Forbidden light detection from single molecules

We present a new concept for ultrasensitive detection of surface-generated fluorescence which is made possible by a new optical module. The detection method leads to an enhancement in fluorescence collection efficiency to more than 65% of the total of emitted light, whereas high-aperture microscope objectives are able to collect 44% at best. Moreover, by employing this new optical module, the detection volume can be restricted to approximately 10(-17) L. This allows for an exceptional discrimination of bulk-generated against surface-generated fluorescence, which may be of great value when surface-binding processes are monitored. We demonstrate the performance of the new detection system by detecting single fluorescent molecules and by determining antigen concentrations down to 5 fmol.     

Microchannel avalanche photodiode with broad linearity range

Design and operation principles of a new microchannel avalanche photodiode with an avalanche multiplication coefficient of up to 10⁵ and a linearity range expanded by an order of magnitude compared to the existing analogs are described. A distinctive feature of the new device design is that the forward-biased p_n junctions (playing the role of individual quenching resistors) are situated under each pixel. This circumstance ensures an increase in the density of multiplication channels up to 40000 mm-2 at a 100% sensitive device area.     

Ultrasensitive coincidence fluorescence detection of single DNA molecules

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.     

Detecting intramolecular conformational dynamics of single molecules in short distance range with subnanometer sensitivity

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nanoparticles and nanodevices with nanometer spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3-8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to subnanometer distance changes in a FRET-insensitive short-range at the single molecule level. A ParM protein mutant labeled with two rhodamines works as a single molecule adenosine 5'-diphosphate (ADP) sensor that has 20 times brighter fluorescence signal in the ADP bound state than the unbound state. Single molecule time trajectories show discrete transitions between fluorescence on and off states that can be directly ascribed to ADP binding and dissociation events. The conformational changes observed with 20:1 contrast are only 0.5 nm in magnitude and are between crystallographic distances of 1.6 and 2.1 nm, demonstrating exquisite sensitivity to short distance scale changes. The systems also allowed us to gain information on the photophysics of self-quenching induced by rhodamine stacking: (1) photobleaching of either of the two rhodamines eliminates quenching of the other rhodamine fluorophore and (2) photobleaching from the highly quenched, stacked state is only 2-fold slower than from the unstacked state.     

Simultaneous high gain and wide dynamic range in a model of bacterial chemotaxis

Mathematical modelling and sensitivity analysis of the signal transduction pathway underlying chemotaxis in Escherichia coli suggests a mechanism for high sensitivity over a dynamic range of five orders of magnitude. The analysis reveals that the enhancement in sensing ability occurs in the signal receiving module that is comprised of ligand binding, change of occupancy and change of receptor activities. The clustering of receptors contributes to the signal capability by exploiting interactions between receptors for the activity change. The role of the autophosphorylation of the histidine kinase CheA and the phosphotransfer to the response regulator protein CheY is to relay the signal to the cell's motor apparatus at little expense to the sensitivity at low stimuli. The results also provide insight on the values of kinetic parameters that maximise the efficiency of the signalling pathway.     

Rapid and efficient detection of single chromophore molecules in aqueous solution

The first experiments on the detection of single fluorescent molecules in a flowing stream of an aqueous solution with high total efficiency are reported. A capillary injection system for sample delivery causes all the dye molecules to pass in a diffusion-broadened stream within a fast-moving sheath flow, through the center of the tightly focused laser excitation beam. Single-molecule detection with a transit time of ~1 ms is accomplished with a high-quantum-efficiency single-photon avalanche diode and a low dead-time time-gating circuit for discrimination of Raman-scattered light from the solvent.     

Vibration-frequency transducer for pressure with a broad measuring range

Measurement Techniques -     

Compact and high-precision range finder with wide dynamic range andits application

Described is a new range finder using a self-mixing laser diode (SM-LD). The range finder has a high accuracy of ±0.15% and a wide dynamic range of 0.2-1 m using only one sensor head. Compared to ultrasonic range finders, the light beam of this laser range finder can be focused and scanned. The feasibility study shows a possible application of the range finder to a visual sensor of a robot. The proposed range finder has been successfully applied as an infrared (IR) active type range finder of a single-lens reflex camera     

Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores

Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of beta-human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.     

Biofabrication of broad range antibacterial and antibiofilm silver nanoparticles

Silver nanoparticles (AgNPs) were biosynthesized via a green route using ten different plants extracts (GNP1‐ Caryota urens, GNP2‐Pongamia glabra, GNP3‐ Hamelia patens, GNP4‐Thevetia peruviana, GNP5‐Calendula officinalis, GNP6‐Tectona grandis, GNP7‐Ficus petiolaris, GNP8‐ Ficus busking, GNP9‐ Juniper communis, GNP10‐Bauhinia purpurea). AgNPs were tested against drug resistant microbes and their biofilms. These nanoparticles (NPs) were characterised using UV‐vis spectroscopy, transmission electron microscope, Fourier transform infrared spectroscopy, X‐ray diffraction and Image J software. Most of the AgNPs were distributed over a range of 1 of 60 nm size. The results indicated that AgNPs were antibacterial in nature without differentiating between resistant or susceptible strains. Moreover, the effect was more prominent on Gram negative bacteria then Gram positive bacteria and fungus. AgNPs inhibited various classes of microbes with different concentration. It was also evident from the results that the origin or nature of extract did not affect the activity of the NPs. Protein and carbohydrate leakage assays confirmed that the cells lysis is one of the main mechanisms for the killing of microbes by green AgNPs. This study suggests that the action of AgNPs on microbial cells resulted into cell lysis and DNA damage. Excellent microbial biofilm inhibition was also seen by these green AgNPs. AgNPs have proved their candidature as a potential antibacterial and antibiofilm agent against MDR microbes.Inspec keywords: silver, nanoparticles, antibacterial activity, nanofabrication, microorganisms, ultraviolet spectra, visible spectra, transmission electron microscopy, Fourier transform infrared spectra, X‐ray diffraction, proteins, DNA, nanomedicine, biomedical materials, cellular biophysicsOther keywords: biofabrication, broad range antibacterial nanoparticles, antibiofilm silver nanoparticles, plant extract contribution, drug resistant microbes, UV‐vis spectroscopy, transmission electron microscope, Fourier transform infrared spectroscopy, X‐ray diffraction, Image J software, resistant strains, susceptible strains, Gram positive bacteria, fungus, protein leakage assays, carbohydrate leakage assays, cell lysis, DNA damage, Ag     

Coupling stimuli-responsive magnetic nanoparticles with antibody-antigen detection in immunoassays

Because current homogeneous immunoassays show some limitations, particularly low sensitivity, we developed a new immunoassay to overcome these limitations. The approach was based on magnetic nanoparticles with a thermoresponsive polymer layer, a negatively charged polymer, and streptavidin-biotin-based antibody-antigen detection and yielded higher sensitivity than commonly used heterogeneous immunoassays. Because no special equipment is needed, it can be applied to currently available absorbance-based systems for high-throughput assays.     

Ultrasensitive fluorescence detection of single protein molecules manipulated electrically on Au nanowire

Proteins assembled on an Au nanowire are manipulated by an electrical potential applied on the nanowire, which leads to the modulation of molecular fluorescence. The molecular modality can be unequivocally correlated with the modulated fluorescence, which enables the specific fluorescence from a single target protein to be unambiguously distinguished from background noise and nonspecific fluorescence. As demonstrated through a thrombin assay, this simple method can significantly improve the sensitivity and specificity of the protein detection down to the single molecule level.     

Label-free detection of single protein molecules using deep UV fluorescence lifetime microscopy

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.     

Ion-exchanged glass waveguides with low birefringence for a broad range of waveguide widths

Optical communications networks require integrated photonic components with negligible polarization dependence, which typically means that the waveguides must feature very low birefringence. Recent studies have shown that waveguides with low birefringence can be obtained, e.g., by use of silica-on-silicon waveguides or buried ion-exchanged glass waveguides. However, many integrated photonic circuits consist of waveguides with varying widths. Therefore low birefringence is consequently required for waveguides having different widths. This is a difficult task for most waveguide fabrication technologies. We present experimental results on waveguide birefringence for buried silver-sodium ion-exchanged glass waveguides. We show that the waveguide birefringence of the order of 10(-6) for waveguide mask opening widths ranging from 2 to 10 microm can be obtained by postprocessing the sample through annealing at an elevated temperature. The measured values are in agreement with the values calculated with our modeling software for ion-exchanged glass waveguides. This unique feature of ion-exchanged waveguides may be of significant importance in a wide variety of integrated photonic circuits requiring polarization-independent operation.     

Calibration method using a single retarder to simultaneously measure polarization and fully characterize a polarimeter over a broad range of wavelengths

A method has been developed to improve the accuracy with which the polarization state of light can be characterized by the rotating quarter-wave plate technique. Through detailed analysis, verified by experiment, we determine the positions of the optic axes of the retarder and linear polarizer, and the wave plate retardance, to better than 1° for typical signal-to-noise ratios. Accurate determination of the Stokes parameters can be achieved using a single wave plate for a wide range of optical wavelengths using this technique to determine the precise retardance at each of the wavelengths of interest.     

Recapturing and trapping single molecules with a solid-state nanopore

The development of solid-state nanopores, inspired by their biological counterparts, shows great potential for the study of single macromolecules. Applications such as DNA sequencing and the exploration of protein folding require control of the dynamics of the molecule's interaction with the pore, but DNA capture by a solid-state nanopore is not well understood. By recapturing individual molecules soon after they pass through a nanopore, we reveal the mechanism by which double-stranded DNA enters the pore. The observed recapture rates and times agree with solutions of a drift-diffusion model. Electric forces draw DNA to the pore over micrometer-scale distances, and upon arrival at the pore, molecules begin translocation almost immediately. Repeated translocation of the same molecule improves measurement accuracy, offers a way to probe the chemical transformations and internal dynamics of macromolecules on sub-millisecond time and sub-micrometre length scales, and demonstrates the ability to trap, study and manipulate individual macromolecules in solution.     

Scattering phase function measurements on single particles and on particle ensembles

We present measurements of the light-scattering phase function of selected carbon and ash particles in the geometric-optics regime in which the particle diameter is much larger than the wavelength of the light source. Measurements were performed on both single particles and particle ensembles. This was accomplished with two separate methodologies: an electrodynamic levitator for single-particle measurements and a particle feeder for the ensemble measurements. For each methodology, two irradiation sources were utilized: an argon-ion laser (lambda = 496 nm) and a xenon lamp. Results of the normalized phase functions are presented.     

Simultaneous topographic and fluorescence imaging of single DNA molecules for DNA analysis with a scanning near-field optical/atomic force microscope.

High-resolution fluorescence imaging of lambda-phage DNA molecules, intercalated with the dye YOYO-1, has been performed by a SNOM/AFM based on a bent-type optical fiber probe. A modified design of the optical probe has been made, and successful near-field optical resolution has been obtained for the strongly stretched lambda-phage DNA molecules. The best optical resolution was estimated at 45 nm for the dye-intercalated single lambda-DNA molecules by a mean width evaluation. In our comparison between the far-field fluorescence and high-resolution near-field fluorescence images for the DNA, it has been found that the near-field images much better defined the intercalation state of the dye. Finally, the relation between the DNA shapes and the dye distribution states, and the discrimination between the double-stranded and single-stranded DNA molecules, are discussed by comparing the topography and fluorescence images of the SNOM/AFM.     

Heteroepitaxial growth of GaSb nanotrees with an ultra-low reflectivity in a broad spectral range

We report on the growth of GaSb nanotrees on InAs { ?1 ?1 ?1}(B) substrates by chemical beam epitaxy. GaSb nanotrees form by the nucleation of Ga droplets on the surface of < ?1 ?1 ?1>(B) oriented GaSb nanowires followed by the epitaxial growth of branches catalyzed by these Ga droplets. In the tip region, the trunks of the GaSb nanotrees are periodically twinned, which is attributed to a change of the effective V/III ratio at the later stage of growth as a consequence of the change in surface structure. The reflectivity of a forest of nanotrees was measured for a broad spectral range and compared to the reflectivity of a GaSb ( ?1 ?1 ?1)(B) wafer and of GaSb nanowires. At wavelengths from 500 to 1700 nm, the presence of GaSb nanotrees decreased the reflection by three orders of magnitude compared to a blank GaSb substrate.