Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues

Role of carbon dioxide and ion transport in the formation of sub-embryonic fluid by the blastoderm of the Japanese quail

1. The explanted blastoderm of the Japanese quail was used to explore the role of ions and carbon dioxide in determining the rate of sub-embryonic fluid (SEF) production between 54 and 72 h of incubation. 2. Amiloride, an inhibitor of Na+/H+ exchange, at concentrations of 10-3 to 10-6 M substantially decreased the rate of SEF production when added to the albumen culture medium. N-ethylmaleimide, an inhibitor of V type H+ ATPase, also decreased this rate but only to a small extent at the highest dose applied, 10-3 M. Both inhibitors had no effect on SEF production when added to the SEF. 3. The inhibitors of cellular bicarbonate and chloride exchange, 4-acetamido-4-'isothiocyano-2, 2-'disulphonic acid (SITS) and 4,4'diisothiocyanostilbene-2,2-'disulphonic acid (DIDS), had no effect upon SEF production. 4. Ouabain, an inhibitor of Na+/K+ ATPase, decreased SEF production substantially at all concentrations added to the SEF (10-3 to 10-6 M). Three sulphonamide inhibitors of carbonic anhydrase, acetazolamide, ethoxzolamide and benzolamide, decreased SEF production when added to the SEF at concentrations of 10-3 to 10-6 M. Benzolamide was by far the most potent. Neither ouabain nor the sulphonamides altered SEF production when added to the albumen culture medium. 5. Using a cobalt precipitation method, carbonic anhydrase activity was localised to the endodermal cells of the area vasculosa. The carbonic anhydrase activity was primarily associated with the lateral plasma membranes, which together with the potent inhibitory effect of benzolamide, suggests the carbonic anhydrase of these cells is the membrane-associated form, CA IV. 6. The changes in SEF composition produced by inhibitors were consistent with the production of SEF by local osmotic gradients. 7. It is concluded that a Na+/K+ ATPase is located on the basolateral membranes of the endodermal cells of the area vasculosa , and that a sodium ion/hydrogen ion exchanger is located on their apical surfaces. Protons for this exchanger would be provided by the hydration of CO2 catalysed by the membrane-associated carbonic anhydrase. Furthermore, it is proposed that the prime function of the endodermal cells of the area vasculosa is the production of SEF

An efficient method for the incompressible Navier-Stokes equations on irregular domains with no-slip boundary conditions, high order up to the boundary

Common efficient schemes for the incompressible Navier-Stokes equations, such as projection or fractional step methods, have limited temporal accuracy as a result of matrix splitting errors, or introduce errors near the domain boundaries (which destroy uniform convergence to the solution). In this paper we recast the incompressible (constant density) Navier-Stokes equations (with the velocity prescribed at the boundary) as an equivalent system, for the primary variables velocity and pressure. We do this in the usual way away from the boundaries, by replacing the incompressibility condition on the velocity by a Poisson equation for the pressure. The key difference from the usual approaches occurs at the boundaries, where we use boundary conditions that unequivocally allow the pressure to be recovered from knowledge of the velocity at any fixed time. This avoids the common difficulty of an, apparently, over-determined Poisson problem. Since in this alternative formulation the pressure can be accurately and efficiently recovered from the velocity, the recast equations are ideal for numerical marching methods. The new system can be discretized using a variety of methods, in principle to any desired order of accuracy. In this work we illustrate the approach with a 2-D second order finite difference scheme on a Cartesian grid, and devise an algorithm to solve the equations on domains with curved (non-conforming) boundaries, including a case with a non-trivial topology (a circular obstruction inside the domain). This algorithm achieves second order accuracy (in L-infinity), for both the velocity and the pressure. The scheme has a natural extension to 3-D.Comment: 50 pages, 14 figure

Continuous Wavelets on Compact Manifolds

Let $\bf M$ be a smooth compact oriented Riemannian manifold, and let $\Delta_{\bf M}$ be the Laplace-Beltrami operator on ${\bf M}$. Say 0 \neq f \in \mathcal{S}(\RR^+), and that $f(0) = 0$. For $t > 0$, let $K_t(x,y)$ denote the kernel of $f(t^2 \Delta_{\bf M})$. We show that $K_t$ is well-localized near the diagonal, in the sense that it satisfies estimates akin to those satisfied by the kernel of the convolution operator $f(t^2\Delta)$ on \RR^n. We define continuous ${\cal S}$-wavelets on ${\bf M}$, in such a manner that $K_t(x,y)$ satisfies this definition, because of its localization near the diagonal. Continuous ${\cal S}$-wavelets on ${\bf M}$ are analogous to continuous wavelets on \RR^n in \mathcal{S}(\RR^n). In particular, we are able to characterize the H$\ddot{o}$lder continuous functions on ${\bf M}$ by the size of their continuous ${\mathcal{S}}-$wavelet transforms, for H$\ddot{o}$lder exponents strictly between 0 and 1. If $\bf M$ is the torus \TT^2 or the sphere $S^2$, and $f(s)=se^{-s}$ (the ``Mexican hat'' situation), we obtain two explicit approximate formulas for $K_t$, one to be used when $t$ is large, and one to be used when $t$ is small

Two-Loop ϕ4\phi^4-Diagrams from String Theory

Using the {\em cutting and sewing} procedure we show how to get Feynman diagrams, up to two-loop order, of $\Phi^{4}$-theory with an internal SU(N) symmetry group, starting from tachyon amplitudes of the open bosonic string theory. In a properly defined field theory limit, we easily identify the corners of the string moduli space reproducing the correctly normalized field theory amplitudes expressed in the Schwinger parametrization.Comment: 28 pages, 12 figure

Non-local SFT Tachyon and Cosmology

Cosmological scenarios built upon the generalized non-local String Field Theory and $p$-adic tachyons are examined. A general kinetic operator involving an infinite number of derivatives is studied as well as arbitrary parameter $p$. The late time dynamics of just the tachyon around the non-perturbative vacuum is shown to leave the cosmology trivial. A late time behavior of the tachyon and the scale factor of the FRW metric in the presence of the cosmological constant or a perfect fluid with $w>-1$ is constructed explicitly and a possibility of non-vanishing oscillations of the total effective state parameter around the phantom divide is proven.Comment: 17 pages, LaTeX; v2: JHEP3 class is used, references adde

Perturbative Computation of the Gluonic Effective Action via Polyaokov's World-Line Path Integral

The Polyakov world-line path integral describing the propagation of gluon field quanta is constructed by employing the background gauge fixing method and is subsequently applied to analytically compute the divergent terms of the one (gluonic) loop effective action to fourth order in perturbation theory. The merits of the proposed approach is that, to a given order, it reduces to performing two integrations, one over a set of Grassmann and one over a set of Feynman-type parameters through which one manages to accomodate all Feynman diagrams entering the computation at once.Comment: 21 page