Victor P. Ostanin

Nanoscale live cell imaging using hopping probe ion conductance microscopy

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.

Direct and sensitive detection of a human virus by rupture event scanning

We have developed a sensitive, economical method that directly detects viruses by making use of the interaction between type 1 herpes simplex virus (HSV1) and specific antibodies covalently attached to the oscillating surface of a quartz crystal microbalance (QCM). The virions were detached from the surface by monotonously increasing the amplitude of oscillation of the QCM, while using the QCM to sensitively detect the acoustic noise produced when the interactions were broken. We term this process rupture event scanning (REVS). The method is quantitative over at least six orders of magnitude, and its sensitivity approaches detection of a single virus particle.

Noncontact Measurement of the Local Mechanical Properties of Living Cells Using Pressure Applied via a Pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.

Pipette-surface interaction: current enhancement and intrinsic force.

There is an intrinsic repulsion between glass and cell surfaces that allows noninvasive scanning ion conductance microscopy (SICM) of cells and which must be overcome in order to form the gigaseals used for patch clamping investigations of ion channels. However, the interactions of surfaces in physiological solutions of electrolytes, including the presence of this repulsion, for example, do not obviously agree with the standard Derjaguin-Landau-Verwey-Overbeek (DLVO) colloid theory accurate at much lower salt concentrations. In this paper we investigate the interactions of glass nanopipettes in this high-salt regime with a variety of surfaces and propose a way to resolve DLVO theory with the results. We demonstrate the utility of this understanding to SICM by topographically mapping a live cells cytoskeleton. We also report an interesting effect whereby the ion current though a nanopipette can increase under certain conditions upon approaching an insulating surface, rather than decreasing as would be expected. We propose that this is due to electroosmotic flow separation, a high-salt electrokinetic effect. Overall these experiments yield key insights into the fundamental interactions that take place between surfaces in strong solutions of electrolytes.

Hydrodynamic trapping of molecules in lipid bilayers

In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the hydrodynamic drag forces arising from a flow through a conical pipette with a tip radius of 1–1.5 μm, placed approximately 1 μm above the investigated SLB. This results in a localized forcefield that acts on molecules protruding from the SLB, yielding a hydrodynamic trap with a size approximately given by the size of the pipette tip. We demonstrate this concept by trapping the protein streptavidin, bound to biotin receptors in the SLB. It is also shown how static and kinetic information about the intermolecular interactions in the lipid bilayer can be obtained by relating how the magnitude of the hydrodynamic forces affects the accumulation of protein molecules in the trap.

Simultaneous readout of multiple microcantilever arrays with phase-shifting interferometric microscopy.

A complete system for the simultaneous monitoring of multiple cantilever sensors from different sensor arrays has been developed and tested for gas- and liquid-phase applications. The cantilever sensors are operated in static-deflection mode and the readout is achieved with phase-shifting interferometric microscopy (PSIM). In contrast to existing cantilever-sensor readout methods, PSIM is not dependent on alignment and allows the monitoring of the entire displacement profiles of all cantilevers within the field of view, using just one light source. To complement the PSIM readout, we have developed a sample cell, which can hold multiple cantilever-array chips, allows for very fast and reproducible sensor-chip replacement, has very low sample-volume requirements, and allows for individual or common addressing of all chips in the sample cell. We demonstrate the functionality of our microcantilever sensor system with a setup that can monitor eight cantilevers from four different sensor chips simultaneously.

A hybrid scanning mode for fast scanning ion conductance microscopy (SICM) imaging

We have developed a new method of controlling the pipette for scanning ion conductance microscopy to obtain high-resolution images faster. The method keeps the pipette close to the surface during a single line scan but does not follow the exact surface topography, which is calculated by using the ion current. Using an FPGA platform we demonstrate this new method on model test samples and then on live cells. This method will be particularly useful to follow changes occurring on relatively flat regions of the cell surface at high spatial and temporal resolutions.

Anharmonic surface interactions for biomolecular screening and characterization

The acoustic response of conventional mechanical oscillators, such as a piezoelectric crystal, is predominantly harmonic at modest amplitudes. However, here, we observe from the electrical response that significant motional anharmonicity is introduced in the presence of attached analyte. Experiments were conducted with streptavidin-coated polystyrene microbeads of various sizes attached to a quartz crystal resonator via specific and nonspecific molecular tethers in liquid. Quantitative analysis reveals that the deviation of odd Fourier harmonics of the response caused by introduction of microbeads as a function of oscillation amplitude presents a unique signature of the molecular tether. Hence, the described anharmonic detection technique (ADT) based on this function allows screening of biomolecules and provides an additional level of selectivity in receptor-based detection that is often associated with nonspecific interactions. We also propose methods to extract mechanical force-extension characteristics of the molecular tether and activation energy using this technique.

Lightweight dew-/frost-point hygrometer based on a surface-acoustic-wave sensor for balloon-borne atmospheric water vapor profile sounding

The design of a very lightweight dew-/frost-point hygrometer for balloon-borne atmospheric water vapor profiling is described. The instrument is based on a surface-acoustic-wave sensor. The low instrument weight is a key feature, allowing flights on meteorological balloons which brings many more flight opportunities. The hygrometer shows consistently good performance in the troposphere and while water vapor measurements near the tropopause and in the stratosphere are possible with the current instrument, the long-time response in these regions hampers realistic measurements. The excellent intrinsic sensitivity of the surface-acoustic-wave sensor should permit considerable improvement in the hygrometer performance in the very dry regions of the atmosphere.

Anharmonic interaction signals for acoustic detection of analyte.

The challenges with frequency-based acoustic detection systems in sensitive, selective, and reliable quantitative estimation of surface-bound analyte are well-known. These systems are traditionally used in their linear incarnations; i.e., the measurement frequency is the same as the driving frequency. However, it was found in this work that interactions of adsorbents with sensor surface show significant anharmonicity even at low drive amplitudes. In particular, using streptavidin-coated polystyrene microbeads on an oscillating quartz surface in air, it has been demonstrated through modeling and experiments that the anharmonic signal from microparticle to surface interaction is significantly higher relative to that from bare quartz and orders of magnitude higher than relative shifts in resonant frequency. The signal is proportional to the number of microparticles and holds a well-defined functional relationship with the amplitude of oscillation, distinct to the nature of interaction with the surface for a given analyte. This approach, thus, can be used for ultrasensitive and quantitative detection of surface adsorbents and characterization of different kinds of surface interactions, distinguishing specific from nonspecific adsorbents. The modeling also reveals a direct functional relationship between the measured anharmonic signal and the interaction potential of the adsorbent with the surface.