A versatile and compact surface plasmon resonance spectrometer based on single board computer.

The widespread diffusion of low-cost but high-performance hardware is enhancing the realization of scientific equipment with features at the research laboratory level. In this paper, we demonstrate hardware implementation of a surface plasmon resonance compact device with high accuracy and measurement times appropriate for many applications. Image acquisition is realized by a Raspberry Pi single board computer with a camera module, and a Python code is used to process data. A flexible optical setup can work in two different configurations, namely, the inspection mode and angle resolved measurement mode. The inspection mode is used to precisely locate the light-emitting diode interrogation beam on the sample, avoiding uneven or faulty regions. The measurement mode allows us to monitor in real time the position of the minimum reflectivity with subpixel resolution. Performance tests show a resolution in the bulk refractive index of 4.9 × 10-6 refractive index units for 10 s acquisition time.

Opto-mechanical probe for combining atomic force microscopy and optical near-field surface analysis.

We have developed a new easy-to-use probe that can be used to combine atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). We show that, using this device, the evanescent field, obtained by total internal reflection conditions in a prism, can be visualized by approaching the surface with the scanning tip. Furthermore, we were able to obtain simultaneous AFM and SNOM images of a standard test grating in air and in liquid. The lateral resolution in AFM and SNOM mode was estimated to be 45 and 160 nm, respectively. This new probe overcomes a number of limitations that commercial probes have, while yielding the same resolution.

Diffusion of two molecular species in a crowded environment: theory and experiments.

Diffusion of a two component fluid is studied in the framework of differential equations, but where these equations are systematically derived from a well-defined microscopic model. The model has a finite carrying capacity imposed upon it at the mesoscopic level and this is shown to lead to nonlinear cross diffusion terms that modify the conventional Fickean picture. After reviewing the derivation of the model, the experiments carried out to test the model are described. It is found that it can adequately explain the dynamics of two dense ink drops simultaneously evolving in a container filled with water. The experiment shows that molecular crowding results in the formation of a dynamical barrier that prevents the mixing of the drops. This phenomenon is successfully captured by the model. This suggests that the proposed model can be justifiably viewed as a generalization of standard diffusion to a multispecies setting, where crowding and steric interferences are taken into account.

Disclosing and overcoming the trade-off between noise and scanning speed in atomic force microscopy.

Traditional dynamic modalities for atomic force microscopy imaging suffer from a stringent trade-off between fast scanning speed, weak interaction forces and accurate topography reconstruction. Finding an effective compromise between these aspects is often challenging, especially for soft biological samples for which stringent requirements hold when imaged in vivo. In this paper the main causes of this undesired trade-off in standard systems are analyzed and the exploitation of the intrinsic dynamics of the cantilever through a nonlinear control strategy is proposed as a method to overcome this limitation. A direct application to imaging of biological samples is reported to validate the results and show the effectiveness of the proposed technique.

ß-Connectin studies by small-angle x-ray scattering and single-molecule force spectroscopy by atomic force microscopy.

The three-dimensional structure and the mechanical properties of a ß-connectin fragment from human cardiac muscle, belonging to the I band, from I(27) to I(34), were investigated by small-angle x-ray scattering (SAXS) and single-molecule force spectroscopy (SMFS). This molecule presents an entropic elasticity behavior, associated to globular domain unfolding, that has been widely studied in the last 10 years. In addition, atomic force microscopy based SMFS experiments suggest that this molecule has an additional elastic regime, for low forces, probably associated to tertiary structure remodeling. From a structural point of view, this behavior is a mark of the fact that the eight domains in the I(27)-I(34) fragment are not independent and they organize in solution, assuming a well-defined three-dimensional structure. This hypothesis has been confirmed by SAXS scattering, both on a diluted and a concentrated sample. Two different models were used to fit the SAXS curves: one assuming a globular shape and one corresponding to an elongated conformation, both coupled with a Coulomb repulsion potential to take into account the protein-protein interaction. Due to the predominance of the structure factor, the effective shape of the protein in solution could not be clearly disclosed. By performing SMFS by atomic force microscopy, mechanical unfolding properties were investigated. Typical sawtooth profiles were obtained and the rupture force of each unfolding domain was estimated. By fitting a wormlike chain model to each peak of the sawtooth profile, the entropic elasticity of octamer was described.

The induction of α-helical structure in partially unfolded HypF-N does not affect its aggregation propensity.

The conversion of proteins into structured fibrillar aggregates is a central problem in protein chemistry, biotechnology, biology and medicine. It is generally accepted that aggregation takes place from partially structured states of proteins. However, the role of the residual structure present in such conformational states is not yet understood. In particular, it is not yet clear as to whether the α-helical structure represents a productive or counteracting structural element for protein aggregation. We have addressed this issue by studying the aggregation of pH-unfolded HypF-N. It has previously been shown that the two native α-helices of HypF-N retain a partial α-helical structure in the pH-unfolded state and that these regions are also involved in the formation of the cross-ß structure of the aggregates. We have introduced mutations in such stretches of the sequence, with the aim of increasing the α-helical structure in the key regions of the pH-unfolded state, while minimizing the changes of other factors known to influence protein aggregation, such as hydrophobicity, ß-Sheet propensity, etc. The resulting HypF-N mutants have higher contents of α-helical structure at the site(s) of mutation in their pH-unfolded states, but such an increase does not correlate with a change of aggregation rate. The results suggest that stabilisation of α-helical structure in amyloidogenic regions of the sequence of highly dynamic states does not have remarkable effects on the rate of protein aggregation from such conformational states. Comparison with other protein systems indicate that the effect of increasing α-helical propensity can vary if the stabilised helices are in non-amyloidogenic stretches of initially unstructured peptides (accelerating effect), in amyloidogenic stretches of initially unstructured peptides (no effect) or in amyloidogenic stretches of initially stable helices (decelerating effect).

Fibre-top atomic force microscope probe with optical near-field detection capabilities.

We present a fibre-top probe fabricated by carving a tipped cantilever on an optical fibre, with the tip machined in correspondence of the fibre core. When approached to an optical prism illuminated under total internal reflection conditions, the tip of the cantilever detects the optical tunnelling signal, while the light coupled from the opposite end of the fibre measures the deflection of the cantilever. Our results suggest that fibre-top technology can be used for the development of a new generation of hybrid probes that can combine atomic force microscopy with scanning near field optical microscopy.

High-Q polymer-coated microspheres for immunosensing applications.

Homogeneous polymeric thin layers have been used as functionalizing agents on silica microspherical resonators in view of the implementation of an immunosensor. We have characterized the microspheres functionalized with poly-L-lactic acid and Eudragit L100, as an alternative to the commonly used 3-Aminopropyltrimethoxysilane. It is shown that polymeric functionalization does not affect the high quality factor (Q greater than 10(7)) of the silica microspheres, and that the Q factor is about 3 x 10(5) after chemical activation and covalent binding of immunogammaglobulin (IgG). This functionalizing process of the microresonator constitutes a promising step towards the achievement of an ultra sensitive immunosensor.

Dynamic light scattering and atomic force microscopy imaging on fragments of beta-connectin from human cardiac muscle.

In order to investigate the protein folding-unfolding process, dynamic light scattering (DLS) and atomic force microscopy (AFM) imaging were used to study two fragments of the muscle cardiac protein beta-connectin, also known as titin. Both fragments belong to the I band of the sarcomer, and they are composed of four domains from I(27) to I(30) (tetramer) and eight domains from I(27) to I(34) (octamer). DLS measurements provide the size of both fragments as a function of temperature from 20 up to 86 degrees C, and show a thermal denaturation due to temperature increase. AFM imaging of both fragments in the native state reveals a homogeneous and uniform distribution of comparable structures. The DLS and AFM techniques turn out to be complementary for size measurements of the fragments and fragment aggregates. An unexpected result is that the octamer folds into a smaller structure than the tetramer and the unfolded octamer is also smaller than the unfolded tetramer. This feature seems related to the significance of the hydrophobic interactions between domains of the fragment. The longer the fragment, the more easily the hydrophobic parts of the domains interact with each other. The fragment aggregation behavior, in particular conditions, is also revealed by both DLS and AFM as a process that is parallel to the folding-unfolding transition.

Modelling and analysis of autonomous micro-cantilever oscillations.

Tapping mode atomic force microscopy provides good resolution in imaging applications, but it still requires a time-consuming initial configuration and features quite low scanning velocity. In this paper we present a new dynamic mode in which the cantilever gets excited by a feedback loop containing a saturation function. The proposed scheme is then analysed in the frequency domain and simulated against the standard set-up, showing good performance and elimination of some of the known drawbacks. Preliminary results in experiments confirm the effectiveness of this operating mode.

Atomic force microscopy images suggest aggregation mechanism in cerato-platanin.

Cerato-platanin (CP), the first member of the "cerato-platanin family", is a moderately hydrophobic protein produced by Ceratocystis fimbriata, the causal agent of a severe plant disease called "canker stain". The protein is localized in the cell wall of the fungus and it seems to be involved in the host-plane interaction and induces both cell necrosis and phytoalexin synthesis (one of the first plant defence-related events). Recently, it has been determined that CP, like other fungal surface protein, is able to self assemble in vitro. In this paper we characterize the aggregates of CP by Atomic Force Microscopy (AFM) images. We observe that CP tends to form early annular-shaped oligomers that seem to constitute the fundamental bricks of a hierarchical aggregation process, eventually resulting in large macrofibrillar assemblies. A simple model, based on the hypothesis that the aggregation is energetically favourable when the exposed surface is reduced, is compatible with the measured aggregates' shape and size. The proposed model can help to understand the mechanism by which CP and many other fungal surface proteins exert their effects.

Atomic force microscopy of histological sections using a chemical etching method.

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.

The orientation of the sandhopper Talitrus saltator during a partial solar eclipse.

To acquire more information about the identification and use of the sun and other celestial cues in the sea-land orientation of the sandhopper Talitrus saltator, we carried out releases in a confined environment during a partial solar eclipse and at sunset. The sandhoppers were unable to identify the sun (86% covered) during the eclipse nor to use other celestial compass factors of orientation. This was probably due to the low level of light intensity (close to the minimum level for orientation recorded at sunset) and to the variations in intensity and pattern of skylight polarization.

Ultracompact autocorrelator for multiphoton microscopy.

Pulse temporal characterization is a fundamental task when operating a Ti:Sapphire ultrafast laser system for multiphoton microscopy applications. In the present report, an ultracompact autocorrelator setup and a simple procedure is reported to perform pulse width measurements at the focal plane of the microscope objective without the need of any further instrumentation, aside from a few optical elements, since the confocal microscope, detection, data acquisition, processing, and displaying capabilities are used.

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

Ca+2 homeostasis and cytoskeletal rearrangement operated by sphingosine 1-phosphate in C2C12 myoblastic cells.

In hypogravity conditions unloading of skeletal muscle fibres causes alterations in skeletal muscle structure and functions including growth, gene expression, cell differentiation, cytoskeletal organization, contractility and plasticity. Recent studies have identified sphingosine I -phosphate (SPP) as a lipid mediator capable of eliciting intracellular Ca2+ transients, cell proliferation, differentiation, suppression of apoptosis, as well as cell injury repair. The aim of this research is to evaluate a possible involvement of SPP in skeletal muscle cells differentiation and repair from space-flight damage. Particularly, we investigated the Ca2+ sources and the changes on the cytoskeletal rearrangement induced by SPP in a mouse skeletal (C2C12) myoblastic cell line. Confocal fluorescence imaging revealed that SPP elicited Ca2+ transients which propagated throughout the cytosol and nucleus. This response required extracellular and intracellular Ca2+ mobilization. SPP also induced cell contraction through a Ca2(+)- independent/Rho-dependent pathway. The nuclear Ca2+ transients are suggestive for an action of SPP in the differentiation program and damage repair.

Fast one-dimensional profilometer with a compact disc pickup.

The use of compact disc (CD) pickups in optical profilometry is a well-established practice. The instruments currently available on the market are, however, expensive both to purchase and to maintain. This expense is mainly due to the high cost of the scanning system, and it makes the use of low-cost pickups fruitless. Moreover, translation stages are bulky, slow, and in most applications neither necessary nor desirable. We present a one-dimensional profilometer, which uses a CD pickup as both the sensor and the actuator. Beam scanning of the sample is in fact performed by the objective lens tracking motor. The device is cheap, fast, compact, light, and a valuable solution for fluid and hard-to-access surface profiling.

Interferometry with optical pickups.

We describe an interferometer that makes use of an optical pickup. This widespread consumer electronics component has a high degree of technological content. A typical head contains a remarkable, highly integrated sample of optoelectronic laboratory equipment. The application that we report is a significant and novel example of the potential exploitation of the unique features of such a device for scientific aims. Many interferometric configurations can be envisaged, depending on the specific pickup design. We present a Fizeau multiphase homodyne interferometer that makes use of an astigmatic-focus-detection pickup. Its quadrant detector provides four photocurrent signals whose phase delays can be easily controlled. This allows us to apply phase-shifting interferometry algorithms for data reduction.

Optomechanics with LEGO.

The basic elements of a fairly complete optomechanical kit based on the use of LEGO are presented. Taking advantage of the great variety of standard LEGO elements, and adding a few custom components made of Plexiglas, we show how most of the mechanical parts of an optical setup can be built with little effort and at an extremely reduced cost. Several systems and experiments are presented, mainly in the fields of optical filtering and interferometry, to show that the proposed mounts are excellent for didactic purposes and often perfectly suitable even in applied research.

Laser Doppler velocimetry with a compact disc pickup.

A laser Doppler velocimeter employing a compact disc pickup for both fringe projection and signal detection is described. The spectrum of the recorded signal gives the information about the speed of the object. The device takes advantage of the Talbot effect to project the grating contained in the pickup onto a moving target, so that no imaging system is required. The peculiar imaging technique allows for the exploitation of several optical configurations and permits the manipulation of the intensity profile of the projected grating. The instrument was used to measure the velocity of dust particles on a solid substrate in the 1-m/s range but could also find an application to the study of liquid flow.