Mass Measurements of Focal Adhesions in Single Cells Using High Resolution Surface Plasmon Resonance Microscopy.

Surface plasmon resonance microscopy (SPRM) is a powerful label-free imaging technique with spatial resolution approaching the optical diffraction limit. The high sensitivity of SPRM to small changes in index of refraction at an interface allows imaging of dynamic protein structures within a cell. Visualization of subcellular features, such as focal adhesions (FAs), can be performed on live cells using a high numerical aperture objective lens with a digital light projector to precisely position the incident angle of the excitation light. Within the cell-substrate region of the SPRM image, punctate regions of high contrast are putatively identified as the cellular FAs. Optical parameter analysis is achieved by application of the Fresnel model to the SPRM data and resulting refractive index measurements are used to calculate protein density and mass. FAs are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPRM with fluorescence images of antibody stained for vinculin, a component in FAs, reveals similar measurements of FA size. In addition, a positive correlation between FA size and protein density is revealed by SPRM. Comparing SPRM images for two cell types reveals a distinct difference in the protein density and mass of their respective FAs. Application of SPRM to quantify mass can greatly aid monitoring basic processes that control FA mass and growth and contribute to accurate models that describe cell-extracellular interactions.

Agglomeration of Escherichia coli with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard Caenorhabditis elegans Toxicity Assay.

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard Caenorhabditis elegans ( C. elegans) toxicity assay containing an Escherichia coli ( E. coli) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with E. coli cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on C. elegans growth and reproduction. When the ENPs were tested in two alternate C. elegans assays that did not contain E. coli, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays.

Surface plasmon resonance microscopy: Achieving a quantitative optical response.

Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based figuration. We carry out SPR imaging on a microscope by launching light into a sample and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy.

High resolution surface plasmon resonance imaging for single cells.

BACKGROUND: Surface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface. We have previously used SPRI to study the dynamics of cell-substratum interactions. However, characterization of spatial resolution in 3 dimensions is necessary to quantitatively interpret SPR images. Spatial resolution is complicated by the asymmetric propagation length of surface plasmons in the x and y dimensions leading to image degradation in one direction. Inferring the distance of intracellular organelles and other subcellular features from the interface by SPRI is complicated by uncertainties regarding the detection of the evanescent wave decay into cells. This study provides an experimental basis for characterizing the resolution of an SPR imaging system in the lateral and distal dimensions and demonstrates a novel approach for resolving sub-micrometer cellular structures by SPRI. The SPRI resolution here is distinct in its ability to visualize subcellular structures that are in proximity to a surface, which is comparable with that of total internal reflection fluorescence (TIRF) microscopy but has the advantage of no fluorescent labels. RESULTS: An SPR imaging system was designed that uses a high numerical aperture objective lens to image cells and a digital light projector to pattern the angle of the incident excitation on the sample. Cellular components such as focal adhesions, nucleus, and cellular secretions are visualized. The point spread function of polymeric nanoparticle beads indicates near-diffraction limited spatial resolution. To characterize the z-axis response, we used micrometer scale polymeric beads with a refractive index similar to cells as reference materials to determine the detection limit of the SPR field as a function of distance from the substrate. Multi-wavelength measurements of these microspheres show that it is possible to tailor the effective depth of penetration of the evanescent wave into the cellular environment. CONCLUSION: We describe how the use of patterned incident light provides SPRI at high spatial resolution, and we characterize a finite limit of detection for penetration depth. We demonstrate the application of a novel technique that allows unprecedented subcellular detail for SPRI, and enables a quantitative interpretation of SPRI for subcellular imaging.

Cell spreading and proliferation in response to the composition and mechanics of engineered fibrillar extracellular matrices.

The extracellular matrix (ECM) consists of a complex mixture of biochemical and physical stimuli that together regulate cell behavior. In this study, we engineer a model ECM consisting of fibrillar Type-1 collagen plus fibronectin that allows systematic examination of the effects of matrix composition and mechanics on cells. On this combined protein matrix, cells exhibit intermediate degrees of spreading and proliferation compared to their responses on collagen or fibronectin alone. Adhesion to the combination matrix could be blocked by peptides containing the sequence arginine-glycine-aspartic acid (RGD) and by antibodies against α1 integrin, suggesting cell-matrix engagement was mediated by a combination of integrin receptors that recognize fibronectin and collagen. Regardless of integrin engagement, cells were sensitive to the mechanical properties of the combination ECM, suggesting that cells could process biochemical and mechanical cues simultaneously and independently.

ATP binding to hemoglobin response gene 1 protein is necessary for regulation of the mating type locus in Candida albicans.

HBR1 (hemoglobin response gene 1) is an essential gene in Candida albicans that positively regulates mating type locus MTLα gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with K(d) ∼2 µM. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its γ-phosphate. ATP bound somewhat more avidly than ATPγS to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTlα2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.

Using surface plasmon resonance imaging to probe dynamic interactions between cells and extracellular matrix.

Spatially resolved details of the interactions of cells with a fibronectin modified surface were examined using surface plasmon resonance imaging (SPRI). SPRI is a label-free technique that is based on the spatial measurement of interfacial refractive index. SPRI is sensitive to short range interactions between cells and their substratum. The high contrast in SPR signal between cell edges and substratum facilitates identification of cell edges and segmentation of cell areas. With this novel technique, we demonstrate visualization of cell-substratum interactions, and how cell-substratum interactions change over time as cells spread, migrate, and undergo membrane ruffling.

Selective binding of RNase B glycoforms by polydopamine-immobilized concanavalin A.

Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the alpha-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the alpha-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.

Surface plasmon resonance imaging of cells and surface-associated fibronectin.

BACKGROUND: A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells. RESULTS: Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm2 of protein was deposited by cells in 24 h. CONCLUSION: SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.

Hybridization of mismatched or partially matched DNA at surfaces.

We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.