Towards Easy-to-Use Bacteria Sensing: Modeling and Simulation of a New Environmental Impedimetric Biosensor in Fluids.

Conventional pathogenic bacteria-detection methods are lab-bound, time-consuming and need trained personnel. Microelectrodes can be used to recognize harmful microorganisms by dielectric impedance spectroscopy. However, crucial for this spectroscopy method are the spatial dimensions and layout of the electrodes, as the corresponding distribution of the electric field defines the sensor system parameters such as sensitivity, SNR, and dynamic range. Therefore, a variety of sensor models are created and evaluated. FEM simulations in 2D and 3D are conducted for this impedimetric sensor. The authors tested differently shaped structures, verified the linear influence of the excitation amplitude and developed a mathematical concept for a quality factor that practically allows us to distinguish arbitrary sensor designs and layouts. The effect of guard electrodes blocking outer influences on the electric field are investigated, and essential configurations are explored. The results lead to optimized electronic sensors in terms of geometrical dimensions. Possible material choices for real sensors as well as design and layout recommendations are presented.

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components.

Using an optical system made from fused silica catalogue optical components, third-order nonlinear microscopy has been enabled on conventional Ti:sapphire laser-based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ∼300 nm and in multimodal nonlinear optical imaging experiments using third-order sum frequency generation and coherent anti-Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ∼300 nm to ∼660 nm.

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

We report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 µm, while in the transverse direction it is ∼1-15 µm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nano-cylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced.

Extracorporeal life support and left ventricular unloading in a non-intubated patient as bridge to heart transplantation.

INTRODUCTION: Veno-arterial extracorporeal life support (ECLS) is a well-established bridging therapy in patients with cardiac or pulmonary failure to maintain organ function and is frequently performed in patients who are not intubated. However, severly impaired cardiac function can occur pulmonary edemy in these patients, necessitating left ventricular unloading. METHODS AND RESULTS: In this study we report a 37-year old female patient with familiar dilated cardiomyopathy suffering from acute biventricular heart failure. After implantation of a peripheral ECLS, the decreased ventricular led to refractory pulmonary edema. To unload the left ventricle, an percutaneous balloon atrioseptostomy was performed without intubating the patient. The left ventricle was vented by the venous cannula resting inside the atrioseptostomy. After twelve days on ECLS, the patient underwent orthotopic heart transplantation. The postoperative course was uneventful and the patient discharged from intensive care unit four days after surgery. CONCLUSIONS: In this report we present a patient in which the hybrid technique of ECLS with secondary left ventricular unloading was successfully used as a bridge to transplant therapy. This procedure may offer an alternative bridge-to-decision options in selected patients, including those that were not intubated or anaesthetized.

3 µm CW lasers for myringotomy and microsurgery.

This paper describes the development and implementation of 3 µm lasers for myringotomy and microsurgery. Two different lasers were investigated. The first, an Er-doped, CW zirconate glass fiber laser optically pumped by a 970 nm diode laser, emitted > 1 W of CW power at 2.76 µm with concomitant green incoherent emission that served as a convenient visible illumination beam. The second, a 1 W CW Er:YAG solid-state laser also optically pumped by a 970 nm diode laser, emitted > 1 W of CW power at 2.94 µm, coincident with the strongest infrared water absorption peak. Running CW, both lasers are expected to avoid the loud acoustical shocks associated with pulsed lasers. Myringotomies were carried out with the Er:YAG laser on anaesthetized guinea pigs and the effects of the laser were documented. Laser ablated samples of tympanic membrane, soft tissue and bone were histologically examined. Histology results indicated that the CW Er:YAG laser is a potential candidate for a new myringotomy tool and possibly for otologic microsurgery, but deliverable power levels need to be increased to the 2 W (or higher) level. This work was funded under NIH SBIR Grant No. 5R44DC004899.

Filamentous bacteria transport electrons over centimetre distances.

Oxygen consumption in marine sediments is often coupled to the oxidation of sulphide generated by degradation of organic matter in deeper, oxygen-free layers. Geochemical observations have shown that this coupling can be mediated by electric currents carried by unidentified electron transporters across centimetre-wide zones. Here we present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with electric currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, electrical cables add a new dimension to the understanding of interactions in nature and may find use in technology development.

Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy.

We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.

Two-dimensional nanoscale structural and functional imaging in individual collagen type I fibrils.

The piezoelectric properties of single collagen type I fibrils in fascia were imaged with sub-20 nm spatial resolution using piezoresponse force microscopy. A detailed analysis of the piezoresponse force microscopy signal in controlled tip-fibril geometry revealed shear piezoelectricity parallel to the fibril axis. The direction of the displacement is preserved along the whole fiber length and is independent of the fiber conformation. It is shown that individual fibrils within bundles in skeletal muscle fascia can have opposite polar orientations and are organized into domains, i.e., groups of several fibers having the same polar orientation. We were also able to detect piezoelectric activity of collagen fibrils in the high-frequency range up to 200 kHz, suggesting that the mechanical response time of biomolecules to electrical stimuli can be approximately 5 micros.

The structural origin of second harmonic generation in fascia.

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.

Diversities of coral-associated bacteria differ with location, but not species, for three acroporid corals on the Great Barrier Reef.

Patterns in the diversity of bacterial communities associated with three species of Acropora (Acropora millepora, Acropora tenuis and Acropora valida) were compared at two locations (Magnetic Island and Orpheus Island) on the Great Barrier Reef to better understand the nature and specificity of coral-microbial symbioses. Three culture-independent techniques demonstrated consistent bacterial communities among replicate samples of each coral species, confirming that corals associate with specific microbiota. Profiles were also conserved among all three species of Acropora within each location, suggesting that closely related corals of the same genus harbor similar bacterial types. Bacterial community profiles of A. millepora at Orpheus Island were consistent in samples collected throughout the year, indicating a stable community despite temporal changes. However, DGGE and T-RFLP profiles differed on corals from different reefs. Nonmetric multidimensional scaling of T-RFLP profiles showed that samples grouped according to location rather than coral species. Although similar sequences were retrieved from clone libraries of corals at both Magnetic and Orpheus Island, differences in the relative dominant bacterial ribotypes within the libraries drive bacterial community structure at different geographical locations. These results indicate certain bacterial groups associated specifically with corals, but the dominant bacterial genera differ between geographically-spaced corals.

Multimodal nonlinear optical imaging of collagen arrays.

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.

The role of backscattering in SHG tissue imaging.

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.

Second harmonic generation imaging of fascia within thick tissue block.

Comparing the SHG image formation for thin sections of tail tendon fascia and skeletal muscle fascia, we demonstrate that the forward (F) and backward (B) SHG images are vastly different. In addition, despite the different arrangement of the collagen Type I fibrillar architecture forming these two fascias, their ratios of forward over backward signal (F/B) are nearly equal. SHG images of thick tissue blocks of the fascia-muscle unit show only backward features, as opposed to SHG images of tissue blocks of the fascia-tendon unit. These images are an amalgamation of forward and backward features due to the backscattering of forward components within tendon. These forward features disappear when this tissue block is immersed in glycerol as backscattering is hereby suppressed.

Processing, localization, and requirement of human separase for normal anaphase progression.

In all eukaryotes, anaphase is triggered by the activation of a protease called separase. Once activated, separase cleaves a subunit of cohesin, a complex that links replicated chromatids before anaphase. Separase and cohesin are conserved from yeasts to humans. Although the machinery for dissolving sister cohesion is conserved, the regulation of this process appears to be more complex in higher eukaryotes than in yeast. Here we report the cloning of full-length human separase cDNA and the characterization of the encoded protein. Human separase was observed at the poles of the mitotic spindle until anaphase, at which time its association with the mitotic spindle was abruptly lost. The dynamic pattern of localization of human separase during cell cycle progression differs from that of fungal separases. Human separase also appears to undergo an autocatalytic processing on anaphase entry. The processed forms of human separase were isolated and the identity of the cleavage sites was determined by N-terminal sequencing and site-directed mutagenesis. The processed catalytic domain was found to be stably associated with the processed N-terminal fragment. Finally, by depletion of endogenous separase with antisense oligonucleotides, we report direct evidence that separase is required for high-fidelity chromosome separation in human cells.