Abstract
BackgroundSurface 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.ResultsAn 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2121-15-35) contains supplementary material, which is available to authorized users.
Highlights
Surface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface
We ask: how far can we detect the evanescent field propagation into the cell, and how close are these intracellular features to the substratum from which the evanescent wave originates? We demonstrate a method for using polymer microspheres to effectively measure the evanescent wave penetration depth in the imaging mode
The microscope objective and inverted microscope body were designed for total internal reflection fluorescence microscopy (TIRFM) [14] of cellular features
Summary
Surface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface. When used in an imaging mode, SPR is an attractive technique for quantifying and visualizing cells and their extracellular environment because it is label-free, requires low levels of incident light to achieve good contrast, and is able to quantitatively report on very small amounts (3 ng/cm2) of biological material [11,12]. Our work in this field has advanced the spatial resolution of SPRI to unprecedented levels
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