Michael Berndt

TIRF microscopy evanescent field calibration using tilted fluorescent microtubules.

Total internal reflection fluorescence microscopy has become a powerful tool to study the dynamics of sub‐cellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field, that is, the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Here, we present a novel method to quantitatively characterize the illumination in total internal reflection fluorescence microscopy using tilted, fluorescently labelled, microtubules. We find that the evanescent field is well described by a single exponential function, with a penetration depth close to theoretically predicted values. The use of in vitro reconstituted microtubules as nanoscale probes results in a minimal perturbation of the evanescent field; excitation light scattering is eliminated and the refractive index of the sample environment is unchanged. The presented method has the potential to provide a generic tool for in situ calibration of the evanescent field.

Axial Nanometer Distances Measured by Fluorescence Lifetime Imaging Microscopy.

We present a novel fluorescence lifetime imaging microscopy technique to measure absolute positions of fluorescent molecules within 100 nm above a metalized surface based on distance-dependent fluorescence lifetime modulations. We apply this technique to fluorescently labeled microtubules as optical probes with various unlabeled proteins attached. By measuring the fluorescence lifetimes, we obtain the position of the microtubules and therefore determine the geometrical size of the attached proteins with nanometer precision.

Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy

Illumination based on objective-type total internal reflection (TIR) is nowadays widely used in high-performance fluorescence microscopy. However, the desirable application of such setups for dark-field imaging of scattering entities is cumbersome due to the spatial overlap of illumination and detection light, which cannot be separated spectrally. Here, we report a novel TIR approach based on a parabolically shaped quartz prism that allows for the detection of single-molecule fluorescence as well as single-particle scattering with high signal-to-noise ratios. We demonstrate homogeneous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, we quantitatively compare the fluorescence performance of our setup to objective-type TIR and demonstrate sub-nanometer localization accuracies for the scattering of 40 nm gold nanoparticles (AuNPs). When bound to individual kinesin-1 motors, the AuNPs reliably report on the characteristic 8 nm stepping along microtubules.

Tailoring surface plasmon polariton propagation via specific symmetry properties of nanostructures

We report on an experimental investigation on surface plasmon polariton (SPP) propagation and interaction on two-dimensional arrays of differing symmetry properties. Providing the required symmetry variations and forming the basis of the arrays are tailor designed nanostructures. We demonstrate that as a result of a 120° symmetry presence, our triquetra-rotor nanostructures can be used for SPP guiding and propagation direction control. As a result, the polarization angle at which the far field SPP related minimum reflectivity occurs can be predetermined by design characteristics and orientation of the nanostructures.

Control and gating of kinesin-microtubule motility on electrically heated thermo-chips.

First lab-on-chip devices based on active transport by biomolecular motors have been demonstrated for basic detection and sorting applications. However, to fully employ the advantages of such hybrid nanotechnology, versatile spatial and temporal control mechanisms are required. Using a thermo-responsive polymer, we demonstrated a temperature controlled gate that either allows or disallows the passing of microtubules through a topographically defined channel. The gate is addressed by a narrow gold wire, which acts as a local heating element. It is shown that the electrical current flowing through a narrow gold channel can control the local temperature and as a result the conformation of the polymer. This is the first demonstration of a spatially addressable gate for microtubule motility which is a key element of nanodevices based on biomolecular motors.

Surface plasmon polaritons on arrays of nanostructures with three‐fold symmetry

The influence of nano‐holes with three‐fold symmetry on the excitation of surface plasmon polaritons (SPPs) at metallic nano‐hole arrays is studied numerically for a quadratic array of rotor‐shaped nano‐holes cut into a silver film. It is found that the SPP‐related minimum of the far‐field reflectivity shifts as a function of the polarization angle of the incident light compared to rotational invariant hole‐shapes. This was also reported in recent experiments. On contrast, the polarization angle for most efficient SPP‐excitation is found to be independent of the nano‐hole shape. We discuss optical near‐ and far‐field properties of the considered structures.