Nanoplasmonics-enhanced label-free imaging of endothelial cell monolayer integrity.

Surface plasmon resonance imaging (SPRI) is a powerful label-free imaging modality for the analysis of morphological dynamics in cell monolayers. However, classical plasmonic imaging systems have relatively poor spatial resolution along one axis due to the plasmon mode attenuation distance (tens of µm, typically), which significantly limits their ability to resolve subcellular structures. We address this limitation by adding an array of nanostructures onto the metal sensing surface (25 nm thick, 200 nm width, 400 nm period grating) to couple localized plasmons with propagating plasmons, thereby reducing attenuation length and commensurately increasing spatial imaging resolution, without significant loss of sensitivity or image contrast. In this work, experimental results obtained with both conventional unstructured and nanostructured gold film SPRI sensor chips show a clear gain in spatial resolution achieved with surface nanostructuring. The work demonstrates the ability of the nanostructured SPRI chips to resolve fine morphological detail (intercellular gaps) in experiments monitoring changes in endothelial cell monolayer integrity following the activation of the cell surface protease-activated receptor 1 (PAR1) by thrombin. In particular, the nanostructured chips reveal the persistence of small intercellular gaps (<5 µm2) well after apparent recovery of cell monolayer integrity as determined by conventional unstructured surface based SPRI. This new high spatial resolution plasmonic imaging technique uses low-cost and reusable patterned substrates and is likely to find applications in cell biology and pharmacology by allowing label-free quantification of minute cell morphological activities associated with receptor dependent intracellular signaling activity.

Spatial resolution versus contrast trade-off enhancement in high-resolution surface plasmon resonance imaging (SPRI) by metal surface nanostructure design.

Surface plasmon resonance imaging (SPRI) is an optical near-field method used for mapping the spatial distribution of chemical/physical perturbations above a metal surface without exogenous labeling. Currently, the majority of SPRI systems are used in microarray biosensing, requiring only modest spatial resolution. There is increasing interest in applying SPRI for label-free near-field imaging of biological cells to study cell/surface interactions. However, the required resolution (sub-µm) greatly exceeds what current systems can deliver. Indeed, the attenuation length of surface plasmon polaritons (SPP) severely limits resolution along one axis, typically to tens of µm. Strategies to date for improving spatial resolution result in a commensurate deterioration in other imaging parameters. Unlike the smooth metal surfaces used in SPRI that support purely propagating surface modes, nanostructured metal surfaces support "hybrid" SPP modes that share attributes from both propagating and localized modes. We show that these hybrid modes are especially well-suited to high-resolution imaging and demonstrate how the nanostructure geometry can be designed to achieve sub-µm resolution while mitigating the imaging parameter trade-off according to an application-specific optimum.

Label-free visualization and quantification of single cell signaling activity using metal-clad waveguide (MCWG)-based microscopy.

Label-free biosensing methods are very effective for studying cell signaling cascade activation induced by external stimuli. Assays generally involve a large number of cells and rely on the underlying assumption that cell response is homogeneous within a cell population. However, there is an increasing body of evidence showing that cell behavior may vary significantly even among genetically identical cells. In this paper, we demonstrate the use of metal-clad waveguide (MCWG)-based microscopy for label-free real-time monitoring of signaling activity and morphology changes in a small population of cells, with the ability to resolve individual cells. We demonstrate the potential of this approach by quantifying apoptosis-induced intracellular activity in individual cells following exposure to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and by visualizing and quantifying extracellular changes in endothelial cell layer integrity following the activation of the proteinase-activated receptor 1 (PAR1) by thrombin. Results show that averaged signals obtained from a cell population may incorrectly reflect the actual distribution of morphology and kinetics parameters across a cell population by a significant margin.

Metal clad waveguide (MCWG) based imaging using a high numerical aperture microscope objective.

Evanescent-field based methods such as surface plasmon resonance (SPR) have been used very effectively for label-free imaging of microscopic biological material in close proximity to a sensing surface. However, the shallow probing depth of SPR (typically less than ~200 nm) can be problematic when imaging relatively thick biological objects such as cells or bacteria. In this paper, we demonstrate how metal-clad waveguides (MCWG) can be used to achieve deeper probing depth compared to SPR while maintaining good imaging spatial resolution. Comparative numerical simulations of imaging spatial resolution versus probing depth are shown for a number of common SPR, long-range SPR, and MCWG configurations, demonstrating that MCWG offer the best compromise between resolution and depth for imaging thick biological objects. Experimental results of synthetic target and live cell imaging are shown that validate the numerical simulations and demonstrate the capabilities of the method.

Virtual reality and neuropsychological assessment: The reliability of a virtual kitchen to assess daily-life activities in victims of traumatic brain injury.

Traumatic brain injury (TBI) causes impairments affecting instrumental activities of daily living (IADL). However, few studies have considered virtual reality as an ecologically valid tool for the assessment of IADL in patients who have sustained a TBI. The main objective of the present study was to examine the use of the Nonimmersive Virtual Coffee Task (NI-VCT) for IADL assessment in patients with TBI. We analyzed the performance of 19 adults suffering from TBI and 19 healthy controls (HCs) in the real and virtual tasks of making coffee with a coffee machine, as well as in global IQ and executive functions. Patients performed worse than HCs on both real and virtual tasks and on all tests of executive functions. Correlation analyses revealed that NI-VCT scores were related to scores on the real task. Moreover, regression analyses demonstrated that performance on NI-VCT matched real-task performance. Our results support the idea that the virtual kitchen is a valid tool for IADL assessment in patients who have sustained a TBI.

Improved resolution in SPR and MCWG microscopy by combining images acquired with distinct mode propagation directions.

In high-resolution surface plasmon (SPR) imaging, lateral resolution is limited along the direction of plasmon propagation by the longitudinal decay length. Though SPR systems can achieve sub-micrometer resolution, the decay length causes a degradation in the images in the direction of plasmon propagation akin to a blurring artifact, with ringing along resonant to nonresonant transition edges. We present a method to significantly reduce this effect based on combining images of a sample acquired with distinct guided-mode propagation directions. As SPR is a special case of the class of optical structures known as metal-clad waveguides (MCWG) that are also affected by the decay length, this work is broadly applicable.