A Map of Functional Synaptic Connectivity in the Mouse Anteroventral Cochlear Nucleus

The cochlear nuclei are the first central processors of auditory information and provide inputs to all the major brainstem and midbrain auditory nuclei. Although the local circuits within the cochlear nuclei are understood at a cellular level, the spatial patterns of connectivity and the connection strengths in these circuits have been less well characterized. We have applied a novel, quantitative approach to mapping local circuits projecting to cells in the mouse anteroventral cochlear nucleus (AVCN) using laser-scanning photostimulation and glutamate uncaging. The amplitude and kinetics of individual evoked synaptic events were measured to reveal the patterns and strengths of synaptic connections. We found that the two major excitatory projection cell classes, the bushy and T-stellate cells, receive a spatially broad inhibition from D-stellate cells in the AVCN, and a spatially confined inhibition from the tuberculoventral cells of the dorsal cochlear nucleus. Furthermore, T-stellate cells integrate D-stellate inhibition from an area that spans twice the frequency range of that integrated by bushy cells. A subset of both bushy and T-stellate cells receives inhibition from an unidentified cell population at the dorsal–medial boundary of the AVCN. A smaller subset of cells receives local excitation from within the AVCN. Our results show that inhibitory circuits can have target-specific patterns of spatial convergence, synaptic strength, and receptor kinetics, resulting in different spectral and temporal processing capabilities

Fast localized wavefront correction using area-mapped phase-shift interferometry

We propose an innovative method for localized wavefront correction based on area-mapped phase-shift (AMPS) interferometry. In this Letter, we present the theory and then experimentally compare it with a previously demonstrated method based on spot-optimized phase-stepping (SOPS) interferometry. We found that AMPS outperforms SOPS interferometry in terms of speed by threefold, although in noisy environments the improvements may be larger. AMPS yielded similar point-spread functions (PSF) as SOPS for moderate system-induced aberrations, but yielded a slightly less ideal PSF for larger aberrations. The method described in this Letter may prove crucial for applications where the phase-stepping solution does not have sufficient speed

Experimental observation of second-harmonic generation and diffusion inside random media

We have experimentally measured the distribution of the second-harmonic intensity that is generated inside a highly-scattering slab of porous gallium phosphide. Two complementary techniques for determining the distribution are used. First, the spatial distribution of second-harmonic light intensity at the side of a cleaved slab has been recorded. Second, the total second-harmonic radiation at each side of the slab has been measured for several samples at various wavelengths. By combining these measurements with a diffusion model for second-harmonic generation that incorporates extrapolated boundary conditions, we present a consistent picture of the distribution of the second-harmonic intensity inside the slab. We find that the ratio $\ell_{2\omega}/L_c$ of the mean free path at the second-harmonic frequency to the coherence length, which was suggested by some earlier calculations, cannot describe the second-harmonic yield in our samples. For describing the total second-harmonic yield, our experiments show that the scattering parameter at the fundamental frequency \k_{1\omega}\ell_{1\omega} is the most relevant parameter in our type of samples.Comment: 10 pages, 7 figure

Second harmonic generating (SHG) nanoprobes for in vivo imaging

Fluorescence microscopy has profoundly changed cell and molecular biology studies by permitting tagged gene products to be followed as they function and interact. The ability of a fluorescent dye to absorb and emit light of different wavelengths allows it to generate startling contrast that, in the best cases, can permit single molecule detection and tracking. However, in many experimental settings, fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation, and tissue autofluorescence. Here, we demonstrate that second harmonic generating (SHG) nanoprobes can be used for in vivo imaging, circumventing many of the limitations of classical fluorescence probes. Under intense illumination, such as at the focus of a laser-scanning microscope, these SHG nanocrystals convert two photons into one photon of half the wavelength; thus, when imaged by conventional two-photon microscopy, SHG nanoprobes appear to generate a signal with an inverse Stokes shift like a fluorescent dye, but with a narrower emission. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and the signal they generate does not saturate with increasing illumination intensity. The resulting contrast and detectability of SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues

Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

<p>Abstract</p> <p>Background</p> <p>Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion.</p> <p>Methods</p> <p>For this investigation we use Second Harmonic Generation (SHG) imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties.</p> <p>Results</p> <p>We find the SHG emission attributes (directionality and relative intensity) and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen.</p> <p>Conclusions</p> <p>Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.</p

Region-Specific Microstructure in the Neonatal Ventricles of a Porcine Model

© 2018, Biomedical Engineering Society. The neonate transitions from placenta-derived oxygen, to supply from the pulmonary system, moments after birth. This requires a series of structural developments to divert more blood through the right heart and onto the lungs, with the tissue quickly remodelling to the changing ventricular workload. In some cases, however, the heart structure does not fully develop causing poor circulation and inefficient oxygenation, which is associated with an increase in mortality and morbidity. This study focuses on developing an enhanced knowledge of the 1-day old heart, quantifying the region-specific microstructural parameters of the tissue. This will enable more accurate mathematical and computational simulations of the young heart. Hearts were dissected from 12, 1-day-old deceased Yorkshire piglets (mass: 2.1–2.4kg, length: 0.38–0.51m), acquired from a breeding farm. Evans blue dye was used to label the heart equator and to demarcate the left and right ventricle free walls. Two hearts were used for three-dimensional diffusion-tensor magnetic resonance imaging, to quantify the fractional anisotropy (FA). The remaining hearts were used for two-photon excited fluorescence and second-harmonic generation microscopy, to quantify the cardiomyocyte and collagen fibril structures within the anterior and posterior aspects of the right and left ventricles. FA varied significantly across both ventricles, with the greatest in the equatorial region, followed by the base and apex. The FA in each right ventricular region was statistically greater than that in the left. Cardiomyocyte and collagen fibre rotation was greatest in the anterior wall of both ventricles, with less dispersion when compared to the posterior walls. In defining these key parameters, this study provides a valuable insight into the 1-day-old heart that will provide a valuable platform for further investigation the normal and abnormal heart using mathematical and computational models

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Order versus Disorder: in vivo bone formation within osteoconductive scaffolds

In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration

Analysis of RNA Binding by the Dengue Virus NS5 RNA Capping Enzyme

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5′ end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the KD for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5′ phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5′ di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented