Topographical depth reveals contact guidance mechanism distinct from focal adhesion confinement.

Cellular response to the topography of their environment, known as contact guidance, is a crucial aspect to many biological processes yet remains poorly understood. A prevailing model to describe cellular contact guidance involves the lateral confinement of focal adhesions (FA) by topography as an underlying mechanism governing how cells can respond to topographical cues. However, it is not clear how this model is consistent with the well-documented depth-dependent contact guidance responses in the literature. To investigate this model, we fabricated a set of contact guidance chips with lateral dimensions capable of confining focal adhesions and relaxing that confinement at various depths. We find at the shallowest depth of 330 nm, the model of focal adhesion confinement is consistent with our observations. However, the cellular response at depths of 725 and 1000 nm is inadequately explained by this model. Instead, we observe a distinct reorganization of F-actin at greater depths in which topographically induced cell membrane deformation alters the structure of the cytoskeleton. These results are consistent with an alternative curvature-hypothesis to explain cellular response to topographical cues. Together, these results indicate a confluence of two molecular mechanisms operating at increased induced membrane curvature that govern how cells sense and respond to topography.

Automated cell segmentation for reproducibility in bioimage analysis.

Live-cell imaging is extremely common in synthetic biology research, but its ability to be applied reproducibly across laboratories can be hindered by a lack of standardized image analysis. Here, we introduce a novel cell segmentation method developed as part of a broader Independent Verification & Validation (IV&V) program aimed at characterizing engineered Dictyostelium cells. Standardizing image analysis was found to be highly challenging: the amount of human judgment required for parameter optimization, algorithm tweaking, training and data pre-processing steps forms serious challenges for reproducibility. To bring automation and help remove bias from live-cell image analysis, we developed a self-supervised learning (SSL) method that recursively trains itself directly from motion in live-cell microscopy images without any end-user input, thus providing objective cell segmentation. Here, we highlight this SSL method applied to characterizing the engineered Dictyostelium cells of the original IV&V program. This approach is highly generalizable, accepting images from any cell type or optical modality without the need for manual training or parameter optimization. This method represents an important step toward automated bioimage analysis software and reflects broader efforts to design accessible measurement technologies to enhance reproducibility in synthetic biology research.

Self-supervised machine learning for live cell imagery segmentation.

Segmenting single cells is a necessary process for extracting quantitative data from biological microscopy imagery. The past decade has seen the advent of machine learning (ML) methods to aid in this process, the overwhelming majority of which fall under supervised learning (SL) which requires vast libraries of pre-processed, human-annotated labels to train the ML algorithms. Such SL pre-processing is labor intensive, can introduce bias, varies between end-users, and has yet to be shown capable of robust models to be effectively utilized throughout the greater cell biology community. Here, to address this pre-processing problem, we offer a self-supervised learning (SSL) approach that utilizes cellular motion between consecutive images to self-train a ML classifier, enabling cell and background segmentation without the need for adjustable parameters or curated imagery. By leveraging motion, we achieve accurate segmentation that trains itself directly on end-user data, is independent of optical modality, outperforms contemporary SL methods, and does so in a completely automated fashion-thus eliminating end-user variability and bias. To the best of our knowledge, this SSL algorithm represents a first of its kind effort and has appealing features that make it an ideal segmentation tool candidate for the broader cell biology research community.

Robust optical flow algorithm for general single cell segmentation.

Cell segmentation is crucial to the field of cell biology, as the accurate extraction of single-cell morphology, migration, and ultimately behavior from time-lapse live cell imagery are of paramount importance to elucidate and understand basic cellular processes. In an effort to increase available segmentation tools that can perform across research groups and platforms, we introduce a novel segmentation approach centered around optical flow and show that it achieves robust segmentation of single cells by validating it on multiple cell types, phenotypes, optical modalities, and in-vitro environments with or without labels. By leveraging cell movement in time-lapse imagery as a means to distinguish cells from their background and augmenting the output with machine vision operations, our algorithm reduces the number of adjustable parameters needed for manual optimization to two. We show that this approach offers the advantage of quicker processing times compared to contemporary machine learning based methods that require manual labeling for training, and in most cases achieves higher quality segmentation as well. This algorithm is packaged within MATLAB, offering an accessible means for general cell segmentation in a time-efficient manner.

Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity.

Surface ligand activity is a key design parameter for successfully interfacing surfaces with cells─whether in the context of in vitro investigations for understanding cellular signaling pathways or more applied applications in drug delivery and medical implants. Unlike other crucial surface parameters, such as stiffness and roughness, surface ligand activity is typically based on a set of assumptions rather than directly measured, giving rise to interpretations of cell adhesion that can vary with the assumptions made. To fill this void, we have developed a concurrent control technique for directly characterizing in vitro ligand surface activity. Pairs of gold-coated glass chips were biofunctionalized with RGD ligand in a parallel workflow: one chip for in vitro applications and the other for surface plasmon resonance (SPR)-based RGD activity characterization. Recombinant αVß3 integrins were injected over the SPR chip surface as mimics of the cellular-membrane-bound receptors and the resulting binding kinetics parameterized to quantify surface ligand activity. These activity measurements were correlated with cell morphological features, measured by interfacing MDA-MB-231 cells with the in vitro chip surfaces on the live cell microscope. We demonstrate how the interpretation of a cell phenotype based on direct activity measurements can vary markedly from interpretations based on assumed activity. The SPR concurrent control approach has multiple advantages due to the fact that SPR is a standardized technique and has the sensitivity to measure ligand activity across the most relevant range of extracellular surface densities, while the in vitro chip design can be used with all commonly used light microscopy modalities (e.g., phase contrast, DIC, and fluorescence) so that a wide range of phenotypic and molecular markers can be correlated to the ligand surface activity.

Mechanisms of cell damage due to mechanical impact: an in vitro investigation.

The dynamic response of cells when subjected to mechanical impact has become increasingly relevant for accurate assessment of potential blunt injuries and elucidating underlying injury mechanisms. When exposed to mechanical impact, a biological system such as the human skin, brain, or liver is rapidly accelerated, which could result in blunt injuries. For this reason, an acceleration of greater than > 150 g is the most commonly used criteria for head injury. To understand the main mechanism(s) of blunt injury under such extreme dynamic threats, we have developed an innovative experimental method that applies a well-characterized and -controlled mechanical impact to live cells cultured in a custom-built in vitro setup compatible with live cell microscopy. Our studies using fibroblast cells as a model indicate that input acceleration ([Formula: see text]) alone, even when it is much greater than the typical injury criteria, e.g., [Formula: see text] g, does not result in cell damage. On the contrary, we have observed a material-dependent critical pressure value above which a sudden decrease in cell population and cell membrane damage have been observed. We have unambiguously shown that (1) this critical pressure is associated with the onset of cavitation bubbles in a cell culture chamber and (2) the dynamics of cavitation bubbles in the chamber induces localized compressive/tensile pressure cycles, with an amplitude that is considerably greater than the acceleration-induced pressure, to cells. More importantly, the rate of pressure change with time for cavitation-induced pressure is significantly faster (more than ten times) than acceleration-induced pressure. Our in vitro study on the dynamic response of biological systems due to mechanical impact is a crucial step towards understanding potential mechanism(s) of blunt injury and implementing novel therapeutic strategies post-trauma.

Problem of Diminished cRGD Surface Activity and What Can Be Done about It.

RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from in vitro experiments probing fundamental molecular mechanisms of cell adhesion to more applied in vivo strategies in medical imaging and cancer therapeutics. To better understand the outcomes of RGD-based approaches, we quantified the degree to which cyclic RGD (cRGD) activity is blocked by nonspecific binding of commonly used medium constituents. First, we show that recombinant αVß3 integrins can be used as a highly sensitive cell-free sensor to quantitatively and reliably characterize the activity of cRGD-functionalized surfaces via surface plasmon resonance (SPR). Next, SPR experiments were utilized to measure the extent of blocking of cRGD-functionalized surfaces by the commonly used agents BSA, PLL-g-PEG, and fetal calf serum (FCS)-supplemented media, using recombinant αVß3 integrin as a probe for cRGD binding activity in the presence of blocking agents. All three additives were highly efficient blockers of cRGD activity, as exemplified by cell culture media containing 1% FCS which reduced the cRGD activity by 33-fold. We then developed a strategy to combat these deleterious effects by employing the recombinant integrins as a protective cap. We show that the unblocked cRGD activity can be preserved in the presence of PLL-g-PEG by employing the αVß3 integrin as a removable protective cap, both in cell-free and in vitro experiments. In vitro studies with MDA-MB-231 cells cultured atop cRGD-functionalized surfaces found that cell adhesion and migration prevented by PLL-g-PEG were restored when this protective cap approach was used.

Nanoplasmonic pillars engineered for single exosome detection.

Exosomes are secreted nanovesicles which incorporate proteins and nucleic acids, thereby enabling multifunctional pathways for intercellular communication. There is an increasing appreciation of the critical role they play in fundamental processes such as development, wound healing and disease progression, yet because of their heterogeneous molecular content and low concentrations in vivo, their detection and characterization remains a challenge. In this work we combine nano- and microfabrication techniques for the creation of nanosensing arrays tailored toward single exosome detection. Elliptically-shaped nanoplasmonic sensors are fabricated to accommodate at most one exosome and individually imaged in real time, enabling the label-free recording of digital responses in a highly multiplexed geometry. This approach results in a three orders of magnitude sensitivity improvement over previously reported real-time, multiplexed platforms. Each nanosensor is elevated atop a quartz nanopillar, minimizing unwanted nonspecific substrate binding contributions. The approach is validated with the detection of exosomes secreted by MCF7 breast adenocarcinoma cells. We demonstrate the increasingly digital and stochastic nature of the response as the number of subsampled nanosensors is reduced from four hundred to one.

Quantifying time-varying cellular secretions with local linear models.

Extracellular protein concentrations and gradients initiate a wide range of cellular responses, such as cell motility, growth, proliferation and death. Understanding inter-cellular communication requires spatio-temporal knowledge of these secreted factors and their causal relationship with cell phenotype. Techniques which can detect cellular secretions in real time are becoming more common but generalizable data analysis methodologies which can quantify concentration from these measurements are still lacking. Here we introduce a probabilistic approach in which local-linear models and the law of mass action are applied to obtain time-varying secreted concentrations from affinity-based biosensor data. We first highlight the general features of this approach using simulated data which contains both static and time-varying concentration profiles. Next we apply the technique to determine concentration of secreted antibodies from 9E10 hybridoma cells as detected using nanoplasmonic biosensors. A broad range of time-dependent concentrations was observed: from steady-state secretions of 230 pM near the cell surface to large transients which reached as high as 56 nM over several minutes and then dissipated.

Improving biosensing activity to carcinoembryonic antigen with orientated single domain antibodies.

Carcinoembryonic antigen (CEA), also referred as CEACAM5, is integral to the adhesion process during cancer invasion and metastasis and is one of the most widely used tumor markers for assisting the diagnosis of cancer recurrence and cancer metastasis. Antibodies against CEA molecules have been developed for detection and diagnostic applications following tumor removal. Single domain antibodies (sdAbs) against CEA isolated from dromedary and llama exhibited high specificity in binding to tumor cells. However, because these CEA sdAbs were not designed to be orientated when conjugated to surface sensors, there is potential for significant improvements in their activity and limit of detection. Herein we modified the CEA sdAbs with two different C-terminal fusions designed to aid with orientation by way of the tail's charge and biotin binding. A fusion which incorporated the C-terminus addition of a positively charged tail (B5-GS3K) improved biosensor sensitivity to CEA while also retaining the sub-nanomolar binding affinity and thermal stability of the unmodified sdAb. Using our fabricated surfaces on bare gold chips and a multiplexed surface plasmon resonance imager (SPRi), we quantified the specific binding activities, defined as the percentage of bound epitopes to the total immobilized, of the sdAb fusions and anti-CEA mAb. Our results demonstrate that monovalent B5-GS3K exhibited significantly improved binding activity, approximately 3-fold higher than bivalent mAb.

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required. In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging. A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Optimizing Nanoplasmonic Biosensor Sensitivity with Orientated Single Domain Antibodies.

Localized surface plasmon resonance (LSPR) spectroscopy and imaging are emerging biosensor technologies which tout label-free biomolecule detection at the nanoscale and ease of integration with standard microscopy setups. The applicability of these techniques can be limited by the restrictions that surface-conjugated ligands must be both sufficiently small and orientated to meet analyte sensitivity requirements. We demonstrate that orientated single domain antibodies (sdAb) can optimize nanoplasmonic sensitivity by comparing three anti-ricin sdAb constructs to biotin-neutravidin, a model system for small and highly orientated ligand studies. LSPR imaging of electrostatically orientated sdAb exhibited a ricin sensitivity equivalent to that of the biotinylated LSPR biosensors for neutravidin. These results, combined with the facts that sdAb are highly stable and readily produced in bacteria and yeast, build a compelling case for the increased utilization of sdAbs in nanoplasmonic applications.

Quantitative imaging of protein secretions from single cells in real time.

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.

Quantitative LSPR imaging for biosensing with single nanostructure resolution.

Localized surface plasmon resonance (LSPR) imaging has the potential to map complex spatio-temporal variations in analyte concentration, such as those produced by protein secretions from live cells. A fundamental roadblock to the realization of such applications is the challenge of calibrating a nanoscale sensor for quantitative analysis. Here, we introduce a new, to our knowledge, LSPR imaging and analysis technique that enables the calibration of hundreds of individual gold nanostructures in parallel. The calibration allowed us to map the fractional occupancy of surface-bound receptors at individual nanostructures with nanomolar sensitivity and a temporal resolution of 225 ms. As a demonstration of the technique's applicability to molecular and cell biology, the calibrated array was used for the quantitative LSPR imaging of anti-c-myc antibodies harvested from a cultured 9E10 hybridoma cell line without the need for further purification or processing.

A new methodology for quantitative LSPR biosensing and imaging.

A new quantitative analysis methodology for localized surface plasmon resonance (LSPR) biosensing which determines surface-receptor fractional occupancy, as well as an LSPR imaging technique for the spatiotemporal mapping of binding events, is presented. Electron beam nanolithography was used to fabricate 20 × 20 arrays of gold nanostructures atop glass coverslips. A single biotinylated array was used to measure the association kinetics of neutravidin to the surface by spectroscopically determining the fractional occupancy as a function of time. By regenerating the same array, a reliable comparison of the kinetics could be made between control samples and neutravidin concentrations ranging from 1 µM to 50 nM. CCD-based imagery of the array, taken simultaneously with the spectroscopic measurements, reveals the binding of neutravidin to the surface as manifested by enhanced scattering over the majority of the resonance peak. The temporal resolution of the LSPR imaging technique was 200 ms and the spatial resolution was 8 µm(2).

Iminobiotin binding induces large fluorescent enhancements in avidin and streptavidin fluorescent conjugates and exhibits diverging pH-dependent binding affinities.

The pH-dependent binding affinity of either avidin or streptavidin for iminobiotin has been utilized in studies ranging from affinity binding chromatography to dynamic force spectroscopy. Regardless of which protein is used, the logarithmic dependence of the equilibrium dissociation constant (K(d)) on pH is assumed conserved. However a discrepancy has emerged from a number of studies which have shown the binding affinity of streptavidin for iminobiotin in solution to be unexpectedly low, with the K(d) at values usually associated with non-specific binding even at strongly basic pH levels. In this work we have utilized a Bodipy fluorescent conjugate of avidin and an Oregon Green fluorescent conjugate of streptavidin to determine the K(d) of the complexes in solution in the pH range of 7.0 to 10.7. The study was made possible by the remarkable fluorescent enhancement of the two fluorescent conjugates (greater than 10 fold) upon saturation with iminobiotin. The streptavidin-iminobiotin interaction exhibited almost no pH dependence over the range studied, with K(d) consistently on the order of 10(-5) M. In contrast, under identical experimental conditions the avidin-iminobiotin interaction exhibited the expected logarithmic dependence on pH. We discuss the possible origins for why these two closely related proteins would diverge in their binding affinities for iminobiotin as a function of pH.

Magnetic moment degradation of nanowires in biological media: real-time monitoring with SQUID magnetometry.

Magnetic nanoparticles are used throughout biology for applications from targeted drug and gene delivery to the labeling of cells. These nanoparticles typically react with the biological medium to which they are introduced, resulting in a diminished magnetic moment. The rate at which their magnetic moment is diminished limits their utility for targeting and can signal the unintended release of surface-functionalized biomolecules. A foreknowledge of the time-dependent degradation of the magnetic moment in a given medium can aid in the selection of the optimal buffering solution and in the prediction of a reasonable experimental time frame. With this goal in mind, we have developed a SQUID magnetometer based methodology for measuring the saturation magnetic moment of nanoparticles in real time while immersed in a biological medium. Measurements on Co and Ni nanowires in a variety of commonly used buffered salines demonstrated that the technique has the dynamic range and sensitivity to detect the rapid reduction in moment due to active corrosion as well as much more subtle changes from the formation of a passivating surface oxide layer. In order to correlate the magnetic moment reductions to these specific chemical processes, samples were additionally characterized using x-ray photoelectron spectroscopy, inductively coupled plasma spectroscopy and scanning electron microscopy. The most reactive buffers studied were found to be phosphate and carbonate based, which caused active corrosion of the Co nanowires but only a comparatively slow passivation of the Ni nanowires by oxidation.

The use of DNA molecular beacons as nanoscale temperature probes for microchip-based biosensors.

Today's biosensors and drug delivery devices are increasingly incorporating lithographically patterned circuitry that is placed within microns of the biological molecules to be detected or released. Elevated temperatures due to Joule heating from the underlying circuitry cannot only reduce device performance, but also alter the biological activity of such molecules (i.e. binding, enzymatic, folding). As a consequence, biochip design and characterization will increasingly require local measurements of the temperature and temperature gradients on the biofunctionalized surface. We have developed a technique to address this challenge based on the use of DNA molecular beacons as a nanoscale temperature probe. The surface of fused-silica chips with lithographically patterned, current-carrying gold rings have been functionalized with a layer of molecular beacons. We utilize the temperature dependence of the molecular beacons to calibrate the temperature at the center of the rings as a function of applied current from 25 to 50 degrees C. The fluorescent images of the rings reveal the extent of heating to the surrounding chip due to the applied current while resolving temperature gradients over length scales of less than 500nm. Finite element analysis and analytic calculations of the distribution of heat in the vicinity of the current-carrying rings agree well with the experimental results. Thus, molecular beacons are shown to be a viable tool for temperature calibration of micron-sized circuitry and the visualization of submicron temperature gradients.