Reflection Interference Contrast Research Articles

Profiling contact ridge of soft substrates with metallic thin films using a novel interference technique

HypothesisAs technology moves toward flexibility, embedded systems, and miniaturization, which often have metallic elements integrated with the functional substrates, understanding the contact deformation of such soft substrates coated with thin metallic films is crucial. However, probing nanoscale deformations optically, typically in a few hundred nanometers, poses challenges. Dual Wavelength - Reflectance Interference Contrast Microscopy (DW-RICM) holds promise in unveiling nanoscale deformations. However, when paired with conventional glass probes to measure deformation of underlying soft substrates, DW-RICM faces limitations due to light reflection at the glass-air interface, impeding accurate determination of the contact zone (e.g., contact area, contact ridge). Here we hypothesize that using alternative probes to eliminate unwanted reflections can enhance understanding of soft substrate deformation with thin metallic films (bilayer substrates). ExperimentsIn this work, we investigate the deformation profile of viscoelastic soft substrates with sputter-coated gold thin films (referred to herein as bilayer substrates) using DW-RICM. We employ millimeter-sized black ink-coated glass spheres as probes, absorbing most light under DW-RICM. We focus our laser beams associated with DW-RICM at the contact zone between the probe and the underlying soft substrates. FindingsOur new technique of using black ink-coated glass probes in contact with soft bilayer substrates enables us to accurately extract the deformation profile, particularly the contact ridge. With the increase in the gold sputter coating time, the contact area and contact ridge of gold-polymer bilayer substrates decrease. Further, long-term contact analysis of gold-coated soft substrates reveals a steady increase in the contact ridge height over time, unlike the gradual decrease observed in their bare counterparts until reaching a steady state.

Visualizing a Nanoscale Lubricant Layer under Blood Flow.

Tethered-liquid perfluorocarbons (TLPs) are a class of liquid-infused surfaces with the ability to reduce blood clot formation (thrombosis) on blood-contacting medical devices. TLP comprises a tethered perfluorocarbon (TP) infused with a liquid perfluorocarbon (LP); this LP must be retained to maintain the antithrombotic properties of the layer. However, the stability of the LP layer remains in question, particularly for medical devices under blood flow. In this study, the lubricant thickness is spatially mapped and quantified in situ through confocal dual-wavelength reflection interference contrast microscopy. TLP coatings prepared on glass substrates are exposed to the flow of 37% glycerol/water mixtures (v/v) or whole blood at a shear strain rate of around 2900 s-1 to mimic physiological conditions (similar to flow conditions found in coronary arteries). Excess lubricant (>2 μm film thickness) is removed upon commencement of flow. For untreated glass, the lubricant is completely depleted after 1 min of shear flow. However, on optimized TLP surfaces, nanoscale films of lubricants (thickness between 100 nm and 2 μm) are retained over many tens of minutes of flow. The nanoscale films conform to the underlying structure of the TP layer and are sufficient to prevent the adhesion of red blood cells and platelets.

Determining effective refractive index and elasticity of nanoscale metal - thin, soft polymer bilayers using interference signatures of a glass probe

HypothesisThin metal films like silver (Ag) and gold (Au) on PDMS are perfect candidates for surface-enhanced Raman spectroscopy (SERS) active substrates whose optical and mechanical properties are often subjected to precise tuning. Measurement of such properties often requires multiple sophisticated experimental techniques and numerical analysis. When depositing a nanoscale thin metal film like Ag or Au on the polymer surface, the refractive index and elastic modulus of this bilayer structure could vary with the metal deposition time and properties of soft substrates. ExperimentsIn this work, we demonstrate a single experimental methodology to measure the refractive index and elasticity of thin metal films coated on PDMS. Using Dual-Wavelength Reflection Interference Contrast Microscopy (DW-RICM) measurements of a spherical glass probe of known shape, the refractive indices of Ag/PDMS and Au/PDMS substrates of varying metal deposition time, PDMS elasticity, and PDMS coating thickness are extracted corresponding to two different wavelengths. FindingsThe extracted refractive indices of metal/PDMS substrates decrease with increasing metal deposition time, decreasing PDMS elasticity, and increasing PDMS coating thickness. The extracted refractive indices are found to be in good agreement with corresponding UV/VIS spectroscopy measurements. Lastly, their elastic moduli are also obtained using Hertz's theory on the observed contact radius of the glass sphere on the aforementioned substrates.

Critical analysis of adhesion work measurements from AFM-based techniques for soft contact

HypothesisThe work of adhesion is a thermodynamic quantity that is frequently measured by atomic force microscopy (AFM). Determination of the work of adhesion requires quasi-equilibrium measurements, where we address the question of to what extent atomic force microscopy qualifies for quasi-equilibrium measurements. ExperimentTo measure the work of adhesion, we combined soft colloidal probe AFM (SCP AFM) with reflection interference contrast microscopy (RICM). This allowed us to extract the work of adhesion either from the pull-off force or from the contact radius. With these methods, we investigated the adhesion behavior of poly(N-isopropylacrylamide) (PNIPAM) polymer brushes in the swollen and solvent-induced collapsed state by systematically analyzing contact radii and adhesive forces. FindingsIn the swollen state, the adhesion to the PNIPAM brush was fivefold larger and exhibited significant time dependencies when measured with SCP AFM. A strong rate dependence of the pull-off force method was indicative of a non-equilibrium process. In order to reliably determine the equilibrium work of adhesion, the contact radius method was found to be the better because it is not rate dependent. Our work highlights the important benefits of using optical measurements to determine the contact radius when deriving the works of adhesion between colloidal probes and polymer brush surfaces.

Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces

Complex motions of immune cells are an integral part of diapedesis, chemotaxis, phagocytosis, and other vital processes. To better understand how immune cells execute such motions, we present a detailed analysis of phagocytic spreading of human neutrophils on flat surfaces functionalized with different densities of immunoglobulin G (IgG) antibodies. We visualize the cell-substrate contact region at high resolution and without labels using reflection interference contrast microscopy and quantify how the area, shape, and position of the contact region evolves over time. We find that the likelihood of the cell commitment to spreading strongly depends on the surface density of IgG, but the rate at which the substrate-contact area of spreading cells increases does not. Validated by a theoretical companion study, our results resolve controversial notions about the mechanisms controlling cell spreading, establishing that active forces generated by the cytoskeleton rather than cell-substrate adhesion primarily drive cellular protrusion. Adhesion, on the other hand, aids phagocytic spreading by regulating the cell commitment to spreading, the maximum cell-substrate contact area, and the directional movement of the contact region.

Wetting behavior of a water droplet on a mutual periodic micro-patterned ternary texture structure

Wetting behavior of a water droplet at a hydrophilic ternary texture structured (TTS) surface is unclear and rarely reported. Herein, wetting behavior of a water droplet was examined for the first time at a micro-patterned hydrophilic TTS-based moisture sensor chip (TTS@MSC) composed of confined silica gap between gold and aluminum arrays. Macroscopic and microscopic behaviors of water droplet were examined using contact angle measurement and reflection interference contrast microscopy (RICM), respectively. Experimental results revealed that water droplet exhibits Cassie state when it was observed parallel (║) to arrays due to existence of air pocket between water droplet and TTS@MSC surface. Moreover, parallel (║) observation to arrays indicated better adhesion stability of water droplet over perpendicular (┴) observation due to existence of arrays that restricted droplet free motion. In addition, volume-based experimental scheme revealed that water droplet loses its adhesion with TTS@MSC surface once its volume exceeds 10 μL. These findings suggested that tiny volume water droplets possess strong contact and adhesion stability at TTS@MSC surface which can feasibly enhance their accurate quantitative and qualitative detection in real practical fields. Present study is believed to open the door for a comprehensive understanding of the wetting behavior at numerous TTS surfaces for various water harvesting and/or repelling-based applications.

Modulation of viscoelasticity and interfacial potential of polyelectrolyte brush by Ion-specific interactions

Polyelectrolyte brushes have drawn increasing attention because their physicochemical properties can be modulated by adjustment of the pH and ion concentration. Here, we report the controlled grafting of poly acrylic acid containing cysteine side chains onto supported lipid membranes to allow for the modulation of viscoelasticity as well as interfacial potential by ion-specific interactions, that is, with cadmium ions. Quartz crystal microbalance with dissipation indicated that the resonance frequency increased and the dissipation decreased as the cadmium concentration increased, attributed to the dehydration of brushes. Systematic variation of the molecular structure demonstrated that the coexistence of thiol and carboxyl moieties is necessary for the viscoelastic response, suggesting that these structural features, common with naturally occurring proteins, form complexes with cadmium ions. Analysis of the height fluctuation of colloidal particles by reflection interference contrast microscopy indicated that the change in the viscoelasticity of the polymer brush layer alters the curvature of the effective interfacial potential. Intriguingly, we found that modulation of the viscoelasticity and interfacial potential caused by calcium ions is weak, suggesting that the interaction is ion-specific. Polymer brushes that can alter the interfacial potential through changes in the degree of hydration opens new avenues for the design of smart, adaptable surfaces.

Probing Liquid Drop Induced Deformation on Soft Solids Using Dual-Wavelength Reflection Interference Contrast Microscopy.

A liquid drop resting on a soft solid deforms the surface at the three-phase contact line. The surface deformation, also called "wetting ridge", varies in size from nanoscales to microscales, depending on the elasticity and thickness of the soft layer. In this work, we probe how surface elasticity and coating thickness influences normal and tangential surface deformation profiles induced by a sessile liquid drop using dual-wavelength reflection interference contrast microscopy. Furthermore, we experimentally verify the appropriate characteristic length scale, which closely describes the ridge profiles on both thick and thin soft layers for two different surface elasticities.

Visualizing and Quantifying Wettability Alteration by Silica Nanofluids.

An aqueous suspension of silica nanoparticles or nanofluid can alter the wettability of surfaces, specifically by making them hydrophilic and oil-repellent under water. Wettability alteration by nanofluids has important technological applications, including for enhanced oil recovery and heat transfer processes. A common way to characterize the wettability alteration is by measuring the contact angles of an oil droplet with and without nanoparticles. While easy to perform, contact angle measurements do not fully capture the wettability changes to the surface. Here, we employed several complementary techniques, such as cryo-scanning electron microscopy, confocal fluorescence and reflection interference contrast microscopy, and droplet probe atomic force microscopy (AFM), to visualize and quantify the wettability alterations by fumed silica nanoparticles. We found that nanoparticles adsorbed onto glass surfaces to form a porous layer with hierarchical micro- and nanostructures. The porous layer can trap a thin water film, which reduces contact between the oil droplet and the solid substrate. As a result, even a small addition of nanoparticles (0.1 wt %) lowers the adhesion force for a 20 μm sized oil droplet by more than 400 times from 210 ± 10 to 0.5 ± 0.3 nN as measured by using droplet probe AFM. Finally, we show that silica nanofluids can improve oil recovery rates by 8% in a micromodel with glass channels that resemble a physical rock network.

Biomimetic estrogen sensor based on soft colloidal probes

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(β-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.

Reflected Laser Interferometry: A Versatile Tool to Probe Condensation of Low-Surface-Tension Droplets.

Experimental investigation of dropwise condensation of low-surface-tension liquids remains prone to error owing to the imaging difficulties caused by the typically low droplet height. Using reflection interference contrast microscopy in confocal mode, we demonstrate a noninvasive framework to accurately capture this condensation dynamics of volatile liquids with low surface tension. The capability of the developed framework is demonstrated in studying the condensation dynamics of acetone, where it accurately describes the growth mechanism of condensed microdroplets with excellent spatiotemporal resolution even for submicron-range drop height and a three-phase contact angle of <5°. From experimentally obtained interferograms, the framework can reconstruct three-dimensional topography of the microdroplets even when the contact line of the droplet is distorted due to strong local pinning. The obtained results exhibit excellent quantitative agreement with several theoretically predicted trends. The proposed protocol overcomes the limitation of conventional techniques (e.g., optical imaging/environmental scanning electron microscopy) and provides an efficient alternative for studying the condensation of low-surface-tension liquids under atmospheric conditions.

ARHGAP45 controls naïve T- and B-cell entry into lymph nodes and T-cell progenitor thymus seeding

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans-endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase-activating proteins (GAPs). T and B cells express several RHO-GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time-lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T-cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T-cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T-cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.

Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Picomolar glyphosate sensitivity of an optical particle-based sensor utilizing biomimetic interaction principles

The continually growing use of glyphosate and its critically discussed health and biodiversity risks ask for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on the remarkably specific recognition of glyphosate by its physiological target enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs). This principle is implemented in an interferometric sensor by using the recently established soft colloidal probe (SCP) technique. EPSPs was site-specifically immobilized on a transparent surface utilizing the self-assembling properties of circadian clock gene 2 hydrophobin chimera and homogeneity of the layer was evidenced by atomic force microscopy. Exposure of the enzyme decorated biochip to glyphosate containing samples causes formation of enzyme-analyte complexes and a competitive loss of available binding sites for glyphosate-functionalized poly(ethylene glycol) SCPs. Functionalization of the SCPs with different types of linker molecules and glyphosate was assessed employing confocal laser scanning microscopy as well as confocal Raman microspectroscopy. Overall, reflection interference contrast microscopy analysis of SCP-biochip interactions revealed a strong influence of linker length and glyphosate coupling position on the sensitivity of the sensor. In employing a combination of pentaglycine linker and tethering glyphosate via its secondary amino group, concentrations in aqueous solutions down to 100 pM could be measured by the differential adhesion between SCP and biochip surface, supported by automated image analysis algorithms. This sensing concept could even prove its exceptional pM sensitivity in combination with a superior discrimination against structurally related compounds.

Recent Advances in the Quantification and Modulation of Hydrophobic Interactions for Interfacial Applications.

Hydrophobic interaction is responsible for a variety of colloidal phenomena, which also plays a key role in achieving the desired characteristics and functionalities for a wide range of interfacial applications. In this feature article, our recent advances in the quantification and modulation of hydrophobic interactions at both solid/water and air/water interfaces in different material systems have been reviewed. On the basis of surface forces apparatus (SFA) measurements of hydrophobic polymers (e.g., polystyrene), a three-regime hydrophobic interaction model that could satisfactorily encompass the hydrophobic interaction with different ranges was proposed. In addition, the atomic force microscope (AFM) coupled with various techniques such as the colloidal probe, the electrochemical process, and force mapping were employed to quantify the hydrophobic interaction from different perspectives. For the hydrophobic interactions involving deformable bubbles, the bubble probe AFM combined with reflection interference contrast microscopy (RICM) was used to simultaneously measure the interaction force and spatiotemporal evolution of the thin film drainage process between air bubbles and hydrophobized mica surfaces in an aqueous medium. The studies on the interactions of air bubbles with self-assembled monolayers (SAMs) demonstrated that the range of hydrophobic interactions does not always increase monotonically with the hydrophobicity of interacting surfaces as characterized by the static water contact angle; viz., surfaces with similar hydrophobicity can exhibit different ranges of hydrophobic interaction, while surfaces with different hydrophobicities can exhibit a similar range of hydrophobic interactions. It is found that the hydrophobic interaction can be modulated by tuning the surface nanoscale structure and chemistry. Moreover, the long-range "hydrophilic" attraction that resembles the hydrophobic interaction was discovered between water droplets and polyelectrolyte surfaces in an oil medium, on the basis of which polyelectrolyte coating materials were designed for oil cleaning, oil/water separation, and demulsification. The interfacial applications, remaining challenges, and future perspectives of hydrophobic interactions are discussed.

Generalized models for advancing and receding contact angles of fakir droplets on pillared and pored surfaces

Understanding how surface properties determine the mobility of a fakir droplet on patterned surfaces, which is typically characterized by advancing and receding contact angles, is important in the design of superhydrophobic surfaces for tailored applications. However, most analytical models of the contact angles are limited to a specific motion direction and surface type, e.g., receding on a pillared surface. Although it was suggested that the contact angles should be determined by the local configuration and dynamics of the droplet boundary, their link remains vague due to the lack of detailed visualizations. In this study, the contact line dynamics of a fakir droplet in both advancing and receding motions on not only micropillared but also micropored surfaces are visualized in reflection interference contrast microscopy along with the contact angle measurement. Based on the energy change induced by the displacement of a contact line and the deformation of a liquid-gas interface in between contact lines, theoretical models of the contact angles to encompass the two different motion directions and surface types are formulated. Moreover, further generalized models are established to serve as effective analytical models to predict the contact angles only based on the given structure dimensions and the inherent surface hydrophobicity.

Radial profile detection of multiple spherical particles in contact with interacting surfaces.

Adhesive interactions of soft materials play an important role in nature and technology. Interaction energies can be quantified by determining contact areas of deformable microparticles with the help of reflection interference contrast microscopy (RICM). For high throughput screening of adhesive interactions, a method to automatically evaluate large amounts of interacting microparticles was developed. An image is taken which contains circular interference patterns with visual characteristics that depend on the probe’s shape due to its surface interaction. We propose to automatically detect radial profiles in images, and to measure the contact radius and size of the spherical probe, allowing the determination of particle-surface interaction energy in a simple and fast imaging and image analysis setup. To achieve this, we analyze the image gradient and we perform template matching that utilizes the physical foundations of reflection interference contrast microscopy.

Effect of calcium ions and pH on the morphology and mechanical properties of hyaluronan brushes.

Hyaluronan (HA) is a linear, regular polysaccharide that plays as a chief structural and functional component in peri- and extracellular matrices, thus contributing significantly to many basic cellular processes. To understand more comprehensively the response of the supramolecular organization of HA polymers to changes in their aqueous environment, we study the effects of Ca2+ concentration and pH on the morphology and rigidity of films of end-grafted HA polymers on planar supports (HA brushes), as a well-defined in vitro model system of HA-rich matrices, by reflection interference contrast microscopy and quartz crystal microbalance. The thickness and softness of HA brushes decrease significantly with Ca2+ concentration but do not change with pH, within the physiological ranges of these parameters. The effect of Ca2+ on HA brush thickness is virtually identical to the effect of Na+ at 10-fold higher concentrations. Moreover, the thickness and softness of HA brushes decrease appreciably upon HA protonation at pH less than 6. Effects of pH and calcium ions are fully reversible over large parameter ranges. These findings are relevant for understanding the supramolecular organization and dynamics of HA-rich matrices in biological systems and will also benefit the rational design of synthetic HA-rich materials with tailored properties.

Effects of Opsonin Density on Phagocytic Behavior of Human Neutrophils

Although many components of the molecular machinery driving phagocytosis in immune cells have been identified, a detailed understanding of the underlying biophysical mechanisms remains elusive. Here, we investigate phagocytic protrusion dynamics in human neutrophils by monitoring mechanical processes and signaling events during phagocytosis of targets coated with varying densities of rabbit immunoglobulin G (IgG). We examine effects of the opsonin density in two complementary experiments: phagocytosis of IgG-coated polystyrene microspheres, and frustrated phagocytic cell spreading on IgG-coated glass coverslips. We facilitate interactions between neutrophils and microspheres using two opposing glass micropipettes to hold and manipulate the cell and target particle; this also allows us to quantify cell morphology and approximate cortical tension during phagocytosis. To analyze frustrated phagocytosis, we image the area of contact between spreading cells and the coverslip with high resolution using reflection interference contrast microscopy (RICM). The frustrated phagocytosis assay revealed that the overall speed of contact-area growth is largely independent of IgG density. However, cells are more likely to spread and reach a larger maximum contact area on higher densities of IgG. Monitoring also the cytosolic calcium concentration, we found that the timing of calcium surges during frustrated phagocytosis depends on IgG density – at lower densities, the cells reach larger contact areas before exhibiting global calcium bursts. Overall, these findings indicate that the speed of cell spreading is determined by the rate of actin protrusion rather than the density of adhesive contacts, while the number of ligands available for binding to Fcγ-receptors impacts both calcium signaling dynamics and the likelihood and extent of cell spreading. This suggests that initial actin polymerization is largely independent of calcium signaling, while later stages of phagocytosis mechanics are regulated by elevated global calcium levels.

An integrated assay to probe endothelial glycocalyx-blood cell interactions under flow in mechanically and biochemically well-defined environments

Cell-cell and cell-glycocalyx interactions under flow are important for the behaviour of circulating cells in blood and lymphatic vessels. However, such interactions are not well understood due in part to a lack of tools to study them in defined environments. Here, we develop a versatile in vitro platform for the study of cell-glycocalyx interactions in well-defined physical and chemical settings under flow. Our approach is demonstrated with the interaction between hyaluronan (HA, a key component of the endothelial glycocalyx) and its cell receptor CD44. We generate HA brushes in situ within a microfluidic device, and demonstrate the tuning of their physical (thickness and softness) and chemical (density of CD44 binding sites) properties using characterisation with reflection interference contrast microscopy (RICM) and application of polymer theory. We highlight the interactions of HA brushes with CD44-displaying beads and cells under flow. Observations of CD44+ beads on a HA brush with RICM enabled the 3-dimensional trajectories to be generated, and revealed interactions in the form of stop and go phases with reduced rolling velocity and reduced distance between the bead and the HA brush, compared to uncoated beads. Combined RICM and bright-field microscopy of CD44+ AKR1 T-lymphocytes revealed complementary information about the dynamics of cell rolling and cell morphology, and highlighted the formation of tethers and slings, as they interacted with a HA brush under flow. This platform can readily incorporate more complex models of the glycocalyx, and should permit the study of how mechanical and biochemical factors are orchestrated to enable highly selective blood cell-vessel wall interactions under flow.