Reflection Interference Contrast Research Articles

Adhesion of Polyelectrolyte Microcapsules through Biotin−Streptavidin Specific Interaction

Adhesion of PAH/PSS and PDADMAC/PSS capsules through electrostatic and specific interactions has been investigated using reflective interference contrast microscopy (RICM). Adhesion of capsules via electrostatic interactions was found to be spontaneous and strong. Capsules functionalized with poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) did not exhibit significant adhesion (as determined by the adhesion area) to streptavidin-coated substrates, whereas capsules functionalized with biotinylated PLL-g-PEG showed a significantly larger adhesion area. Using continuum mechanical models, the total adhesion energies for these cases were calculated and were found to correspond to several tens of individual biotin-streptavidin pairs. The application of specific interactions such as the biotin-streptavidin system for controlled capsule adhesion has been demonstrated in this study.

A Novel Optical Technique to Measure Pad-Wafer Contact Area in Chemical Mechanical Planarization

AbstractReal contact area between a CMP pad and wafer is a key factor in local contact pressure, friction and pad wear, all of which impact material removal and defect formation. A new optical method is introduced that quantifies the real contact area during polishing. Confocal reflectance interference contrast microscopy (C-RICM) uses a single focal plane to image the pad-wafer contact interface. A sapphire cover slip is used to provide optical transparency and to match the refractive index of the pad. Imaging the pad surface through the cover slip reveals areas of no reflection (pad-cover slip contact), areas of reflection (non-contact), and interference fringes (near contact). The C-RICM method was validated using micro-fabricated pads having uniform arrays of cylindrical surface structures of known contact area. Experiments conducted on porous polyurethane pads revealed that the real contact area is less than 10% of the total presented area. However Greenwood-Williamson (G-W) theory, widely used in CMP material removal models, predicts a contact area at least a factor of ten smaller. The discrepancy was found to result at least in part because the individual contact zones are not elliptical as assumed in G-W theory. In fact many contacting structures are crescents located at the perimeter of individual pores. These findings underscore the need for accurate control of pore density and morphology in polishing pads, in both initial manufacture and surface conditioning during CMP. The C-RICM method allows non-destructive benchmarking of polishing media in terms of pad-wafer contact, essential for developing improved pad architectures that achieve lower CMP defect levels.

Activation-Independent Platelet Adhesion and Aggregation under Elevated Shear Stress.

The response of platelets to vascular injury involves adhesion onto reactive substrates followed by activation and aggregation, the latter mediated by the integrin αIIbβ3 with bound adhesive proteins such as fibrinogen and von Willebrand factor (VWF). Hemostasis and pathological arterial occlusion, however, occur in distinct hydrodynamic environments. A 90% lumen reduction in a coronary artery may cause shear rates of 20,000–40,000 s−1, values that are 10-fold higher than in arterioles where platelets participate in hemostasis after trauma. How hemodynamic forces influence platelet function is not fully understood. We have now found that platelets adherent to an immobilized VWF surface in a flow field can promote aggregation without activation, provided that soluble VWF be present and the shear rate exceed 10,000 s−1. In these studies blood was treated with 10 μM prostaglandin (PG) E1, thus the definition of aggregation is extended to a process of platelet cohesion mediated by physiologic blood components even in the absence of activation. Upon blood perfusion, individual rolling platelets covered the VWF surface at the shear rate of 3,000 s−1. In contrast, at 20,000 s−1 rolling platelets formed aggregates within 100–200 μm from the point of initial contact with immobilized VWF, equivalent to a few seconds. These aggregates became larger with varying shape and velocity while translocating in the direction of flow, but were predominantly elongated during periods of prolonged arrest. We then used plasma-free blood cells suspended in buffer to evaluate whether addition of soluble VWF was sufficient to mediate activation-independent platelet aggregation. Single platelet adhesion to immobilized VWF was the same with or without soluble VWF at the shear rate of 3,000 s−1. At 24,000 s−1, in contrast, adhesion was minimal without soluble VWF, but in its presence activation-independent aggregates formed as in whole blood. To elucidate the nature of the links that support activation-independent platelet aggregation, we used reflection interference contrast microscopy (RICM). This showed that, from a discrete point of tight adhesion and over a period of several seconds, the platelet body was stretched by the fluid drag into a structure reaching lengths of 20 μm or more but less than 1 μm thick at the narrowest point. Additional platelets then adhered to these elongated ones, and some that were stretched by the fluid drag contributed to the increasing length of string-like formations, while many retained their discoid morphology. In the end, stretched platelets joined to one another formed continuous structures that reached lengths of 100–200 μm and remained attached to immobilized VWF for minutes, while hundreds of discoid platelets adhered to them and to one another with arrest times of variable duration until they eventually detached as rolling aggregates. Thus, stretching of platelets by fluid drag and binding of soluble VWF are required to form elastic links that connect platelets into activation independent aggregates.

Recognition‐Induced Polymersomes: Structure and Mechanism of Formation

Random polystyrene copolymers grafted with complementary recognition elements were combined in chloroform producing vesicular aggregates, that is, recognition-induced polymersomes (RIPs). Reflection interference contrast microscopy (RICM) in solution, coupled with optical microscopy (OM) and atomic force microscopy (AFM) on solid substrates, were used to determine the wall thickness of the RIPs. Rather than a conventional mono- or bilayer structure (approximately 10 or approximately 20 nm, respectively) the RIP membrane was 43+/-7 nm thick. Structural arrangement of the polymer chains on the RIP wall were characterized by using angle-resolved X-ray photoelectron spectroscopy (AR-XPS). The interior portion of the vesicle membrane was found to be more polar, containing more recognition units, than the exterior part. This gradient suggests that a rapid self-sorting of polymers takes place during the formation of RIPs, providing the likely mechanism for vesicle self-assembly.

Adhesion Contact Dynamics of Fibroblasts on Biomacromolecular Surfaces

Biomacromolecules like gelatin and chitosan have emerged as highly versatile biomimetic coatings for applications in tissue engineering. The elucidation of the interfacial kinetics of cell adhesion on biomacromolecular surfaces will pave the way for the rational design of chitosan/gelatin-based systems for cell regeneration. Biomacromolecular ultra-thin films, chemically immobilized on fused silica are ideal experimental models for determining the effect of surface properties on the biophysical cascades following cell seeding. In this study, confocal reflectance interference contrast microscopy (C-RICM), in conjunction with phase contrast microscopy and fluorescence confocal microscopy, was applied to detect the adhesion contact dynamics of 3T3 fibroblasts on chitosan and gelatin ultrathin films. X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of chitosan or gelatin on the silanized glass surface. Both the initial cell deformation rate and the change of two-dimensional spread area of the 3T3 fibroblasts are higher on gelatin-modified surfaces than on chitosan surfaces. The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. Immuno-staining of actin further demonstrates the different organization of cytoskeleton, likely induced by the change in cell signaling mechanism on the two biomacromolecular surfaces. The better attachment of 3T3 fibroblast to gelatin is postulated to be caused by the presence of adhesive domains on gelatin.

Adhesion Dynamics, Morphology, and Organization of 3T3 Fibroblast on Chitosan and Its Derivative: The Effect of O-Carboxymethylation

Chitosan and O-carboxymethylchitosan (OCMCS) have been proved to have biocompatibility and have been extensively researched in the field of biomaterials. In this study, Confocal-reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast imaging was used to investigate the adhesion contact dynamics of 3T3 fibroblasts on chitoan and OCMCS surface-modified silica coverslips. The C-RICM results demonstrate that the weak cell contact forms on OCMCS surface while a much stronger contact area forms on the chitosan surface. 3T3 fibroblasts are found to spread randomly with spindlelike morphology on the chitosan surface, while they exhibit elongated morphology and align on the OCMCS surface. It is believed that fibroblast behaviors such as migration, spreading with an elongated morphology, and alignment on the OCMCS surface are correlated with the weak cell contact. The mechanisms to form cell adhesion contact on chitosan and OCMCS were discussed.

Electrostatically Self‐Assembled Multilayers of Chitosan and Xanthan Studied by Atomic Force Microscopy and Micro‐Interferometry

AbstractThe thickness and surface morphology of electrostatically self‐assembled films of chitosan and xanthan (persistence length of ∼120nm) have been studied using dual‐wavelength Reflection Interference Contrast Microscopy (DW‐RICM) and tapping mode Atomic Force Microscopy (AFM). The multilayers were prepared at two ionic strengths (5mM and 150mM). When the multilayers were assembled at 150 mM a network like morphology was observed after one bilayer. This structure was found to be of large influence in the further growth of the multilayers, with the same kind of network structure being observed at all number of bilayers. A lack of swelling behaviour, as well as the network structure and the poresize of the network, is suggested to originate from the high chain stiffness of xanthan.

Many-Particle Tracking with Nanometer Resolution in Three Dimensions by Reflection Interference Contrast Microscopy

We have developed and characterized a method, based on reflection interference contrast microscopy, to simultaneously determine the three-dimensional positions of multiple particles in a colloidal monolayer. To evaluate this method, the interaction of 6.8 microm (+/-5%) diameter lipid-derivatized silica microspheres with an underlying planar borosilicate substrate is studied. Measured colloidal height distributions are consistent with expectations for an electrostatically levitated colloidal monolayer. The precision of the method is analyzed using experimental techniques in addition to computational bootstrapping algorithms. In its present implementation, this technique achieves 16 nm lateral and 1 nm vertical precision.

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm 2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm 2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R∝ t 0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R∝ t 0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R∝t0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R∝t0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.

Microinterferometric Study of the Structure, Interfacial Potential, and Viscoelastic Properties of Polyelectrolyte Multilayer Films on a Planar Substrate

Thin films of polyelectrolyte multilayers composed of alternating layers of poly(styrene sulfonate) and poly(allylamine hydrochloride) were characterized in terms of their structure, surface potential, and viscosity, by monitoring the Brownian height fluctuations of colloidal beads placed on the films using dual wavelength−reflection interference contrast microscopy (DW−RICM). Special attention was directed toward characterizing the surface properties of the probe beads. The effect of coating the beads with differently charged polymers and of having differently charged polymers as the topmost layer of the film was investigated. It was found that, unless the coated beads and the topmost layer were similarly charged, the beads were present at a lower height than expected and did not exhibit any Brownian fluctuations. In the case of well-fluctuating beads, the film thickness obtained by DW−RICM agreed well with that obtained by optical waveguide lightmode spectroscopy. The height fluctuations of the beads were analyzed to yield information about the bead/film interaction potential and the viscosity of the films. Both the viscosity and the stiffness of the potential decrease as the film thickness increases. We also report an unexpected power law dependence of the stiffness of the bead/substrate interaction potential on the height of the beads.

Absolute interfacial distance measurements by dual-wavelength reflection interference contrast microscopy.

Dual-wavelength reflection interference contrast microscopy (DW-RICM) is established as a microinterferometric technique to measure absolute optical distances between transparent planar substrates and hard or soft surfaces such as colloidal beads or artificial and biological membranes, which hover over the substrate. In combination with a fast image processing algorithm the technique was applied to analyze the trajectories of colloidal beads sedimenting under gravity. As the beads approach the surface of the substrate, they slow down because of hydrodynamic coupling of the bead motion to the substrate. The effective surface friction coefficients were measured as a function of the absolute distance of the beads from the surface. The height dependence of the friction coefficient was found to be in quantitative agreement with previous theoretical predictions. Furthermore, we demonstrate that the DW-RICM technique allows the determination of the height of membranes above substrates and the amplitude and direction of height fluctuations. Without any further need to label the membrane the unambiguous reconstruction of the surface profile of soft surfaces is possible.

A quantitative contour analysis of axisymmetric vesicles spontaneously adhering onto a substrate

The determination of membrane-substrate profile for adherent vesicle using confocal reflectance interference contrast microscopy (C-RICM) has pushed for the need of advanced mechanics model for interpreting adhesion mechanisms. In this work, a model for vesicles or cells adhesion is established, the governing equation is derived from the variation of the potential energy at the cohesive zone. A closed-form solution is found for vesicle spontaneously adheres to a substrate when its shear modulus, μ vanishes. Based on the model and C-RICM experiments the magnitude of the adhesion force is calculated for a lipid vesicle adheres to a glass substrate.

Optical Techniques for Imaging Membrane Topography

In recent years three powerful optical imaging techniques have emerged that provide nanometer-scale information about the topography of membrane surfaces, whether cellular or artificial: intermembrane fluorescence resonance energy transfer (FRET), fluorescence interference contrast microscopy (FLIC), and reflection interference contrast microscopy (RICM). In intermembrane FRET, the sharp distance dependence of resonant energy transfer between fluorophores allows topographic measurements in the Angstrom to few-nanometer range. In FLIC and RICM, interference between light from a membrane (either from fluorescent probes, or reflected illumination) and light reflected by a planar substrate provide spatial sensitivity in the few to hundreds of nanometer range, with few-nanometer resolution. All of these techniques are fairly easy to implement. We discuss the physics and optics behind each of these tools, as well as practical concerns regarding their uses. We also provide examples of their application in imaging molecular-scale structures at intermembrane junctions.

Bioadhesive characterization of poly(methylidene malonate 2.12) microparticle on model extracellular matrix

The efficacy of a drug delivery system is predicated on its retention in the target tissue. Microparticle is one of the most popular and effective drug delivery configurations. Recently, it has been shown that the interaction between drug-loaded microparticles and tissues is related to the effectiveness of paclitaxel delivery to the bladder wall of mice for treating superficial bladder cancer. In this study, the adhesive interaction between poly(methylidene malonate 2.12) or PMM 2.1.2 microparticles and collagen, which serves as the model extracellular matrix for bladder wall, was probed with confocal reflectance interference contrast microscopy (C-RICM), single-particle compressive force measurement and contact mechanics theory. Young's modulus of single PMM 2.1.2 microparticle was determined as 1.56±0.25×10 4 N/m 2. For plain PMM 2.1.2 microparticle in water (pH 5.5), the degree of deformation ( a/ R) on collagen coated substrate decreased from 0.77 to 0.26 against the increase of mid-plane diameter from 2 to 18 μm. The adhesion energy of PMM 2.1.2 microparticle was determined from Maguis-JKR theory and remained at around 1.5 mJ/m 2 against the increase of particle diameter. At pH 4, the average degree of particle deformation and adhesion energy was increased by 11% and 32%, respectively, in comparison with that at pH 5.5. The loading of paclitaxel in PMM 2.1.2 microspheres enhanced the deformation and adhesion of microspheres at pH 5.5. It is hypothesized that the electrostatic repulsion between paclitaxel and collagen at pH 4 reduces the adhesion energy of PMM 2.1.2-paclitaxel microsphere. This study may offer insight for design of future microparticulate delivery systems by providing the experimental and theoretical tools to study the bioadhesive interaction between drug-loaded microparticles and model extracellular matrices.

Elastic properties of polyelectrolyte capsules studied by atomic-force microscopy and RICM

Mechanical properties of polyelectrolyte multilayer capsules were studied using a new method combining atomic-force microscopy and reflection interference contrast microscopy. By measuring the force vs. deformation for poly(styrene sulfonate)/poly(allylamine) capsules the existence of different deformation regimes depending on the applied deformation was shown. The present paper focuses on the small-deformation regime. The elastic response of the deformed capsule was studied as a function of the wall thickness and the capsule size, and showed the theoretically expected variations. The Young modulus obtained from the experiments ranges between 1.3 and 1.9 GPa.

The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion

AbstractPlatelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collagen-induced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VInull platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VInull or FcR-γnull animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VInull platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils.

Interaction of liposome with immobilized chitosan during main phase transition.

It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on amino-silanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 degrees C and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems.

2] Reflection interference contrast microscopy

This chapter describes reflection interference contrast microscopy (RICM). The term “RICM” is used in the chapter to cover all optical configurations and practical applications of the method. The method by which the light reflected from a semitransparent, epi-illuminated specimen contributes to the image formation in a microscope has been given different names, depending on details of the optical configuration of the microscope and on the particular application. It was introduced into cell biology by Curtis using the name “interference reflection microscopy” (IRM), and this name, or its variant, “reflection interference microscopy” (RIM), is often used in studies of adhesion of living cells. Ploem used the term “reflection contrast microscopy” (RCM) on the introduction of the antiflex method to increase image contrast. In RICM of living cells, reflections from the cell plasma membrane and intracellular membranes contribute to the image formation by interfering with each other and with the light reflected from the interface between the underlying glass surface and the liquid medium.

Adhesion of Arg-Gly-Asp (RGD) Peptide Vesicles onto an Integrin Surface: Visualization of the Segregation of RGD Ligands into the Adhesion Plaques by Fluorescence

Integrins are adhesion receptors that mediate cell adhesion and play an important function in many biological processes such as morphogenesis and tissue remodeling. These membrane proteins specifically interact with a short tripeptide sequence, RGD (Arg-Gly-Asp), present in numerous extracellular macromolecules. Model systems have been developed in order to understand how membrane adhesion is induced by this specific RGD peptide ligand/integrin recognition system. We have previously shown that RGD giant vesicles selectively adhere to endothelial cells by formation of pinning centers. Nevertheless, the nature of the lipids located in the adhesion contact zone is unknown. One hypothesis is that the lipidic ligands migrate to the contact zone where they are confined after binding to the receptor. To study the possible formation of ligand domains within the vesicle bilayer, we synthesized a fluorescently labeled RGD lipid that can be easily incorporated in giant vesicles. Adhesion of giant RGD vesicles onto an integrin-functionalized surface was followed simultaneously by reflection interference contrast microscopy and fluorescence microscopy. For the first time, it was possible to observe the microsegregation of RGD lipids in the contact zone during adhesion. Additionally, we observed interesting photosensitive properties of the chalcone chromophore that could lead to a new method of analyzing the lipid organization within the membrane during adhesion and to the design of new ligand lipids and vesicle vectors for cell targeting.