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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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Spatial resolution of fluorescent (FL) and SPR imaging using fluorescent point-source nanospheres. A) Transparent coverslip a 0.17 μm diameter particle fluorescing at 515 nm peak emitting wavelength in epi-fluorescent mode. Line scan plot next to image is used to determine full width half-maximum (FWHM) at 0.29 μm for a 1.65 NA objective. B) SPR image of nanoparticle at 620 nm shows a FWHM of 0.3 μm in the x-direction (red) and 0.6 μm in the y-direction using the 1.65 NA objective. The scale bar of 2 μm applies to A and B. Nanoparticle measurements made under water media, and fluorescence emission collected with 530 nm bandpass filter.
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Fig4: Spatial resolution of fluorescent (FL) and SPR imaging using fluorescent point-source nanospheres. A) Transparent coverslip a 0.17 μm diameter particle fluorescing at 515 nm peak emitting wavelength in epi-fluorescent mode. Line scan plot next to image is used to determine full width half-maximum (FWHM) at 0.29 μm for a 1.65 NA objective. B) SPR image of nanoparticle at 620 nm shows a FWHM of 0.3 μm in the x-direction (red) and 0.6 μm in the y-direction using the 1.65 NA objective. The scale bar of 2 μm applies to A and B. Nanoparticle measurements made under water media, and fluorescence emission collected with 530 nm bandpass filter.

Mentions: The theoretical diffraction-limited resolution for a 1.65 NA microscope objective is 0.23 μm and 0.20 μm for 620 nm and 530 nm light, respectively. The theoretical propagation length for 620 nm light of the surface plasmon in the direction parallel to the surface plasmon excitation is ≈ 3 μm [21]. We measured the spatial resolution of our microscope using fluorescent nanoparticles to determine the point spread function in both epifluorescence and SPR imaging mode. We chose the fluorescence wavelength to be distinct in both excitation and emission wavelengths from the SPR imaging wavelength, and we chose the SPR wavelength for its observable lateral decay length. The fluorescent nanoparticles were measured at 530 nm emission under water in epi-fluorescent mode (Figure 4A), and a line profile plot was used to determine a full width at half maximum (FWHM) value of 0.29 μm, very close to the theoretical limit. For the second measurement, the SPR excitation light was set to 620 nm with ≈ 53.5° incident angle and the nanoparticle bead image was obtained, Figure 4B, with the corresponding line profile resulting in a FWHM of 0.30 μm in the x-direction perpendicular to the surface plasmon propagation vector, and 0.60 μm in the y-direction, parallel to the surface plasmon propagation. The asymmetry in the x- versus y- resolution arises from the surface plasmon leakage radiation decay in the direction parallel to the excitation light [22].Figure 4


High resolution surface plasmon resonance imaging for single cells.

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

Spatial resolution of fluorescent (FL) and SPR imaging using fluorescent point-source nanospheres. A) Transparent coverslip a 0.17 μm diameter particle fluorescing at 515 nm peak emitting wavelength in epi-fluorescent mode. Line scan plot next to image is used to determine full width half-maximum (FWHM) at 0.29 μm for a 1.65 NA objective. B) SPR image of nanoparticle at 620 nm shows a FWHM of 0.3 μm in the x-direction (red) and 0.6 μm in the y-direction using the 1.65 NA objective. The scale bar of 2 μm applies to A and B. Nanoparticle measurements made under water media, and fluorescence emission collected with 530 nm bandpass filter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Spatial resolution of fluorescent (FL) and SPR imaging using fluorescent point-source nanospheres. A) Transparent coverslip a 0.17 μm diameter particle fluorescing at 515 nm peak emitting wavelength in epi-fluorescent mode. Line scan plot next to image is used to determine full width half-maximum (FWHM) at 0.29 μm for a 1.65 NA objective. B) SPR image of nanoparticle at 620 nm shows a FWHM of 0.3 μm in the x-direction (red) and 0.6 μm in the y-direction using the 1.65 NA objective. The scale bar of 2 μm applies to A and B. Nanoparticle measurements made under water media, and fluorescence emission collected with 530 nm bandpass filter.
Mentions: The theoretical diffraction-limited resolution for a 1.65 NA microscope objective is 0.23 μm and 0.20 μm for 620 nm and 530 nm light, respectively. The theoretical propagation length for 620 nm light of the surface plasmon in the direction parallel to the surface plasmon excitation is ≈ 3 μm [21]. We measured the spatial resolution of our microscope using fluorescent nanoparticles to determine the point spread function in both epifluorescence and SPR imaging mode. We chose the fluorescence wavelength to be distinct in both excitation and emission wavelengths from the SPR imaging wavelength, and we chose the SPR wavelength for its observable lateral decay length. The fluorescent nanoparticles were measured at 530 nm emission under water in epi-fluorescent mode (Figure 4A), and a line profile plot was used to determine a full width at half maximum (FWHM) value of 0.29 μm, very close to the theoretical limit. For the second measurement, the SPR excitation light was set to 620 nm with ≈ 53.5° incident angle and the nanoparticle bead image was obtained, Figure 4B, with the corresponding line profile resulting in a FWHM of 0.30 μm in the x-direction perpendicular to the surface plasmon propagation vector, and 0.60 μm in the y-direction, parallel to the surface plasmon propagation. The asymmetry in the x- versus y- resolution arises from the surface plasmon leakage radiation decay in the direction parallel to the excitation light [22].Figure 4

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