Ben Ovryn

Evaporatively-driven Marangoni instabilities of volatile liquid films spreading on thermally conductive substrates

Laser confocal microscopy is used to non-invasively investigate the steady and unsteady evolution of viscous microdroplets on solid substrates. Three characteristic dynamical regimes of spreading drops (viscous-capillary, viscous-inertia-capillary, and inertia-capillary) are studied using this non-invasive optical technique. It is shown that the dynamics of each regime depend on the Ohnesorge number, Oh =/(R) 1 2, and on the relative magnitudes of the droplet height, radius, compared with the capillary length, lcap=/g. The power-law relationships between the extent of spreading and elapsed time that are extracted from the experiments are in excellent agreement with available analytical results. We also study the onset and evolution of surface instabilities of the slightly volatile liquid films as they spread across the thermally-conductive surfaces. When the fluid droplet is a volatile silicone oil and the surface is a smooth silicon wafer, an evaporatively-driven thermocapillary instability leads to onset of a time-dependent free surface motion. Below a certain critical thickness (20 m), waves can be observed on the free surface of the film, and the confocal technique is used to measure the amplitude, the frequency, and non-linear evolution of these waves. We interpret these waves in terms of evaporatively-driven Marangoni instabilities induced by surface tension gradients close to the moving contact line. Experiments show that the amplitude and the critical onset thickness of the disturbances vary with the viscosity and the volatility of the liquid, and also with the surface roughness and thermal diffusivity of the substrate. The critical onset conditions for this evaporatively driven instability can be characterized by a dimensionless interfacial thermal resistance, R, which has to be larger than a critical value at the onset of instability. We also demonstrate that this evaporatively-driven Marangoni instability can be eliminated by reducing the volatility of the liquid or the thermal diffusivity of the substrate.

Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. DOI: http://dx.doi.org/10.7554/eLife.10415.001

Tracking Surface Glycans on Live Cancer Cells with Single-Molecule Sensitivity†

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide- or alkyne-tagged glycans, a sufficiently low spatial density of dye-labeled glycans was achieved, enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked N-acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye-labeled glycans in mammary cancer cells revealed constrained diffusion of both N- and O-linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.

Phase-Shifted Laser Feedback Interferometry

We have introduced the techniques of phase-shifting interferometry into a laser feedback interference microscope based on a helium-neon laser. With moderate feedback, multiple reflections between the sample and the laser are shown to be negligible, and the interferometer responds sinusoidally with a well-characterized fringe modulation. One can obtain higher signal-to-noise ratios by determining the number of additional terms required for modeling the effect of multiple reflections on the phase and visibility measurements in the high-feedback regime. Changes in optical path length are determined with nanometer precision without phase averaging or lock-in detection.

Nucleation and Growth of Integrin Adhesions

We present a model that provides a mechanistic understanding of the processes that govern the formation of the earliest integrin adhesions ex novo from an approximately planar plasma membrane. Using an analytic analysis of the free energy of a dynamically deformable membrane containing freely diffusing receptors molecules and long repeller molecules that inhibit integrins from binding with ligands on the extracellular matrix, we predict that a coalescence of polymerizing actin filaments can deform the membrane toward the extracellular matrix and facilitate integrin binding. Monte Carlo simulations of this system show that thermally induced membrane fluctuations can either zip-up and increase the radius of a nucleated adhesion or unzip and shrink an adhesion, but the fluctuations cannot bend the ventral membrane to nucleate an adhesion. To distinguish this integrin adhesion from more mature adhesions, we refer to this early adhesion as a nouveau adhesion.

Imaging of gene expression in living cells and tissues

It is possible to observe gene expression within single cells using a tetracycline inducible promoter for activation. Transcription can be observed by using a fluorescent fusion protein to bind nascent RNA. Ultimately, it is desirable to activate a reporter gene within a single cell with only photons. This is achieved by preparing a chemically altered transcription factor that is functionally unable to activate a reporter gene until it is exposed to photon excitation. We apply two-photon imaging to visualize tumor cells expressing a transgene and ultimately this approach will provide the means to activate a specific gene within a single cell within any tissue to ultimately observe its functional significance in situ.

Reflectivity and topography of cells grown on glass-coverslips measured with phase-shifted laser feedback interference microscopy

In spite of the advantages associated with the molecular specificity of fluorescence imaging, there is still a significant need to augment these approaches with label-free imaging. Therefore, we have implemented a form of interference microscopy based upon phase-shifted, laser-feedback interferometry and developed an algorithm that can be used to separate the contribution of the elastically scattered light by sub-cellular structures from the reflection at the coverslip-buffer interface. The method offers an opportunity to probe protein aggregation, index of refraction variations and structure. We measure the topography and reflection from calibration spheres and from stress fibers and adhesions in both fixed and motile cells. Unlike the data acquired with reflection interference contrast microscopy, where the reflection from adhesions can appear dark, our approach demonstrates that these regions have high reflectivity. The data acquired from fixed and live cells show the presence of a dense actin layer located ≈ 100 nm above the coverslip interface. Finally, the measured dynamics of filopodia and the lamella in a live cell supports retrograde flow as the dominate mechanism responsible for filopodia retraction.

Measurement of changes in optical path length and reflectivity with phase-shifting laser feedback interferometry

The operating characteristics of a novel phase-shifting interferometer are presented. Interference arises by reflecting the light from a sample back into the cavity of a cw He-Ne laser. Changes in phase and fringe visibility are calculated from an overdetermined set of phase-shifted intensity measurements with the phase shifts being introduced with an electro-optic modulator. The interferometer is sensitive enough to measure displacements below 1 Hz with a rms error of approximately 1 nm from a sample that reflects only 3% of the 28 microW that is incident on its surface. The interferometer is applied to the determination of cantilever bending of a piezoelectric bimorph.

Membrane Deformation at Integrin Adhesions

In order to measure the nucleation of nouveau adhesions on the ventral surface of a cell, we have combined phase shifting laser feedback interferometry with a high numerical aperture inverted fluorescence microscope. We use fluorescence to image molecules at the adhesion site and stage scanning interference microscopy in order to measure the distance between the ventral surface of a cell and the substratum with several nanometer precision. Our analytic and Monte Carlo simulations of integrin mediated adhesions predict several features of these nouveau adhesions. An analysis of the energetics of membrane bending and the effects of a composite system of freely diffusing repellers and receptors and a fixed network of ligands on the extracellular matrix predicts that a small bundle of actin filaments should be able to push the membrane down to the extracellular matrix and nucleate a nouveau adhesion with critical radius below the diffraction limit. We have obtained a map of the reflectivity of the ventral surface of fixed metastatic mammary adenocarcinoma cells and we have shown that the data are correlated with markers for a focal adhesion adaptor protein. We are modeling the interference of the incident electric field with the field reflected from the ventral surface so as to obtain the surface topography at focal adhesions from the optical phase data.

Temporal averaging in a turbulent environment: compensation for phase drifts in phase-shifting interferometry

In an effort to improve the signal to noise in an interference experiment, we have developed a method to remove systematic phase drift between data sets acquired over long time intervals. Using this technique, it is possible to average repeatedly acquired phase measurements and improve the phase estimate without sacrificing spatial resolution. Results from tests using real-time phase stepping holographic interferometry applied to cantilever bending of a piezoelectric bimorph indicate that white noise has been reduced from 3 to less than 1 deg (lambda/360) by averaging 36 phase compensated data sets before object bending and 36 data sets after bending.