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High resolution surface plasmon resonance imaging for single cells.

Peterson AW, Halter M, Tona A, Plant AL - BMC Cell Biol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

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.

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SPR imaging through a microscope objective instrumentation schematic. Incident angle selected light is generated and patterned by a digital light projector that is then collimated, wavelength filtered, linear polarized, and then directed through an inverted microscope platform (shaded in grey). The reflected image is captured on a CCD camera after a switchable lens assembly selects for either the image plane or back focal plane.
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Fig1: SPR imaging through a microscope objective instrumentation schematic. Incident angle selected light is generated and patterned by a digital light projector that is then collimated, wavelength filtered, linear polarized, and then directed through an inverted microscope platform (shaded in grey). The reflected image is captured on a CCD camera after a switchable lens assembly selects for either the image plane or back focal plane.

Mentions: The use of an objective lens for exciting SPR is described schematically in FigureĀ 1. The microscope objective and inverted microscope body were designed for total internal reflection fluorescence microscopy (TIRFM) [14] of cellular features. Here we adapted this configuration for SPRI. For performing SPRI, the microscope objective is used to direct and collect the incident and reflected light. The use of the objective lens and microscope body allows for SPR to be integrated into a device that allows multiple imaging modes.Figure 1


High resolution surface plasmon resonance imaging for single cells.

Peterson AW, Halter M, Tona A, Plant AL - BMC Cell Biol. (2014)

SPR imaging through a microscope objective instrumentation schematic. Incident angle selected light is generated and patterned by a digital light projector that is then collimated, wavelength filtered, linear polarized, and then directed through an inverted microscope platform (shaded in grey). The reflected image is captured on a CCD camera after a switchable lens assembly selects for either the image plane or back focal plane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289309&req=5

Fig1: SPR imaging through a microscope objective instrumentation schematic. Incident angle selected light is generated and patterned by a digital light projector that is then collimated, wavelength filtered, linear polarized, and then directed through an inverted microscope platform (shaded in grey). The reflected image is captured on a CCD camera after a switchable lens assembly selects for either the image plane or back focal plane.
Mentions: The use of an objective lens for exciting SPR is described schematically in FigureĀ 1. The microscope objective and inverted microscope body were designed for total internal reflection fluorescence microscopy (TIRFM) [14] of cellular features. Here we adapted this configuration for SPRI. For performing SPRI, the microscope objective is used to direct and collect the incident and reflected light. The use of the objective lens and microscope body allows for SPR to be integrated into a device that allows multiple imaging modes.Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

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.

Show MeSH
Related in: MedlinePlus