Force-Induced Near-Infrared Chromism of Mechanophore-Linked Polymers.

A near-infrared (NIR) mechanophore was developed and incorporated into a poly(methyl acrylate) chain to showcase the first force-induced NIR chromism in polymeric materials. This mechanophore, based on benzo[1,3]oxazine (OX) fused with a heptamethine cyanine moiety, exhibited NIR mechanochromism in solution, thin-film, and bulk states. The mechanochemical activity was validated using UV-vis-NIR absorption/fluorescence spectroscopies, gel permeation chromatography (GPC), NMR, and DFT simulations. Our work demonstrates that NIR mechanochromic polymers have considerable potential in mechanical force sensing, damage detection, bioimaging, and biomechanics.

Driving complex flow waveforms with a linear voice coil actuator.

Oscillatory and pulsatile fluid flows for use in microfluidic applications were generated using a deformable chamber driven by a low cost linear voice coil actuator. Compliance in the fluidic system originating in the deformable chamber and the fluidic tubing produced a strong frequency dependence in the relationship between the system's input and the output flow rate. The effects of this frequency dependence were overcome by precise system calibration, enabling on-demand generation of sinusoidal oscillations in the fluid flow rate with a controlled amplitude in the range from 0.1 to over 1 ml/min across a frequency range from 0.1 Hz to 10 Hz. The calibration data further enabled the optimization of a multistage exponential smoothing model of the system that allowed the generation of arbitrary complex waveforms. This was demonstrated by combining flow modulation with a constant background flow generated by a syringe pump to mimic the pulsatile flow found in the human vascular system.

Mechanical properties of P-selectin PSGL-1 bonds.

The accurate determination of the mechanical properties of P-selectin and PSGL-1 is crucial for design and optimization of applications utilizing such bonds, e.g. biosensors and targeted drug delivery systems, as adhesion and mechanical interactions play a critical role in several key functions of biological cells. In current work, the spring constant and rupture force of a single P-selectin PSGL-1 ligand receptor bond and the Young's modulus of a layer made of these ligand receptors are reported. The work-of-adhesion of the P-selectin PSGL-1 interface is also characterized. In the reported experiments, PSGL-1 coated particles are deposited on a P-selectin coated substrate and their transient nanometer scale out-of-plane displacements are acquired employing a laser Doppler vibrometer as they are excited by an ultrasonic field. From the spectral response of a single particle, the resonance frequencies of its vibrational motion are identified, and with help of a particle adhesion model, the average rupture force and stiffness of a single P-selectin PSGL-1 ligand receptor are determined as Frupt = 171 ± 56 pN and kb = 0.56 ± 0.04 mN/m, respectively. Furthermore, the Young's modulus and work-of-adhesion of a layer of P-selectin PSGL-1 ligand receptors are extracted as E = 28.74 ± 3.96 MPa and WA = 70.0 ± 8.0 mJ/m2, respectively. Unlike Atomic Force Microscopy (AFM) and other probe-based techniques, the reported approach eliminates the need for direct contact with the sample, which could compromise the accuracy of the results by imposing unspecified additional contact interactions. Further, the current technique can be employed for measurements under various fluid flow conditions.

Adhesion and stiffness of biotin-superavidin bonds.

A non-invasive vibrational spectroscopy technique is introduced and utilized to characterize the average spring constant of a single Superavidin (SAv)-Biotin (Bi).polyethylene glycol (PEG) ligand receptor complex as well as the effective Young's modulus and adhesion of a layer formed by the SAv-Bi.PEG ligand-receptors. In the reported experiments, SAv coated Polystyrene (PS) particles are deposited on a layer of Bi.PEG receptors, bound to a silicon (Si) substrate by silanization. The substrate and the bonded particles are subjected to a pulsed ultrasonic excitation field and their nanometer scale out-of-plane dynamic responses are acquired using a laser vibrometer. The acquired waveforms are processed to obtain the resonance frequencies of the particle motion. Employing a particle adhesion model, the average spring constant of the single ligand-receptor complex and the effective Young's modulus and work-of-adhesion of the SAv-Bi.PEG ligand-receptor layer are extracted from the resonance frequencies. The average spring constant of an individual SAv-Bi.PEG bond is approximated as 0.1-0.4 mN/m. The work-of-adhesion and effective Young's modulus of the SAv-Bi.PEG layer are determined to be 0.54-2.62 J/m2 and 0.15-2.80 MPa, respectively. The compressive Young's modulus of the SAv-Bi.PEG layer is estimated as 31.0-58.0 MPa. The current approach provides a direct non-contact measurement technique for the stiffness of single ligand receptor complexes and the adhesion of their interfaces. SAv-Bi bonds and PEG polymers are among the most widely utilized complexes in the pharmaceutical and biological applications. Understanding the mechanical properties of PEG and SAv-Bi is an important step towards optimization of their utilization in practical applications such as biosensors and targeted drug delivery.

Optimizing likelihood models for particle trajectory segmentation in multi-state systems.

Particle tracking offers significant insight into the molecular mechanics that govern the behavior of living cells. The analysis of molecular trajectories that transition between different motive states, such as diffusive, driven and tethered modes, is of considerable importance, with even single trajectories containing significant amounts of information about a molecule's environment and its interactions with cellular structures. Hidden Markov models (HMM) have been widely adopted to perform the segmentation of such complex tracks. In this paper, we show that extensive analysis of hidden Markov model outputs using data derived from multi-state Brownian dynamics simulations can be used both for the optimization of likelihood models describing the states of the system and for characterization of the technique's failure mechanisms. The major drivers of HMM failure were found to be likelihood overlap, which was visualized using the Bhattacharyya coefficient, and state mixing caused by state transitions that occur between time points in a particle's trajectory both of which are intrinsically associated with the multi-state nature of the data. This approach provides critical information for the visualization of HMM failure and successful design of particle tracking experiments where trajectories contain multiple mobile states.

Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix.

The pericellular matrix is a robust, hyaluronan-rich polymer brush-like structure that controls access to the cell surface, and plays an important role in cell adhesion, migration, and proliferation. We report the observation of single bottlebrush proteoglycan dynamics in the pericellular matrix of living chondrocytes. Our investigations show that the pericellular matrix undergoes gross extension on the addition of exogenous aggrecan, and that this extension is significantly in excess of that observed in traditional particle exclusion assays. The mean-square displacement of single, bound proteoglycans increases with distance to cell surface, indicating reduced confinement by neighboring hyaluronan-aggrecan complexes. This is consistent with published data from quantitative particle exclusion assays that show openings in the pericellular matrix microstructure ranging from ∼150 nm near the cell surface to ∼400 nm near the cell edge. In addition, the mobility of tethered aggrecan drops significantly when the cell coat is enriched with bottlebrush proteoglycans. Single-molecule imaging in this thick polysaccharide matrix on living cells has significant promise in the drive to elucidate the role of the pericellular coat in human health.

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.

Fast weighted centroid algorithm for single particle localization near the information limit.

A simple weighting scheme that enhances the localization precision of center of mass calculations for radially symmetric intensity distributions is presented. The algorithm effectively removes the biasing that is common in such center of mass calculations. Localization precision compares favorably with other localization algorithms used in super-resolution microscopy and particle tracking, while significantly reducing the processing time and memory usage. We expect that the algorithm presented will be of significant utility when fast computationally lightweight particle localization or tracking is desired.

Speed dependence of thermochemical nanolithography for gray-scale patterning.

Thermochemical nanolithography (TCNL) is a high-resolution lithographic technique and, owing to its fast speed, versatility, and unique ability to fabricate arbitrary, gray-scale nanopatterns, this scanning probe technique is relevant both for fundamental scientific research as well as for nanomanufacturing applications. In this work, we study the dependence of the TCNL driven chemical reactions on the translation speed of the thermal cantilever. The experimental data compares well with a model of the chemical kinetics for a first-order reaction. The impact of higher order reactions on the optimization of TCNL is addressed. The reported quantitative description of the speed dependence of TCNL is exploited and illustrated by fabricating controlled gradients of chemical concentration.

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.

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 (V(H)) and tail domains. V(H) 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; V(H) 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 V(H), correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.

Spatial organization and mechanical properties of the pericellular matrix on chondrocytes.

A voluminous polymer coat adorns the surface of many eukaryotic cells. Although the pericellular matrix (PCM) often extends several microns from the cell surface, its macromolecular structure remains elusive. This massive cellular organelle negotiates the cell's interaction with surrounding tissue, influencing important processes such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. Investigations of the PCM's architecture and function have been hampered by the difficulty of visualizing this invisible hydrated structure without disrupting its integrity. In this work, we establish several assays to noninvasively measure the ultrastructure of the PCM. Optical force probe assays show that the PCM of rat chondrocyte joint (RCJ-P) cells easily reconfigures around optically manipulated microparticles, allowing the probes to penetrate into rather than compress the matrix. We report distinct changes in forces measured from PCMs treated with exogenous aggrecan, illustrating the assay's potential to probe proteoglycan distribution. Measurements reveal an exponentially increasing osmotic force in the PCM arising from an inherent concentration gradient. With this result, we estimate the variation of the PCM's mesh size (correlation length) to range from ∼100 nm at the surface to 500 nm at its periphery. Quantitative particle exclusion assays confirm this prediction and show that the PCM acts like a sieve. These assays provide a much-needed tool to study PCM ultrastructure and its poorly defined but important role in fundamental cellular processes.

Aberration correction in wide-field fluorescence microscopy by segmented-pupil image interferometry.

We present a new technique for the correction of optical aberrations in wide-field fluorescence microscopy. Segmented-Pupil Image Interferometry (SPII) uses a liquid crystal spatial light modulator placed in the microscope's pupil plane to split the wavefront originating from a fluorescent object into an array of individual beams. Distortion of the wavefront arising from either system or sample aberrations results in displacement of the images formed from the individual pupil segments. Analysis of image registration allows for the local tilt in the wavefront at each segment to be corrected with respect to a central reference. A second correction step optimizes the image intensity by adjusting the relative phase of each pupil segment through image interferometry. This ensures that constructive interference between all segments is achieved at the image plane. Improvements in image quality are observed when Segmented-Pupil Image Interferometry is applied to correct aberrations arising from the microscope's optical path.

Nonperturbative chemical modification of graphene for protein micropatterning.

Graphene's 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).

A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning.

Cells naturally exist in a dynamic chemical environment, and therefore it is necessary to study cell behaviour under dynamic stimulation conditions in order to understand the signalling transduction pathways regulating the cellular response. However, until recently, experiments looking at the cellular response to chemical stimuli have mainly been performed by adding a stress substance to a population of cells and thus only varying the magnitude of the stress. In this paper we demonstrate an experimental method enabling acquisition of data on the behaviour of single cells upon reversible environmental perturbations, where microfluidics is combined with optical tweezers and fluorescence microscopy. The cells are individually selected and positioned in the measurement region on the bottom surface of the microfluidic device using optical tweezers. The optical tweezers thus enable precise control of the cell density as well as the total number of cells within the measurement region. Consequently, the number of cells in each experiment can be optimized while clusters of cells, that render subsequent image analysis more difficult, can be avoided. The microfluidic device is modelled and demonstrated to enable reliable changes between two different media in less than 2 s. The experimental method is tested by following the cycling of GFP-tagged proteins (Mig1 and Msn2, respectively) between the cytosol and the nucleus in Saccharomyces cerevisiae upon changes in glucose availability.

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.

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.

Live cell lithography: using optical tweezers to create synthetic tissue.

We demonstrate a new method for creating synthetic tissue that has the potential to capture the three-dimensional (3D) complexity of a multi-cellular organism with submicron precision. Using multiple laminar fluid flows in a microfluidic network, we convey cells to an assembly area where multiple, time-shared optical tweezers are used to organize them into a complex array. The cells are then encapsulated in a 30 microm x 30 microm x 45 microm volume of photopolymerizable hydrogel that mimicks an extra-cellular matrix. To extend the size, shape and constituency of the array without loss of viability, we then step to an adjacent location while maintaining registration with the reference array, and repeat the process. Using this step-and-repeat method, we formed a heterogeneous array of E. coli genetically engineered with a lac switch that is functionally linked to fluorescence reporters. We then induced the array using ligands through a microfluidic network and followed the space-time development of the fluorescence to evaluate viability and metabolic activity.

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.

Laser surgery and optical trapping in a laser scanning microscope.

Optical manipulation opens up many new possibilities for experiments in the field of microbiology and is a very powerful tool for investigating cellular structure. In this emerging field imaging retains an important role, and systems that combine advanced imaging techniques with optical manipulation tools, such as laser scalpels or optical tweezers, are an important starting point for researchers. We present a flexible experimental platform that contains both a laser scalpel and optical tweezers, in combination with confocal and multiphoton microscopy. A simple manipulation of the external optics is used to retain the three-dimensional imaging capabilities of the microscopes. Two applications of the system are presented. In the first, the laser scalpel is used to initiate diffusion of a fluorescent dye through Escherichia coli mutants, which exhibit abnormal cell division, forming filaments, or chains of bacteria. The diffusion assay is used to assess the potential for the exchange of cytoplasmic material between neighboring cells. The second application investigates the binding of endoplasmic reticulum (ER) to chloroplasts in Pisum sativum (garden pea). Individual plant protoplasts are ruptured using the laser scalpel, allowing individual chloroplasts to be trapped and manipulated. Strands of the ER which are attached to the chloroplast are identified. The magnitude and nature of the binding between the chloroplast and the ER are investigated.