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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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Related in: MedlinePlus

Measurement of SPR evanescent wave (EW) penetration depth by geometric relationship of measured radii of polymer microspheres. A) Bright-field and SPR images (for 5 wavelengths) of a polymethylmethacrylate (PMMA) microsphere in water. B) Diagram of a microsphere at the SPR sensor interface showing that only a fraction of the bead lies within the EW. The equation shows the relationship between the EW penetration depth (d) and the radius of the sphere measured by bright field (r1) and SPRI (r2). The overlay shows a layer model of the interface; the water layer decreases thickness as the bead moves toward the surface and into the EW. C) SPR image and insert of a microsphere used to illustrate the image analysis procedure for measuring the value of r2, the SPRI detected radius. The standard deviation (σ) of background intensity is determined by the annulus-shaped ROI in the primary image, and in the image insert a value of 3σ is set as the threshold with pixels values above threshold colored red; the radius of the bead (r2 in blue) is determined from the area of circle (green dashed circle) computed from the threshold. D) Exponential decay from surface into media of the SPR generated EW calculated as field intensity versus distance from surface (nm). Two values are labeled: the 1/e decay at 37% field strength commonly referred to as the “penetration depth”, and the 5% field strength value, which is the theoretical detection limit. E) Extent of the EW field depth (Lp) measured for several polymer microspheres as a function of excitation wavelength, along with theoretical values of the penetration depth (1/e) and detection limit (5% field strength). Within the standard deviation of the measurement error, the decay values agree for all microspheres and the calculated penetration depth at 1/e field decay.
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Fig5: Measurement of SPR evanescent wave (EW) penetration depth by geometric relationship of measured radii of polymer microspheres. A) Bright-field and SPR images (for 5 wavelengths) of a polymethylmethacrylate (PMMA) microsphere in water. B) Diagram of a microsphere at the SPR sensor interface showing that only a fraction of the bead lies within the EW. The equation shows the relationship between the EW penetration depth (d) and the radius of the sphere measured by bright field (r1) and SPRI (r2). The overlay shows a layer model of the interface; the water layer decreases thickness as the bead moves toward the surface and into the EW. C) SPR image and insert of a microsphere used to illustrate the image analysis procedure for measuring the value of r2, the SPRI detected radius. The standard deviation (σ) of background intensity is determined by the annulus-shaped ROI in the primary image, and in the image insert a value of 3σ is set as the threshold with pixels values above threshold colored red; the radius of the bead (r2 in blue) is determined from the area of circle (green dashed circle) computed from the threshold. D) Exponential decay from surface into media of the SPR generated EW calculated as field intensity versus distance from surface (nm). Two values are labeled: the 1/e decay at 37% field strength commonly referred to as the “penetration depth”, and the 5% field strength value, which is the theoretical detection limit. E) Extent of the EW field depth (Lp) measured for several polymer microspheres as a function of excitation wavelength, along with theoretical values of the penetration depth (1/e) and detection limit (5% field strength). Within the standard deviation of the measurement error, the decay values agree for all microspheres and the calculated penetration depth at 1/e field decay.

Mentions: Theory predicts that the wavelength of incident light used to excite surface plasmons determines the evanescent wave penetration depth [21]. To directly measure the sensitivity of the SPR field as a function of distance from the surface, we employed measurements of polymer microspheres with known refractive index and diameter, and incident light of several wavelengths. Images of a representative poly (methyl methacrylate) (PMMA) microsphere are taken in bright field as well as with SPR imaging at several wavelengths (Figure 5A). The radius of the microsphere r1, measured in the bright field image, can be related to the radius observed in the SPR image, r2, and to the measured detectable penetration depth, d with the following equation, r12 = r22 + (r2 - d)2, which is derived from the geometric Pythagoras theorem (Figure 5B). The physical picture is that only a small portion of the bead is in contact with the gold sensor surface, while most of the bead resides above the surface, and above the surface plasmon generated evanescent wave. As the evanescent wave extends into the water media, the bead, which has a measurably different refractive index from water, is partially sampled by the evanescent wave. The distance at which the refractive index change due to the presence of the bead is detectable above the background is the threshold which we interpret as the detectable extent of the surface plasmon penetration depth. Figure 5C shows a plot summarizing the image analysis routine, (and which is described in further detail in the Methods), to determine the penetration depth values. Essentially, for each SPR image, the background region of the image is used to determine the standard deviation (σ) of background noise. Intensity values of 3σ from the average background intensity are considered to be signal resulting from detectable bead material in the evanescent field. The r2 value is obtained from the apparent area of the object in the SPR image estimated as a circle. The SPR penetration depth for each wavelength is determined using the r2 value obtained for each SPR image, along with the r1 value obtained for the bead diameter measured from the bright field image. Figure 5D shows a theoretically calculated SPR evanescent wave intensity decay, described by a single exponential decay function, as a function of distance from the surface for 620 nm excitation light (see Methods). This plot also indicates the distance from the surface where the field decays to 1/e of its original intensity or 37% field intensity, and where the field decays to 5% field intensity, which represents the theoretically maximum distance to detect a change of refractive index. Finally, Figure 5E shows a compilation plot that depicts the calculated penetration depth and detection threshold for a range of SPR excitation wavelengths, and is also plotted with the measured detectable penetration depths for several types of polymer microspheres over the same range of wavelengths. As can be seen, the measured limit of detection, within the experimental noise, seems to be described well by the theoretically determined distance at which the evanescent field decays to 1/e (63%). This occurs for a variety of polymer microspheres with differing refractive index values (n = 1.345 to 1.59). This implies that subcellular structures observed in the SPR images (Figure 3A) are likely to reside within a distance from the surface up to a maximum amount as described by the 1/e distance threshold. This is expected to be true for all the observable cellular components (membrane, fibrillar structures, etc.) since the refractive indices of these components will be within the range of refractive indices we probed with the polymeric beads.Figure 5


High resolution surface plasmon resonance imaging for single cells.

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

Measurement of SPR evanescent wave (EW) penetration depth by geometric relationship of measured radii of polymer microspheres. A) Bright-field and SPR images (for 5 wavelengths) of a polymethylmethacrylate (PMMA) microsphere in water. B) Diagram of a microsphere at the SPR sensor interface showing that only a fraction of the bead lies within the EW. The equation shows the relationship between the EW penetration depth (d) and the radius of the sphere measured by bright field (r1) and SPRI (r2). The overlay shows a layer model of the interface; the water layer decreases thickness as the bead moves toward the surface and into the EW. C) SPR image and insert of a microsphere used to illustrate the image analysis procedure for measuring the value of r2, the SPRI detected radius. The standard deviation (σ) of background intensity is determined by the annulus-shaped ROI in the primary image, and in the image insert a value of 3σ is set as the threshold with pixels values above threshold colored red; the radius of the bead (r2 in blue) is determined from the area of circle (green dashed circle) computed from the threshold. D) Exponential decay from surface into media of the SPR generated EW calculated as field intensity versus distance from surface (nm). Two values are labeled: the 1/e decay at 37% field strength commonly referred to as the “penetration depth”, and the 5% field strength value, which is the theoretical detection limit. E) Extent of the EW field depth (Lp) measured for several polymer microspheres as a function of excitation wavelength, along with theoretical values of the penetration depth (1/e) and detection limit (5% field strength). Within the standard deviation of the measurement error, the decay values agree for all microspheres and the calculated penetration depth at 1/e field decay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Measurement of SPR evanescent wave (EW) penetration depth by geometric relationship of measured radii of polymer microspheres. A) Bright-field and SPR images (for 5 wavelengths) of a polymethylmethacrylate (PMMA) microsphere in water. B) Diagram of a microsphere at the SPR sensor interface showing that only a fraction of the bead lies within the EW. The equation shows the relationship between the EW penetration depth (d) and the radius of the sphere measured by bright field (r1) and SPRI (r2). The overlay shows a layer model of the interface; the water layer decreases thickness as the bead moves toward the surface and into the EW. C) SPR image and insert of a microsphere used to illustrate the image analysis procedure for measuring the value of r2, the SPRI detected radius. The standard deviation (σ) of background intensity is determined by the annulus-shaped ROI in the primary image, and in the image insert a value of 3σ is set as the threshold with pixels values above threshold colored red; the radius of the bead (r2 in blue) is determined from the area of circle (green dashed circle) computed from the threshold. D) Exponential decay from surface into media of the SPR generated EW calculated as field intensity versus distance from surface (nm). Two values are labeled: the 1/e decay at 37% field strength commonly referred to as the “penetration depth”, and the 5% field strength value, which is the theoretical detection limit. E) Extent of the EW field depth (Lp) measured for several polymer microspheres as a function of excitation wavelength, along with theoretical values of the penetration depth (1/e) and detection limit (5% field strength). Within the standard deviation of the measurement error, the decay values agree for all microspheres and the calculated penetration depth at 1/e field decay.
Mentions: Theory predicts that the wavelength of incident light used to excite surface plasmons determines the evanescent wave penetration depth [21]. To directly measure the sensitivity of the SPR field as a function of distance from the surface, we employed measurements of polymer microspheres with known refractive index and diameter, and incident light of several wavelengths. Images of a representative poly (methyl methacrylate) (PMMA) microsphere are taken in bright field as well as with SPR imaging at several wavelengths (Figure 5A). The radius of the microsphere r1, measured in the bright field image, can be related to the radius observed in the SPR image, r2, and to the measured detectable penetration depth, d with the following equation, r12 = r22 + (r2 - d)2, which is derived from the geometric Pythagoras theorem (Figure 5B). The physical picture is that only a small portion of the bead is in contact with the gold sensor surface, while most of the bead resides above the surface, and above the surface plasmon generated evanescent wave. As the evanescent wave extends into the water media, the bead, which has a measurably different refractive index from water, is partially sampled by the evanescent wave. The distance at which the refractive index change due to the presence of the bead is detectable above the background is the threshold which we interpret as the detectable extent of the surface plasmon penetration depth. Figure 5C shows a plot summarizing the image analysis routine, (and which is described in further detail in the Methods), to determine the penetration depth values. Essentially, for each SPR image, the background region of the image is used to determine the standard deviation (σ) of background noise. Intensity values of 3σ from the average background intensity are considered to be signal resulting from detectable bead material in the evanescent field. The r2 value is obtained from the apparent area of the object in the SPR image estimated as a circle. The SPR penetration depth for each wavelength is determined using the r2 value obtained for each SPR image, along with the r1 value obtained for the bead diameter measured from the bright field image. Figure 5D shows a theoretically calculated SPR evanescent wave intensity decay, described by a single exponential decay function, as a function of distance from the surface for 620 nm excitation light (see Methods). This plot also indicates the distance from the surface where the field decays to 1/e of its original intensity or 37% field intensity, and where the field decays to 5% field intensity, which represents the theoretically maximum distance to detect a change of refractive index. Finally, Figure 5E shows a compilation plot that depicts the calculated penetration depth and detection threshold for a range of SPR excitation wavelengths, and is also plotted with the measured detectable penetration depths for several types of polymer microspheres over the same range of wavelengths. As can be seen, the measured limit of detection, within the experimental noise, seems to be described well by the theoretically determined distance at which the evanescent field decays to 1/e (63%). This occurs for a variety of polymer microspheres with differing refractive index values (n = 1.345 to 1.59). This implies that subcellular structures observed in the SPR images (Figure 3A) are likely to reside within a distance from the surface up to a maximum amount as described by the 1/e distance threshold. This is expected to be true for all the observable cellular components (membrane, fibrillar structures, etc.) since the refractive indices of these components will be within the range of refractive indices we probed with the polymeric beads.Figure 5

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