Device for real-time monitoring of oil-in-water and suspended solids based on thermal lens spectrometry and light scattering.

A novel system suitable for simultaneous monitoring of both oil-in-water and suspended solids based on thermal lens spectroscopy and forward light scattering is presented. The technique measures the concentration of dissolved hydrocarbons and simultaneously detects single oil droplets and suspended particles separately. The device was tested with injection water samples from an on-field water treatment plant, and hydrocarbon concentrations were measured with a precision better than 5% in the range of up to 100 ppm, reaching resolutions as low as 0.03 ppm. Particle detection was tested with model samples of dyed and undyed polystyrene spheres acting as absorption and scattering centers, which simulated oil droplets and suspended solids, respectively. We show that particles of different sizes are distinguished by the magnitude of the perturbations introduced in the signals, and their concentrations can be measured independently of dissolved components.

Laser-based single-axon transection for high-content axon injury and regeneration studies.

The investigation of the regenerative response of the neurons to axonal injury is essential to the development of new axoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that the ability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We show that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely induce damage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axon regeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectories and established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. The characterization of the motile response of axotomized RGC-5 cells showed that cells that were capable of repair or regrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells with long axons could not recover their injured axons, and such cells were much more motile. The platform we describe allows highly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.

Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.

The failure of blood vessels to revascularize ischemic neural tissue represents a significant challenge for vascular biology. Examples include proliferative retinopathies (PRs) such as retinopathy of prematurity and proliferative diabetic retinopathy, which are the leading causes of blindness in children and working-age adults. PRs are characterized by initial microvascular degeneration, followed by a compensatory albeit pathologic hypervascularization mounted by the hypoxic retina attempting to reinstate metabolic equilibrium. Paradoxically, this secondary revascularization fails to grow into the most ischemic regions of the retina. Instead, the new vessels are misdirected toward the vitreous, suggesting that vasorepulsive forces operate in the avascular hypoxic retina. In the present study, we demonstrate that the neuronal guidance cue semaphorin 3A (Sema3A) is secreted by hypoxic neurons in the avascular retina in response to the proinflammatory cytokine IL-1ß. Sema3A contributes to vascular decay and later forms a chemical barrier that repels neo-vessels toward the vitreous. Conversely, silencing Sema3A expression enhances normal vascular regeneration within the ischemic retina, thereby diminishing aberrant neovascularization and preserving neuroretinal function. Overcoming the chemical barrier (Sema3A) released by ischemic neurons accelerates the vascular regeneration of neural tissues, which restores metabolic supply and improves retinal function. Our findings may be applicable to other neurovascular ischemic conditions such as stroke.

Single photon fluorescent microlithography for live-cell imaging.

Using fluorescent dyes to trigger the polymerization of a commercial polyurethane resin allows a rapid fabrication of micrometer and submicrometer sized fluorescent structures by one-photon absorption. Here, we show that standard He-Ne lasers emitting at 632.8 nm can be used to start the photopolymerization and that very low laser power is required. This procedure allows the fabrication of fiduciary fluorescent references on standard glass coverslips, mica sheets, or gold-coated coverslips for laser scanning or standard fluorescent microscopy. The biocompatibility of the polymerized resin with cells in culture was tested by growing Xenopus melanophores and a standard laser scanning microscope was used to demonstrate that it is possible to use equipment readily available in several laboratories. We show that fluorescent structure with less than 10 nm in height may be used as references in fluorescence microscopy allowing a smooth environment for cell growth. Different dyes were tested and the conditions for one-photon polymerization were outlined.

Rapid multicomponent optical protein patterning.

Cells sense spatial distributions of molecules which trigger signal transduction pathways that induce the cell to migrate or extend by remodelling the cytoskeleton. However, the influence of local and small variations of extracellular protein concentration on chemotaxis is not fully understood, due in part to the lack of simple and precise methods to pattern proteins in vitro. We recently developed a new technology to fabricate such patterns which relies on photobleaching fluorophores to adsorb proteins on a cell culture substrate: laser-assisted protein adsorption by photobleaching (LAPAP). Here we report several key improvements to LAPAP: we created arbitrary patterns made of several different proteins simultaneously, we reduced the fabrication time more than one order of magnitude and we used secondary antibodies to significantly enlarge the spectrum of proteins that can be employed. As a result, multicomponent protein gradients can be produced using reagents that are typically available in life science research laboratories on a standard inverted microscope equipped with a camera port.