Robert Molenaar

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.

Acousto-optic-assisted diffuse optical tomography.

We introduce and experimentally demonstrate acousto-optic-assisted diffuse optical tomography (DOT) using a holography-based acousto-optic setup. The method is based on probing a scattering medium with a localized acoustical modulation of the phase of the scattered light. The optical properties of the scattering medium are recovered with ultrasound-limited resolution by applying DOT reconstruction methods on a set of the measured intensities of light, modulated at different locations throughout the medium.

Feasibility of quantitative determination of local optical absorbances in tissue-mimicking phantoms using acousto-optic sensing

We have investigated the application of ultrasound modulation of coherent light for quantitative determination of local absorbances in tissue-mimicking phantoms. An Intralipid-based phantom model, which mimics a blood vessel in human tissue, was used. The detection technique was based on homodyne parallel speckle detection in transmission mode. Based on a comparison of experimental data and Monte Carlo simulations, a quantitative correlation between local absorbances of the phantom and the measured signal has been shown. The use of microsecond pulses of ultrasound and laser light resulted in a spatial resolution of the system of a few millimeters

Millimeter-resolution acousto-optic quantitative imaging in a tissue model system

We have investigated the application of ultrasound modulated coherent light for quantitative determination of the ratio of dye concentrations and total concentration of absorbers in a blood vessel-mimicking sample. A 3-mm-diam tube containing the mixture of dyes inside an Intralipid-based gel with optical properties similar to tissue was interrogated by two different laser wavelengths in combination with intense microsecond ultrasound bursts. The use of calibration curves allowed us to extract quantitative information on the ratio of dye concentrations with the accuracy of better than 15%, as well as on the total concentration. Furthermore, we demonstrated the feasibility to obtain a quantitative 3-D map of the absorbing structure with a spatial resolution of better than 3 mm. These findings give an outlook to apply this technique for noninvasive 3-D mapping of oxygen saturation and total concentration of hemoglobin in tissue.

Towards quantitative acousto-optic imaging in tissue

We have investigated the possibilities and limitations of the application of ultrasound modulated coherent light to obtain quantitative information of local absorbers in light-scattering objects, among which tissue. For all objects studied, the combined use of microsecond ultrasound and light pulses enabled us to construct a 3D map of local absorbers with a spatial resolution of ∼2 mm. Moreover, in relatively homogeneous model systems, mimicking a blood vessel embedded in tissue, the use of a calibration procedure allowed for a determination of the local absorbance. Speckle decorrelation times for real tissue containing blood vessels, in which appreciable motion of scatterers can exist, were found to be smaller than 1ms. These relatively short times present a major challenge for acousto-optics to be applied in living tissue systems.

Acousto-optic spectroscopy as a tool for quantitative determination of chemical compounds in tissue: a model study

We have made a comparison of various detection strategies for detection of acoustically modulated light in a scattering medium. Furthermore we have investigated the possibility to determine the local absorbance in a model system mimicking a blood vessel in tissue.

Characterization of a clinical prototype for photoacoustic mammography and some phantom studies

Performance studies of a clinical prototype for detecting tumors in the breast based on the photoacoustic effect are presented in terms of sensitivity, frequency response and resolution. Some imaging results on well characterized breast tissue phantoms with embedded tumor simulating inserts are also shown.

Microcantilever based distance control between a probe and a surface

We demonstrate a method to accurately control the distance between a custom probe and a sample on a {\mu}m to nm scale. The method relies on the closed-loop feedback on the angular deflection of an in-contact AFM microcantilever. High performance in stability and accuracy is achieved in this method by taking advantage of the small mechanical feedback path between surface and probe. We describe how internal error sources that find their origin in the microcantilever and feedback can be minimized to achieve an accurate and precise control up to 3 nm. In particular, we investigated how hysteresis effects in the feedback caused by friction forces between tip and substrate, can be minimized. By applying a short calibration procedure, distance control from contact to several micrometers probe-sample distance can be obtained with an absolute nanometer-scale accuracy. The method presented is compatible with any probe that can be fixed on a microcantilever chip and can be easily built into existing AFM systems.We demonstrate a method to control the distance between a custom probe and a sample on a μm to nm scale. The method relies on the closed-loop feedback on the angular deflection of an in-contact AFM microcantilever. High performance in stability and accuracy is achieved in this method by taking advantage of the small mechanical feedback path between surface and probe. We describe how internal error sources that find their origin in the microcantilever and feedback can be minimized to achieve an accurate and precise control up to 3 nm. In particular, we investigated how hysteresis effects in the feedback caused by friction forces between tip and substrate can be minimized. By applying a short calibration procedure, distance control from contact to several micrometers probe-sample distance can be obtained with an absolute nanometer-scale accuracy. The method presented is compatible with any probe that can be fixed on a microcantilever chip and can be easily built into existing AFM systems.

Floating and Tether-Coupled Adhesion of Bacteria to Hydrophobic and Hydrophilic Surfaces

Models for bacterial adhesion to substratum surfaces all include uncertainty with respect to the (ir)reversibility of adhesion. In a model, based on vibrations exhibited by adhering bacteria parallel to a surface, adhesion was described as a result of reversible binding of multiple bacterial tethers that detach from and successively reattach to a surface, eventually making bacterial adhesion irreversible. Here, we use total internal reflection microscopy to determine whether adhering bacteria also exhibit variations over time in their perpendicular distance above surfaces. Streptococci with fibrillar surface tethers showed perpendicular vibrations with amplitudes of around 5 nm, regardless of surface hydrophobicity. Adhering, nonfibrillated streptococci vibrated with amplitudes around 20 nm above a hydrophobic surface. Amplitudes did not depend on ionic strength for either strain. Calculations of bacterial energies from their distances above the surfaces using the Boltzman equation showed that bacteria with fibrillar tethers vibrated as a harmonic oscillator. The energy of bacteria without fibrillar tethers varied with distance in a comparable fashion as the DLVO (Derjaguin, Landau, Verwey, and Overbeek)-interaction energy. Distance variations above the surface over time of bacteria with fibrillar tethers are suggested to be governed by the harmonic oscillations, allowed by elasticity of the tethers, piercing through the potential energy barrier. Bacteria without fibrillar tethers “float” above a surface in the secondary energy minimum, with their perpendicular displacement restricted by their thermal energy and the width of the secondary minimum. The distinction between “tether-coupled” and “floating” adhesion is new, and may have implications for bacterial detachment strategies.

Monitoring the Switching of Single BSA-ATTO 488 Molecules Covalently End-Attached to a pH-Responsive PAA Brush

We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the biomolecule can be switched from inside to outside the brush and vice versa by a change in pH. For this, we grafted ultrathin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a bis-N-succinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble, and it is swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface under basic pH conditions, and fluorescence recurred (in-state) when the chains collapsed under acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single-molecule level, and we demonstrate that this switching is fast but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush.