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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 and phase contrast images of five cell types (3T3, HepG2, Vero, A10 and A549) fixed under PBS buffer 72 h after plating on fibronectin coated substrates, and SPR image comparison with fluorescently stained α-vinculin. A) SPR images collected with 100X/1.65 NA objective using 590 nm incident light; phase contrast images acquired with a 20X/0.4 NA objective. The SPR image displays distinct changes in reflectivity for various intracellular components within the evanescent wave such as cell membrane, focal adhesions, and cell nucleus. B) SPR image of A10 cell collected as in A) but contrast adjusted on a linear scale to emphasize the focal adhesions. Subsequently, the sample was immunofluorescently labeled with α-vinculin and imaged with a 63X/1.3 NA objective. A manual intensity threshold of α-vinculin was then overlaid onto the SPR image for comparison. C) Imaging conditions same as in A). A region of intermediate intensity, putative extracellular deposited material, is detected along the cell periphery. A linescan in the SPR image is segregated into four distinct image features: fibronectin (FN) coated substratum, extracellular deposited material (ECM), the basal cell surface, and focal adhesions. The scale bar of 25 μm applies to all images in figure.
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Fig3: SPR and phase contrast images of five cell types (3T3, HepG2, Vero, A10 and A549) fixed under PBS buffer 72 h after plating on fibronectin coated substrates, and SPR image comparison with fluorescently stained α-vinculin. A) SPR images collected with 100X/1.65 NA objective using 590 nm incident light; phase contrast images acquired with a 20X/0.4 NA objective. The SPR image displays distinct changes in reflectivity for various intracellular components within the evanescent wave such as cell membrane, focal adhesions, and cell nucleus. B) SPR image of A10 cell collected as in A) but contrast adjusted on a linear scale to emphasize the focal adhesions. Subsequently, the sample was immunofluorescently labeled with α-vinculin and imaged with a 63X/1.3 NA objective. A manual intensity threshold of α-vinculin was then overlaid onto the SPR image for comparison. C) Imaging conditions same as in A). A region of intermediate intensity, putative extracellular deposited material, is detected along the cell periphery. A linescan in the SPR image is segregated into four distinct image features: fibronectin (FN) coated substratum, extracellular deposited material (ECM), the basal cell surface, and focal adhesions. The scale bar of 25 μm applies to all images in figure.

Mentions: Figure 3A and 3B shows SPR and phase contrast images of five selected cell types: mouse fibroblast cells (3T3), human liver carcinoma cells (HepG2), kidney epithelial cells (Vero), vascular smooth muscle cells (A10) and adenocarcinomic alveolar basal epithelial cells (A549) that were seeded and fixed after 72 h on a fibronectin coated substrate as described in Methods. The left row shows SPR images taken with 590 nm incident light in comparison to the right row using phase contrast. The SPR images show high optical contrast of the cell-substrate interface with a spatial resolution sufficient to reveal a number of subcellular features. The brighter regions of the interest are areas where the density of dielectric material is greater, resulting in greater reflectivity of the incident light due to reduced plasmon coupling. Thus the optical contrast is measured in reflectivity units that have a direct relationship to the mass and refractive index of material within the evanescent wave. Since values used for refractive index for proteins and lipids are usually similar [19], it is likely that the difference in mass, not refractive index, is the primary source of contrast. However, because the thickness of the cell is much larger than the depth of penetration of the evanescent wave, the reflectivity contrast will also be determined by the distance between the cell components and the surface.Figure 3


High resolution surface plasmon resonance imaging for single cells.

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

SPR and phase contrast images of five cell types (3T3, HepG2, Vero, A10 and A549) fixed under PBS buffer 72 h after plating on fibronectin coated substrates, and SPR image comparison with fluorescently stained α-vinculin. A) SPR images collected with 100X/1.65 NA objective using 590 nm incident light; phase contrast images acquired with a 20X/0.4 NA objective. The SPR image displays distinct changes in reflectivity for various intracellular components within the evanescent wave such as cell membrane, focal adhesions, and cell nucleus. B) SPR image of A10 cell collected as in A) but contrast adjusted on a linear scale to emphasize the focal adhesions. Subsequently, the sample was immunofluorescently labeled with α-vinculin and imaged with a 63X/1.3 NA objective. A manual intensity threshold of α-vinculin was then overlaid onto the SPR image for comparison. C) Imaging conditions same as in A). A region of intermediate intensity, putative extracellular deposited material, is detected along the cell periphery. A linescan in the SPR image is segregated into four distinct image features: fibronectin (FN) coated substratum, extracellular deposited material (ECM), the basal cell surface, and focal adhesions. The scale bar of 25 μm applies to all images in figure.
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Related In: Results  -  Collection

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Fig3: SPR and phase contrast images of five cell types (3T3, HepG2, Vero, A10 and A549) fixed under PBS buffer 72 h after plating on fibronectin coated substrates, and SPR image comparison with fluorescently stained α-vinculin. A) SPR images collected with 100X/1.65 NA objective using 590 nm incident light; phase contrast images acquired with a 20X/0.4 NA objective. The SPR image displays distinct changes in reflectivity for various intracellular components within the evanescent wave such as cell membrane, focal adhesions, and cell nucleus. B) SPR image of A10 cell collected as in A) but contrast adjusted on a linear scale to emphasize the focal adhesions. Subsequently, the sample was immunofluorescently labeled with α-vinculin and imaged with a 63X/1.3 NA objective. A manual intensity threshold of α-vinculin was then overlaid onto the SPR image for comparison. C) Imaging conditions same as in A). A region of intermediate intensity, putative extracellular deposited material, is detected along the cell periphery. A linescan in the SPR image is segregated into four distinct image features: fibronectin (FN) coated substratum, extracellular deposited material (ECM), the basal cell surface, and focal adhesions. The scale bar of 25 μm applies to all images in figure.
Mentions: Figure 3A and 3B shows SPR and phase contrast images of five selected cell types: mouse fibroblast cells (3T3), human liver carcinoma cells (HepG2), kidney epithelial cells (Vero), vascular smooth muscle cells (A10) and adenocarcinomic alveolar basal epithelial cells (A549) that were seeded and fixed after 72 h on a fibronectin coated substrate as described in Methods. The left row shows SPR images taken with 590 nm incident light in comparison to the right row using phase contrast. The SPR images show high optical contrast of the cell-substrate interface with a spatial resolution sufficient to reveal a number of subcellular features. The brighter regions of the interest are areas where the density of dielectric material is greater, resulting in greater reflectivity of the incident light due to reduced plasmon coupling. Thus the optical contrast is measured in reflectivity units that have a direct relationship to the mass and refractive index of material within the evanescent wave. Since values used for refractive index for proteins and lipids are usually similar [19], it is likely that the difference in mass, not refractive index, is the primary source of contrast. However, because the thickness of the cell is much larger than the depth of penetration of the evanescent wave, the reflectivity contrast will also be determined by the distance between the cell components and the surface.Figure 3

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