Andreas Bruckbauer

Frequency and Voltage Dependence of the Dielectrophoretic Trapping of Short Lengths of DNA and dCTP in a Nanopipette

The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.

The scanned nanopipette: a new tool for high resolution bioimaging and controlled deposition of biomolecules

The boundary between the physical and biological sciences has been eroded in recent years with new physical methods applied to biology and biological molecules being used for new physical purposes. We have pioneered the application of a form of scanning probe microscopy based on a scanned nanopipette, originally developed by Hansma and co-workers, for reliable non-contact imaging over the surface of a live cell. We have found that the nanopipette can also be used for controlled local voltage-driven application of reagents or biomolecules and this can be used for controlled deposition and the local delivery of probes for mapping of specific species. In this article we review this progress, focussing on the physical principles and new phenomena that we have observed, and then outline the future applications that are now possible.

Bayesian Inference for Improved Single Molecule Fluorescence Tracking

Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images. We describe here a Bayesian-based inference approach, based on a trans-dimensional sequential Monte Carlo method that utilizes both the spatial and temporal information present in the image sequences. We show, using model data, where the real trajectory of the molecule is known, that our method allows accurate tracking of molecules over long trajectories even with low signal/noise ratio and in the presence of fluorescence blinking and photobleaching. The method is then applied to real experimental data.

Tracking diffusion of GM1 gangliosides and zona pellucida binding molecules in sperm plasma membranes following cholesterol efflux

The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing zona-binding molecules and GM1 gangliosides in the plasma membrane of live boar spermatozoa before and after cholesterol reduction. Both GM1 gangliosides and zona-binding molecules partition into a low density Triton X100 resistant phase suggesting their association with lipid rafts. Initially, GM1 and zona-binding molecules localize to the apical ridge on the acrosome but following cholesterol efflux with methyl-beta-cyclodextrin, clusters containing zona-binding molecules diffuse randomly over the acrosomal domain. Diffusing clusters of either type do not access the postacrosome. Spermatozoa agglutinated head-to-head show contact-induced coalescence of GM1 gangliosides (but not zona-binding molecules) suggestive of a specific mechanosensitive response. Thus, cholesterol efflux initiates diffusion (and possibly formation) of novel lipid raft-like structures containing zona-binding molecules over the sperm acrosome. We hypothesise that in combination with contact coalescence, these mechanisms concentrate important molecules to the appropriate site on the sperm surface to mediate zona binding.

A novel light source for SICM-SNOM of living cells

We have developed a novel light source for use in a scanning near‐field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo‐3 and calcium, respectively. This complex is then excited with an Ar+ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high‐resolution topographic and optical images of biological samples in physiological buffer. A resolution of ∼220 nm topographic and ∼190 nm optical was determined through imaging fixed sea‐urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.