A simple method for examining organotypic CNS cultures with nomarski optics

Summary This paper describes a method for examination of living organotypic cultures of CNS with Nomarski differential interference-contrast optics. Cultures grown in Maximow assemblies. which promote the best differentiation of the tissue but are optically faulty, are transferred for Nomarski observation to a simple sandwich chamber which combines the optical perfection of the usual sandwich chamber with the flexibility and safeguarding of sterility characteristic of the Maximow assembly. Thus cultures can be transferred repeatedly between their maintenance and observation chambers. In the resulting microscopic images, it is posible to visualize delicate unmyelinated fibers, myelinated cell bodies and other features which are normally impossible to demonstrate in living cultures as well as to improve the images of other structures such as large neuronal perikarya and myelinated axons. This work was supported in part by NIH Grant NS 11425     

New photocrosslinkable polymers for second-order nonlinear optics

Among all the strategies developed by chemists to stabilize NLO properties of poled amorphous polymeric films, one of the most efficient is the formation of a network into which NLO moieties are covalently bound by both ends after their alignment under a DC electric field. For that purpose, complementary chemical groups can be used, reacting either thermally or photochemically. In our work, the use of photocrosslinkable groups (cinnamate and furylacrylate) has been investigated. In that case, photocrosslinking reactions occur between the photocrosslinkable groups functionalized into NLO molecules and the same photocrosslinkable groups appended via an alkyl spacer group into a polymer acting as the matrix. After synthesis of the different compounds and film realization, the photocrosslinking process was characterized by UV-visible spectroscopy, thus giving evidence of the higher efficiency of the furylacrylate-based system with respect to the cinnamate-based one. The alkyl spacer group was recognized as playing a crucial role in furthering the dimerization reactions between the polymer and the NLO moieties. Evolution of the maximal absorption band corresponding to the NLO moiety itself (around 500 nm) was interpreted through UV-visible spectroscopic studies using different model compounds. This was not due to NLO moiety damage, as first feared, but only to a modification in the surrounding polarity during the crosslinking process. The furylacrylate-based system was then poled and photocrosslinked in the poled state, yielding a material with excellent optical quality and very stable optical nonlinearity.     

Sensitivity of corneal biomechanical and optical behavior to material parameters using design of experiments method

The optical performance of the human cornea under intraocular pressure (IOP) is the result of complex material properties and their interactions. The measurement of the numerous material parameters that define this material behavior may be key in the refinement of patient-specific models. The goal of this study was to investigate the relative contribution of these parameters to the biomechanical and optical responses of human cornea predicted by a widely accepted anisotropic hyperelastic finite element model, with regional variations in the alignment of fibers. Design of experiments methods were used to quantify the relative importance of material properties including matrix stiffness, fiber stiffness, fiber nonlinearity and fiber dispersion under physiological IOP. Our sensitivity results showed that corneal apical displacement was influenced nearly evenly by matrix stiffness, fiber stiffness and nonlinearity. However, the variations in corneal optical aberrations (refractive power and spherical aberration) were primarily dependent on the value of the matrix stiffness. The optical aberrations predicted by variations in this material parameter were sufficiently large to predict clinically important changes in retinal image quality. Therefore, well-characterized individual variations in matrix stiffness could be critical in cornea modeling in order to reliably predict optical behavior under different IOPs or after corneal surgery.     

Enhanced-contrast two-photon optogenetic pH sensing and pH-resolved brain imaging

We present experiments on cell cultures and brain slices that demonstrate two-photon optogenetic pH sensing and pH-resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two-photon fluorescence readout from a high-brightness genetically encoded yellow-fluorescent-protein-based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two-photon-excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways—via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high-contrast optogenetic deep-brain pH sensing and pH-resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain.     

The odyssey of new production

In studies of primary production of the open ocean, the measurement of new production is often considered a measure of the degree of eutrophication. Because of new research this is questionable, which in our opinion, calls for a refinement to the original concept. We believe that the measure of variable fluorescence is pivotal to a new understanding. Our research enforces the growing conviction that the measurement of Fv/Fm can be interpreted as an analogue for nutrient stress. We measured variable fluorescence in axenic cultures at the CCMP at the Bigelow Laboratory. The Fv/Fm of cultures, upon transfer to new media reached a maximum followed by a decline after approximately 30 days. The rate of decline does not appear to be species specific. Most of the clones remained relatively high after 30 days. The high Fv/Fm values observed in nutrient-replete cultures are not characteristic of the oligotrophic surface waters off Florida and the Bahamas, but are approached in the eutrophic waters of the Gulf of Maine. In contrast, Fv/Fm measurements of attached macroalgae and coral zooxanthellae are characteristically much higher than microalgae of either oligotrophic or eutrophic regions. Accordingly, we advance the case for interpretation of Fv/Fm in terms of nutrient stress in ecological studies and advocate that the old concept of new production should be modified.     

Electronic structure and polarizabilities of some heterocycles: I. Hydroxypyrazoles and related systems

The (hyper)polarizabilities of different tautomer forms of hydroxypyrazoles and pyrazolones have been calculated by the finite-field procedure in the MNDO approximation and the sum of states formalism in the PPP approximation, with all singly- and doubly-excited electronic configurations in the CI method. It was shown that while in the ground electronic state the values of the (hyper) polarizabilities are not essentially different, in the first excited singlet Franck-Condon state an increase of the molecular polarizabilities of some tautomers is observed. This increase is attributed to a specific change in the electronic structure of the excited state, demonstrated by the localization of the electronic transition in the different pyrazolone tautomers. The electron-donor capabilities of phenyl-substituted hydroxypyrazoles and pyrazolones are discussed.