Jan Scrimgeour

How vinculin regulates force transmission

Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (VH) and tail domains. VH enhances adhesion strength by increasing ECM-bound integrin–talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; VH has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or VH, correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin’s active conformation to promote force transfer.

Nonperturbative Chemical Modification of Graphene for Protein Micropatterning

Graphenes extraordinary physical properties and its planar geometry make it an ideal candidate for a wide array of applications, many of which require controlled chemical modification and the spatial organization of molecules on its surface. In particular, the ability to functionalize and micropattern graphene with proteins is relevant to bioscience applications such as biomolecular sensors, single-cell sensors, and tissue engineering. We report a general strategy for the noncovalent chemical modification of epitaxial graphene for protein immobilization and micropatterning. We show that bifunctional molecule pyrenebutanoic acid-succinimidyl ester (PYR-NHS), composed of the hydrophobic pyrene and the reactive succinimide ester group, binds to graphene noncovalently but irreversibly. We investigate whether the chemical treatment perturbs the electronic band structure of graphene using X-ray photoemission (XPS) and Raman spectroscopy. Our results show that the sp(2) hybridization remains intact and that the π band maintains its characteristic Lorentzian shape in the Raman spectra. The modified graphene surfaces, which bind specifically to amines in proteins, are micropatterned with arrays of fluorescently labeled proteins that are relevant to glucose sensors (glucose oxidase) and cell sensor and tissue engineering applications (laminin).

Optical manipulation and microfluidics for studies of single cell dynamics

Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

Fiber-based confocal microscope for cryogenic spectroscopy

We describe the design and performance of a fiber-based confocal microscope for cryogenic operation. The microscope combines positioning at low temperatures along three space coordinates of millimeter translation and nanometer precision with high stability and optical performance at the diffraction limit. It was successfully tested under ambient conditions as well as at liquid nitrogen (77 K) and liquid helium (4 K) temperatures. The compact nonmagnetic design provides for long term position stability against helium refilling transfers, temperature sweeps, as well as magnetic field variation between -9 and 9 T. As a demonstration of the microscope performance, applications in the spectroscopy of single semiconductor quantum dots are presented.

Registration of single quantum dots using cryogenic laser photolithography

We have registered the position of single InGaAs quantum dots using a cryogenic laser photolithography technique. This is an important advance towards the reproducible fabrication of solid-state cavity quantum electrodynamic devices, a key requirement for commercial exploitation of quantum information processing. The quantum dot positions were registered with an estimated accuracy of 50 nm by fabricating metal alignment markers around them. Photoluminescence spectra from quantum dots before and after marker fabrication were identical except for a small redshift (~1 nm), probably introduced during the reactive ion etching.

Fabricating Nanoscale Chemical Gradients with ThermoChemical NanoLithography

Production of chemical concentration gradients on the submicrometer scale remains a formidable challenge, despite the broad range of potential applications and their ubiquity throughout nature. We present a strategy to quantitatively prescribe spatial variations in functional group concentration using ThermoChemical NanoLithography (TCNL). The approach uses a heated cantilever to drive a localized nanoscale chemical reaction at an interface, where a reactant is transformed into a product. We show using friction force microscopy that localized gradients in the product concentration have a spatial resolution of ~20 nm where the entire concentration profile is confined to sub-180 nm. To gain quantitative control over the concentration, we introduce a chemical kinetics model of the thermally driven nanoreaction that shows excellent agreement with experiments. The comparison provides a calibration of the nonlinear dependence of product concentration versus temperature, which we use to design two-dimensional temperature maps encoding the prescription for linear and nonlinear gradients. The resultant chemical nanopatterns show high fidelity to the user-defined patterns, including the ability to realize complex chemical patterns with arbitrary variations in peak concentration with a spatial resolution of 180 nm or better. While this work focuses on producing chemical gradients of amine groups, other functionalities are a straightforward modification. We envision that using the basic scheme introduced here, quantitative TCNL will be capable of patterning gradients of other exploitable physical or chemical properties such as fluorescence in conjugated polymers and conductivity in graphene. The access to submicrometer chemical concentration and gradient patterning provides a new dimension of control for nanolithography.

Automated focusing of nuclei for time lapse experiments on single cells using holographic optical tweezers

Experiments on single cells are currently gaining more and more interest. Single cell studies often concerns the spatio-temporal distribution of fluorescent proteins inside living cells, visualized using fluorescence microscopy. In order to extract quantitative information from such experiments it is necessary to image the sample with high spatial and temporal resolution while keeping the photobleaching to a minimum. The analysis of the spatial distribution of proteins often requires stacks of images at each time point, which exposes the sample to unnecessary amounts of excitation light. In this paper we show how holographic optical tweezers combined with image analysis can be used to optimize the axial position of trapped cells in an array in order to bring the nuclei into a single imaging plane, thus eliminating the need for stacks of images and consequently reducing photobleaching. This allows more images to be collected, as well as increasing the time span and/or the time resolution in time lapse studies of single cells.

Microfluidic dialysis cell for characterization of macromolecule interactions

Microfluidic dialysis is a promising tool for characterisation of macromolecule interactions and responsive materials, but until now it has been limited to the characterisation of high molecular weight molecules. Here, we introduce a low molecular weight cut-off dialysis membrane fabricated by coating a rigid membrane filter with a layer of crosslinked hydrogel. The modified hybrid silicone/glass microfluidic cells retain low molecular weight samples at a well-defined concentration for long periods of time, while allowing timely changes to be made in the fluids supporting solvent (i.e. pH, ionic strength, ionic species). Straightforward access to the sample using high-resolution optical microscopy enables in situ characterisation of the samples physical properties in response to changes in solvent conditions. A proof of principle measurement investigating the pH-sensitive binding between the protein/polysaccharide pair, neurocan and hyaluronan, is presented to demonstrate the integration of protein retention, solvent switching and fluorescence recovery after photobleaching (FRAP).

Photobleaching-activated micropatterning on self-assembled monolayers

Functional chemical micropatterns were fabricated by exploiting the photobleaching of dye-coupled species near methacrylate self-assembled monolayers. Using this approach we have demonstrated that multiple chemistries can be coupled to the monolayer using a standard fluorescence microscope. The surface bound functional groups remain active and patterns with feature sizes down to 3 µm can be readily achieved with excellent signal-to-noise ratio. Control over the ligand binding density was demonstrated to illustrate the convenient route provided by this platform for fabricating complex spatial gradients in ligand density.

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans

The hyaluronan-rich pericellular matrix (PCM) plays physical and chemical roles in biological processes ranging from brain plasticity, to adhesion-dependent phenomena such as cell migration, to the onset of cancer. This study investigates how the spatial distribution of the large negatively charged bottlebrush proteoglycan, aggrecan, impacts PCM morphology and cell surface access. The highly localized pericellular milieu limits transport of nanoparticles in a size-dependent fashion and sequesters positively charged molecules on the highly sulfated side chains of aggrecan. Both rat chondrocyte and human mesenchymal stem cell PCMs possess many unused binding sites for aggrecan, showing a 2.5x increase in PCM thickness from ∼7 to ∼18 μm when provided exogenous aggrecan. Yet, full extension of the PCM occurs well below aggrecan saturation. Hence, cells equipped with hyaluronan-rich PCM can in principle manipulate surface accessibility or sequestration of molecules by tuning the bottlebrush proteoglycan content to alter PCM porosity and the number of electrostatic binding sites.