Spectral Phasor Analysis for Brillouin Microspectroscopy

Brillouin Light Scattering (BLS) spectroscopy allows for the all-optical measurement of the hypersonic velocities in a sample, from which one can extract high-frequency elastic moduli. Recent advances in high resolution optical imaging spectrometers have made it conducive to studying live biological samples and further its implementation as an imaging modality for the life sciences. One major challenge in this context is the relatively weak BLS signal together with the subtle BLS spectral variations observed in many biological samples. Here we show that using spectral phasor analysis one can more easily distinguish variations in noisy spectra compared to standard least-squares (LS) fitting. There is no fitting in phasor analysis, and it is both robust in regards to unaccounted for functional variations in the spectra, and orders of magnitude faster than LS-fitting. As such it can prove particularly useful for increasing contrast in Brillouin imaging as well as for high-throughput BLS applications such as cell-sorting and medical diagnostics especially for statistically compromised or noisy data

Defining a Superlens Operating Regime for Imaging Fluorescent Molecules

It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such “lenses” is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix) calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close ( nm) or too far ( nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating structure (such as in the far-field superlens design previously proposed [1]) or a fast near-field scanning probe, it could provide a means for fast fluorescent imaging with sub-diffraction limit resolution

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

Background: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/ antigen binding. Methodology/Principal Findings: We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. Conclusions/Significance: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformationa

Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites

Hormone-regulated expansins : expression, localization, and cell wall biomechanics in Arabidopsis root growth

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young’s modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN: XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fouriertransform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.peer-reviewe

Understanding the insulating phases of disordered materials through crosstalk and Coulomb drag experiments

In this study we experimentally explore how the phenomenon of Coulomb drag and crosstalk can be used to probe the size of charge-density fluctuations and coupling mechanisms of Anderson insulators. We study a doped amorphous semiconductor (silicon-niobium), which from conventional transport measurements is understood to be a Coulomb glass at low dopant concentrations. Calculations and experimental results are presented for the linear-response Coulomb drag coefficients when one or both layers consist of various insulating states. It is found that the screening properties only become weak enough for accurate analysis when the material is deep in the insulating phase, and a linear-response transresistance is only obtainable over a very narrow range of experimental parameters. We uncover an additional regime that becomes dominant at higher driving-currents in which the Coulomb drag coefficient is negative at the lowest temperatures probed. We also present results for the non-linear dependence of the induced field at low driving currents in the silicon-niobium layers of certain bilayer samples. We suggest that this is related to the extended non-linearity range of the Coulomb drag coefficients with respect to the driving current

Field and intensity profiles of superlens images.

<p>(a) Contour plot of the -component of the electric-field amplitude (, normalized to unity) in the image plane for a GFP-like source (dipole axis in -direction). Dipole is located at nm in front of the stacked superlens () discussed in text. The metal filling factor “” has been adjusted so that Eqn. 3 is true for nm. (<b>b</b>) Electric field intensity for the superlens image of a GFP-like source when its dipole axis is parallel (in the -direction) [left] and perpendicular (in -direction) [right]. The intensity profile of the image in the direction at is plotted as a function of the source-superlens distance (). For each constant- slice has been adjusted so that Eqn. 3 is true at the respective .</p

Imaging a GFP-like source (dipole axis in -direction) with an optimized superlens.

<p>(a) Peak image intensity as a function of source-superlens distance , including (<i>open circles</i>) and neglecting (<i>solid circles</i>) modifications to the decay-rate from non-radiative coupling to the superlens. (b) Estimated resolution for the same setup. The dashed vertical line in both plots represents the distance at which Eqn. 3 holds. All superlens parameters are the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007963#pone-0007963-g003" target="_blank">Fig. 3a & b</a>.</p