title: High resolution surface plasmon resonance imaging for single cells.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 8313, Gaithersburg, MD 20899, USA. alexander.peterson@nist.gov.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Peterson
authors.forename: Michael
authors.initials: M
authors.lastname: Halter
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tona
authors.forename: Anne L
authors.initials: AL
authors.lastname: Plant
mesh_terms: D000818:Animals; D002460:Cell Line; D004867:Equipment Design; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000295:instrumentation* / Q000379:methods; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods