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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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Back focal plane (BFP) image of SPR angle reflectance data and demonstration of incident angle control of excitation light by digital light projector (DLP). A) The BFP is fully illuminated, at all incident angles available, by 620 nm excitation light off of a 45 nm gold coated coverslip under water media. The incident light is linearly polarized in the x-direction. The dark crescent shaped regions at the edges of the field are where the incident light passes through the objective lens at a high angle of incidence and is coupled into the surface plasmons of the metal film, thus reducing reflectance. The absorbance occurs at the sides of the field where the incident light is p-polarized, and not at the top and bottom of the field where the incident light is s-polarized. The red line traces the angular distribution of incident light onto the image plane from the center of the BFP (0°) to the periphery (60°). B) A line scan of the red line in A) in the p-polarized direction shows the angular dependence of reflectivity and SPR coupling. The green line depicts the angle of illumination that provides maximum SPR coupling (≈53.5° for 620 nm) to be used for SPR imaging. C) Same BFP image area as shown in A) except here the DLP is used to project a thin arc of light that contains the same radial incident angle (≈53.5° illumination angle shown) as depicted by the green line in B).
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Fig2: Back focal plane (BFP) image of SPR angle reflectance data and demonstration of incident angle control of excitation light by digital light projector (DLP). A) The BFP is fully illuminated, at all incident angles available, by 620 nm excitation light off of a 45 nm gold coated coverslip under water media. The incident light is linearly polarized in the x-direction. The dark crescent shaped regions at the edges of the field are where the incident light passes through the objective lens at a high angle of incidence and is coupled into the surface plasmons of the metal film, thus reducing reflectance. The absorbance occurs at the sides of the field where the incident light is p-polarized, and not at the top and bottom of the field where the incident light is s-polarized. The red line traces the angular distribution of incident light onto the image plane from the center of the BFP (0°) to the periphery (60°). B) A line scan of the red line in A) in the p-polarized direction shows the angular dependence of reflectivity and SPR coupling. The green line depicts the angle of illumination that provides maximum SPR coupling (≈53.5° for 620 nm) to be used for SPR imaging. C) Same BFP image area as shown in A) except here the DLP is used to project a thin arc of light that contains the same radial incident angle (≈53.5° illumination angle shown) as depicted by the green line in B).

Mentions: The spatial location of light at the back focal plane (BFP) of the microscope objective indicates the incident angle with which that light impinges on the sample. The angle of incidence increases approximately linearly with distance from the optical axis of the objective. For example, an illuminated spot in the center of the BFP would irradiate the sample normal to the surface while a spot at the edge of the objective BFP would be illuminating the sample at an angle that is steep enough to achieve total internal reflection. This phenomenon is shown in Figure 2A, which displays the image of the BFP which is fully illuminated by the reflection of 620 nm from a 45 nm thick gold coating on a glass coverslip in buffer. The incident light is linearly polarized, and impinges on the sample at angles that vary from 0° at the center of the BFP to an angle of 60° at the periphery. This angle span is marked by the red line (Figure 2A). The regions of lowest intensity, which appear as the dark ring at the periphery of the BFP, occur at the angle of incidence where the light is maximally coupled with the surface plasmons, and minimum reflectance occurs. This angle is ≈ 54.0°. The strong plasmon absorbance of the p-polarized light occurs in the x-direction and fades away azimuthally as the light becomes entirely s-polarized in the y-direction.Figure 2


High resolution surface plasmon resonance imaging for single cells.

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

Back focal plane (BFP) image of SPR angle reflectance data and demonstration of incident angle control of excitation light by digital light projector (DLP). A) The BFP is fully illuminated, at all incident angles available, by 620 nm excitation light off of a 45 nm gold coated coverslip under water media. The incident light is linearly polarized in the x-direction. The dark crescent shaped regions at the edges of the field are where the incident light passes through the objective lens at a high angle of incidence and is coupled into the surface plasmons of the metal film, thus reducing reflectance. The absorbance occurs at the sides of the field where the incident light is p-polarized, and not at the top and bottom of the field where the incident light is s-polarized. The red line traces the angular distribution of incident light onto the image plane from the center of the BFP (0°) to the periphery (60°). B) A line scan of the red line in A) in the p-polarized direction shows the angular dependence of reflectivity and SPR coupling. The green line depicts the angle of illumination that provides maximum SPR coupling (≈53.5° for 620 nm) to be used for SPR imaging. C) Same BFP image area as shown in A) except here the DLP is used to project a thin arc of light that contains the same radial incident angle (≈53.5° illumination angle shown) as depicted by the green line in B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Back focal plane (BFP) image of SPR angle reflectance data and demonstration of incident angle control of excitation light by digital light projector (DLP). A) The BFP is fully illuminated, at all incident angles available, by 620 nm excitation light off of a 45 nm gold coated coverslip under water media. The incident light is linearly polarized in the x-direction. The dark crescent shaped regions at the edges of the field are where the incident light passes through the objective lens at a high angle of incidence and is coupled into the surface plasmons of the metal film, thus reducing reflectance. The absorbance occurs at the sides of the field where the incident light is p-polarized, and not at the top and bottom of the field where the incident light is s-polarized. The red line traces the angular distribution of incident light onto the image plane from the center of the BFP (0°) to the periphery (60°). B) A line scan of the red line in A) in the p-polarized direction shows the angular dependence of reflectivity and SPR coupling. The green line depicts the angle of illumination that provides maximum SPR coupling (≈53.5° for 620 nm) to be used for SPR imaging. C) Same BFP image area as shown in A) except here the DLP is used to project a thin arc of light that contains the same radial incident angle (≈53.5° illumination angle shown) as depicted by the green line in B).
Mentions: The spatial location of light at the back focal plane (BFP) of the microscope objective indicates the incident angle with which that light impinges on the sample. The angle of incidence increases approximately linearly with distance from the optical axis of the objective. For example, an illuminated spot in the center of the BFP would irradiate the sample normal to the surface while a spot at the edge of the objective BFP would be illuminating the sample at an angle that is steep enough to achieve total internal reflection. This phenomenon is shown in Figure 2A, which displays the image of the BFP which is fully illuminated by the reflection of 620 nm from a 45 nm thick gold coating on a glass coverslip in buffer. The incident light is linearly polarized, and impinges on the sample at angles that vary from 0° at the center of the BFP to an angle of 60° at the periphery. This angle span is marked by the red line (Figure 2A). The regions of lowest intensity, which appear as the dark ring at the periphery of the BFP, occur at the angle of incidence where the light is maximally coupled with the surface plasmons, and minimum reflectance occurs. This angle is ≈ 54.0°. The strong plasmon absorbance of the p-polarized light occurs in the x-direction and fades away azimuthally as the light becomes entirely s-polarized in the y-direction.Figure 2

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