attributed to a progressive depletion of the phosphodiesterase along the rod OS. Moreover we demonstrate that, using restricted spots of light, the duration of the photoresponse along the OS does not increase linearly with the light intensity as with diffuse light.

High aspect ratio silicon nanowires control fibroblast adhesion and cytoskeleton organization

Cell-cell and cell-matrix interactions are essential to the survival and proliferation of most cells, and are responsible for triggering a wide range of biochemical pathways. More recently, the biomechanical role of those interactions was highlighted, showing, for instance, that adhesion forces are essential for cytoskeleton organization. Silicon nanowires (Si NWs) with their small size, high aspect ratio and anisotropic mechanical response represent a useful model to investigate the forces involved in the adhesion processes and their role in cellular development. In this work we explored and quantified, by single cell force spectroscopy (SCFS), the interaction of mouse embryonic fibroblasts with a flexible forest of Si NWs. We observed that the cell adhesion forces are comparable to those found on collagen and bare glass coverslip, analogously the membrane tether extraction forces are similar to that on collagen but stronger than that on bare flat glass. Cell survival did not depend significantly on the substrate, although a reduced proliferation after 36 h was observed. On the contrary both cell morphology and cytoskeleton organization revealed striking differences. The cell morphology on Si-NW was characterized by a large number of filopodia and a significant decrease of the cell mobility. The cytoskeleton organization was characterized by the absence of actin fibers, which were instead dominant on collagen and flat glass support. Such findings suggest that the mechanical properties of disordered Si NWs, and in particular their strong asymmetry, play a major role in the adhesion, morphology and cytoskeleton organization processes. Indeed, while adhesion measurements by SCFS provide out-of-plane forces values consistent with those measured on conventional substrates, weaker in-plane forces hinder proper cytoskeleton organization and migration processes.