Ellipsometry of thin films of biological objects under conditions of total internal reflection

An analysis of the ellipsometric parameters of the reflected light from the prism test material air system is carried out when circularly polarized light is incident on it under the conditions of the onset of the phenomenon of total internal reflection. At the onset of total internal reflection, the ellipsometry parameter shows high variability with the angle of incidence, in contrast to the parameter r0. It is shown that TIR occurs when the angle of incidence is not equal to the critical angle of the adjacent media for two different materials, these angles differ from each other. In the case of a film, the TIR phenomenon occurs at an angle equal to the critical angle at the prism-air interface and does not depend on the film material. The results obtained show the high efficiency of using the ellipsometric method together with circularly polarized incident radiation for diagnostics of thin films made of biological material.

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

Light Scattering by a Subwavelength Plasmonic Array: Anisotropic Model

We calculate the light transmission by a subwavelength plasmonic array using the boundary element method for parallel cylinders with different cross-sections: circular or elliptic with axis ratio 4:1. We demonstrate that plasmonic resonance is sharper for the case of horizontal ellipses. This structure is susceptible to refractive index variations in the media since the high derivatives of reflection and transmission coefficients are near the angle of total internal reflection. To obtain an approximate analytical expression, we used the model of a metallic layer. We explore the “sandwich” structure with an anisotropic film between two dielectrics and demonstrate its quantitative agreement with numerical results.

Counting fluorescently labeled proteins in tissues in the spinning disk microscope using single-molecule calibrations

Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning-disk confocal (SDC) microscopy, commonly used for imaging in tissues, that uses single-step bleaching kinetics to estimate the single-fluorophore intensity. To cross-check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total-internal-reflection (TIRF) and epifluorescence microscopes. We applied this calibration method to quantify the number of EB1-eGFP proteins in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1-eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1-eGFP from the microtubules as wells as the GTP-tubulin cap size. Our results show the unexplored potential of single-molecule imaging using spinning disk confocal microscopy and provide a straight-forward method to count the absolute number of fluorophores in tissues which can be applied to a wide range of biological systems and imaging techniques.

Design and simulation of liquid infiltrated photonic crystal fibre in terahertz frequencies

Ethanol, methanol and water are polar solvents with similar physical properties albeit contrasting chemical properties. Therefore, it is essential to provide accurate and reliable methods for detecting these liquids. In this paper, a novel liquid infiltrated photonic crystal fibre for ethanol, methanol and water sensing is introduced. The novel structure is modelled, simulated and analysed in the terahertz (THz) region using a full vectorial finite element method. It is shown that the THz light, which is guided using modified total internal reflection, is confined within the infiltrated analytes with negligible losses. For the detection of infiltrated liquids at 1.6 THz operating frequency, the proposed fibre demonstrates high sensitivities up to 99.73% and confinement losses in the order of 10−4 dB/m. Manufacturing of the proposed fibre is feasible using existing fabrication technologies and it is envisaged that the fibre may provide a solution to existing challenges in detecting common polar solvents.

Improved immunoassay sensitivity and specificity using single-molecule colocalization

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still suffers some notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background arising from the interaction of detection antibodies with the assay substrate or interferents in the sample matrix. Single-Molecule Colocalization Assay (SiMCA) overcomes this problem by employing total internal reflection fluorescence (TIRF) microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized fluorescent signals, we can essentially eliminate the confounding effects of background produced by non-specific binding of detection antibodies. We further employed a normalization strategy to account for the heterogeneous distribution of the capture antibodies, greatly improving the reproducibility of our measurements. In a series of experiments with TNF-α, we show that SiMCA can achieve a three-fold lower limit of detection compared to conventional single-color assays using the same antibodies and exhibits consistent performance for assays performed in complex specimens such as chicken serum and human blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

On Surface-Plasmon-Polariton Waves Excited in the Turbadar–Otto Configuration

A local minimum in the plot of linear reflectance versus angle of incidence, on its own, is insufficient to identify a surface-plasmon-polariton wave (SPPW). Further checks are required in order to confirm the identity of a SPPW. The wavenumber should be compared with that extracted from the dispersion relation for the corresponding canonical boundary-value problem. Also, for prism-coupled configurations such as the Turbadar–Otto configuration which are based on SPPW-excitation via evanescent waves, the angle of incidence should be greater than the critical angle needed for total internal reflection.

Orbiting Light Around the Outer Surface of Dielectric Microspheres

Circulating light in the total internal reflection within dielectric spheres or disks is called the whispering gallery mode (WGM), which by itself is highly sensitive to its surface and capable of detecting viruses and single atomic ions. The detection site of the sensors using WGM is created by the evanescent light from the circulating light inside spheres. On the other hand, there have been no reports of observation or discussion of the light orbiting outside the surface of dielectric microspheres. Here we report light orbiting the outer surface of a dielectric microspheres different from the WGM. We observed anomalously enhanced Raman spectrum at the periphery of 3 μm diameter polystyrene (PS) microspheres on a silicon nitride (SiN) film using Raman microscopy. The wavelength intensity of this enhanced Raman spectrum was accompanied by periodic changes due to interference. These features may lead to the development of high-sensitive sensors and optical devices.