Serous cutaneous glands in anurans: Fourier transform analysis of the repeating secretory granule substructure.

A combined transmission electron microscopy (TEM) and Fourier transform analysis has been performed on the secretory granules storing active peptides/proteins in serous cutaneous glands of n = 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM findings revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns depending on the degree of subunit clustering. This variety of recurrent patterns has been plotted into a range of frequency spectra. Through this quantitative approach, we found that the varying granule substructure can be reduced to a single mechanism of peptide/protein aggregation.

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.

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.

Altered Cx43 expression during myocardial adaptation to acute and chronic volume overloading.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

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.

High-resolution angular and displacement sensing based on the excitation of surface plasma waves.

The possibility of building angular and displacement sensors based on the phenomenon of attenuated total reflection (ATR) is explored both numerically and experimentally. ATR occurs when a surface wave is excited by an incoming TM electromagnetic wave through a resonant phase-matching process, as in the Kretschmann coupling scheme. The reflected intensity strongly depends on the angle of incidence of the beam. We first show some computations of the sensitivity and the linearity of an ATR-based sensor, then proceed to the experiment, illustrating how an angular resolution of the order of 0.1 arc sec can be obtained with moderate effort. Finally we show how the sensor, combined with a simple optical arrangement, can be used to detect and measure nanometric displacements, as those provided by piezoelectric actuators.

Correlation optical velocimetry with a compact disk pickup.

The compact disk pickup is a consumer electronics component that can be purchased for a low price owing to the great number of units produced every year. A typical three-beam head contains, enclosed in a tiny case, a quite complete sample of what can be found in an optoelectronics laboratory: a coherent source, a grating, a polarizing beam splitter, a fourth-wave plate, solid-state detectors, and much more. Exploiting the unique features of such a device for scientific aims is intriguing from many points of view. We present a straightforward application of a compact disk pickup to optical correlation velocimetry and show how a rather complicated discrete setup can be replaced successfully by this highly integrated component.

Multiphase homodyne interferometry: analysis of some error sources.

Some sources of error with multiphase homodyne interferometry are reviewed. A major advantage over the classic two-channel approach is that the inaccuracies that originate from laser-power fluctuations and drifts are shown to be automatically compensated for by proper adjustment of the light beams.

Characterization of the scattering properties of a mirror by speckle-field statistics.

The statistics of partially developed speckle patterns produced by reflection under coherent illumination can be used to determine the scattering properties of a mirror. The theory of the approach is presented, showing that in proper conditions the standard deviation of the speckle-field distribution expresses the finish grade of the reflecting surface. Interferometric measurements are described, and experiments on selected samples are reported.

Three-channel Homodyne Interferometer.

A three-channel homodyne interferometer based on a structured delay plate is described. Detection and data acquisition are performed with standard optoelectronics in a PC environment. The device, which is used as a displacement sensor, features sub-nanometric sensitivity.

Optimal phase filtering for high-power laser array far-field distribution.

We present numerical simulations of the propagation of the field emitted by laser arrays consisting of a small number of elements. Our computations show how propagation transforms part of an amplitude modulation into a phase modulation. This fact can be used to enhance the central lobe energy in the far field of high-power laser arrays by means of a simple corrective optical element. We discuss optimal filtering conditions and prove that the quarter-Talbot distance generally used in previous studies is not the best choice.

Telescopes of galileo.

The Florentine Istituto e Museo di Storia delta Scienza houses two complete telescopes and a single objective lens (reconstructed from several fragments) that can be attributed to Galileo. These optics have been partially dismantled and made available for optical testing with state-of-the-art equipment. The lenses were investigated individually; the focal length and the radii of curvature were measured, and the optical layout of the instruments was worked out. The optical quality of the surfaces and the overall performance of the two complete telescopes have been evaluated interferometrically at a wavelength of 633 nm (with a He-Ne laser source). It was found in particular that the optics of Galileo came close to attaining diffraction-limited operation.

Denisyuk hologram recording with a laser diode.

Making Denisyuk holograms in the near IR with a laser diode is reported, tuning reconstruction to the visible.