Surface plasmon resonance imaging of cells and surface-associated fibronectin
© Peterson et al; licensee BioMed Central Ltd. 2009
Received: 04 March 2008
Accepted: 26 February 2009
Published: 26 February 2009
A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells.
Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm2 of protein was deposited by cells in 24 h.
SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.
Cellular remodeling of the ECM is a critical factor in wound healing, developmental biology, metastasis of tumor cells, and diseases such as hypertension [1–4]. The study of cell-matrix dynamics and cellular remodeling of the ECM is challenging, and has involved the use of fluorophores, including fluorescent fusion proteins , often using total internal reflection fluorescence microscopy (TIRFM) . We show here that as an alternative, SPRI can be a sensitive, label-free, and low-light optical method that eliminates the requirement for modified biological molecules and transfected cells, and allows for highly sensitive real-time observation of protein deposition and live cell engagement with the ECM.
Surface plasmon resonance (SPR) occurs when light energy couples into the electromagnetic field at a metal-coated surface. The reflectivity of the incident light is inverse to the extent of plasmon resonance, and is determined by the identity and the thickness of the metal layer, the angle of incidence, the wavelength of the incident light, and the refractive index of the medium at the interface. Because the refractive index is proportional to the amount of adsorbate at the surface , SPR has been used as a quantitative, sensitive, and label-free technique for measuring the binding kinetics of proteins , DNA [9, 10], and small molecules [11, 12], to surface immobilized capture agents. Using SPR in an imaging mode, high throughput analysis of proteins and DNA has also been demonstrated [13, 14]. SPR imaging has not previously been considered a useful technique for imaging cell features, largely because of previous assumptions that poor spatial resolution would prevent useful imaging.
In this report, we demonstrate that SPRI contrast allows sensitive measurement of cell-substrate interactions and mass changes at the substrate interface. SPRI allows quantification of cell secreted and deposited material by observing changes in surface protein mass/area as a function of time and location. We use SPRI to observe the initial surface preparation by monitoring the deposition of the extracellular matrix protein fibronectin which serves as the substrate for the cell based measurements. By using different incident wavelengths and image processing routines for SPRI, it is possible to tune the SPRI measurement for sensitivity versus spatial resolution to suit each step of the experiment. In this report, we demonstrate that SPRI is a sensitive interfacial technique that is able to bridge the gap between molecular (protein adsorption) and cellular (cell-substrate) measurements.
Results and discussion
SPRI apparatus and resolution
Figure 1B provides an indication of the lateral resolution of the SPRI instrument. Polydimethylsiloxane (PDMS) samples, photolithographically patterned with 5 μm circles or 2 μm lines, were placed directly onto SF-10 slides coated with gold and mounting onto the SF-10 prism. The 2 μm patterned lines are arranged obliquely to the direction of the surface plasmon propagation. The SPR images (Fig. 1B) arise due to the difference in the refractive index of air and the refractive index of the PDMS in contact with the surface. Images were acquired using 470 nm and 630 nm incident light as indicated. The 5 μm circle images and line scans are normalized and the normalized reflectivities are plotted displaying a similar spatial sensitivity. The images of the 2 μm lines and line scans are displayed using the raw reflectivity scale to show that the 630 nm SPR image has a larger reflectivity response than the 470 nm SPR image. These results also demonstrate the lateral resolution of the SPRI system to be at least 2 μm based on the ability to distinguish 2 μm features spaced 2 μm apart. This value is in agreement with an estimate made in a previous report using a similar prism based configuration and numerical aperture lens .
In this study, we employed the use of both 470 and 630 nm incident light. According to theory, adjusting the wavelength of the incident light affects spatial resolution, refractive index sensitivity, and evanescent wave penetration depth . However, in practice, we could detect little difference in spatial resolution at the two wavelengths. The main difference was that 470 nm SPR images had a larger depth of field and therefore a larger portion of the image was in focus at the maximal spatial resolution. Also, even though Figure 2 indicates that 630 nm light provided greater sensitivity, the sensitivity at 470 nm was maximized by using a gold thickness (30 nm versus 45 nm) more optimal for 470 nm signal response and image analysis was used to average many pixel values into one measurement. This enabled both wavelengths to be sufficiently sensitive and provide comparable results for protein mass/area. For the data shown here, we used 630 nm incident light for monitoring time-dependent deposition of protein coupled with difference imaging to obtain a signal-to-noise of ~3 ng/cm2. For cell measurements we used 470 nm incident light with a signal-to-noise of ~20 ng/cm2. Because the cell mass provides a very large SPR signal, the smaller response of 470 nm light helped insure linearity of SPR signal to mass. The SPR evanescent wave penetration depth (defined as decay of the field to 1/e) for 470 nm light is calculated to be ~60 nm compared to ~150 nm for 630 nm light. Thus, about 63% of the intensity of the field for the 470 nm light is penetrating to a depth of 60 nm above the gold surface. The lower penetration of 470 nm light allowed us to image cell-matrix interactions while minimizing contributions to the SPR signal from the cell interior.
Substrate patterning and fibronectin deposition
Following fibronectin adsorption, the patterned sample was exposed to serum-containing cell culture medium and measured by SPRI at 630 nm for over 400 minutes at 37°C. The kinetics of adsorption of serum components to the fibronectin coated surface is displayed in Figure 3C and shows approximately 280 ng/cm2 of serum proteins added to the surface at saturation; this result is consistent with a previous report that observed serum albumin adsorption onto fibronectin coated substrates . Negligible adsorption of protein to the PEG-thiol coated areas was observed. The SPRI reflectivity signals were converted to mass/area of serum proteins bound based on the partial specific volume for globular proteins [26, 27]. Although SPRI cannot independently identify the specific proteins absorbed, the partial specific volume for globular proteins is highly conserved , and therefore the amount of protein adsorbed can be estimated with good accuracy, without requiring specific knowledge of the composition.
Fibronectin and serum proteins appear to adsorb homogeneously onto the hexadecanethiol patterns as shown in line scans of SPR intensity across these areas (Fig. 3D). This observation is shown here to provide contrast to the non-uniform distribution of deposited protein following the addition of cells to the matrix as shown in following sections.
SPR imaging of cells and patterned fibronectin
The pseudocolor scale in Figure 4 indicates the reflectivity values associated with areas of different mass. It can be seen that the PEG areas have the lowest mass, with a reflectivity on the order of 0.16. The adsorption of protein to the hexadecanethiol areas results in a higher reflectivity ranging between 0.18 and 0.19, and the greatest mass and highest reflectivity values are associated with regions of cell-substrate interactions. Figure 4 shows that a number of cells appear to span the PEG-thiol regions. Since the cell thickness is much larger than the depth of penetration of the plasmon evanescent wave, SPRI contrast is putatively generated by the distance that the cell membrane is from the surface. Thus, for cells that span PEG locations (Fig. 4A, arrow) the region of the cell above the PEG often has less intensity, suggesting that the part of the cell that spans the PEG-thiol is further from the substrate than the rest of the cell.
The optical contrast provided by SPRI allows label-free segmentation of cell areas. Segmentation of cells (Fig. 4C) based on the SPR signal (right) compared to segmentation based on staining with Texas Red maleimide (left) shows that both SPR and fluorescence images provide similar outlines for cell area. Subtle differences are observed, however, in specific areas that provide a strong fluorescence intensity, but little SPR signal (arrow in Fig. 4C). These observations suggest that portions of cells reside farther away from the interface than is detectable by the SPR evanescent wave.
SPRI of cellular deposited material
Additional file 1: Quantifying protein deposition at cell periphery. Movie showing image analysis procedure used to quantify protein deposition at the cell periphery after 24 h in culture by dilating the cell contours to extract protein coverage versus distance-from-cell edge. Also, shows image analysis for low (<15%) and high (>25%) cell density regions. (MOV 1 MB)
While the flat response of the PEG distance-to-cell-edge coverage plot in Figure 5E shows that no measurable cell derived material is being deposited onto the PEG regions, it also serves as an image analysis control to insure influence from edge effects is minimized. This also shows that other effects such as lateral decay of SPR signal are not observed to bleed into low signal regions.
We also observed that in areas of low cell density (<15% of area corresponding to cells) and high cell density (>25% of area corresponding to cells) that there was deposition of protein that was dependent on distance from cell edges (Additional File 1). This distance dependence was observed to be more pronounced when applied to regions of lower cell density. The sometimes more subtle distance dependence at high cell density is perhaps due to the influence of material secreted by neighboring cells.
Our results show that SPR images, obtained without the addition of fluorescent or other labels, can provide information on cell-substrate contacts, ECM deposition and substrate protein patterns. Cells can be observed over long periods of time because cellular damage is minimized at the low light levels that are sufficient for SPRI. A previous report demonstrated the first example of SPR imaging of cells , but the present study describes how the use of appropriate optical architecture, light sources, and image analysis permit a successful application of SPRI. This study demonstrates the use of SPRI to perform cell object segmentation, protein pattern identification, and quantitation of protein deposition by cells. Live-cell measurements by SPRI demonstrate the time-dependent deposition of cellular protein. The data indicate that SPRI can detect deposited protein with a sensitivity of ~20 ng/cm2, and can achieve a lateral resolution of at least 2 μm. While the current study does not attempt to identify the proteins that cells contribute to the matrix, future integration of SPRI with fluorescence based microscopy methods will combine the unique label-free information from SPRI of live cells with complementary biochemical information for a more complete understanding of cell interactions with their extracellular environment.
SF-10 glass slides (25 mm × 25 mm × 1 mm) (Schott, Elmsford, NY) were acid-washed with 7:3 (v/v) H2SO4:H2O2, rinsed with 18 MΩ cm distilled water, rinsed with ethanol, dried under a N2 stream, and then coated with ~1 nm chromium and ~30 nm gold by magnetron sputtering using an Edwards Auto 306 vacuum system (Edwards, Wilmington, MA) at 1 × 10-7 mbar. We selected this thickness to be optimized for 470 nm incident light instead of thicker gold (45 nm would be optimal for coupling 630 nm light) in order to maximize its transmissivity and facilitate the phase contrast and fluorescence measurements.
Polydimethylsiloxane (PDMS) stamps with 300 μm or 500 μm square features spaced 50 μm apart were used for microcontact printing. Masters for casting PDMS, (Sylgard 184, Dow-Corning, Midland, MI) stamps were made using a dry film resist technique and PDMS stamps were cast from these as previously described . Microcontact printing of hexadecanethiol to the gold substrate was performed according to a published procedure  using a 2 mM hexadecanethiol (Aldrich, St. Loius, MO) solution in ethanol. Following stamping with hexadecanethiol, the slides were immersed in a 0.5 mM solution of HS(CH2)11(OCH2CH2)3OH (PEG-thiol)(Asemblon, Seattle, Washington) in ethanol for 12 h in order to coat the exposed gold with protein resistant polyethylene glycol terminated alkanethiol. The hexadecanethiol areas of the patterned substrates were coated with fibronectin by inserting slides into a sterile solution of 25 μg/ml bovine plasma fibronectin (Sigma, St. Louis, MO) in Ca2+-and Mg2+-free Dulbecco's phosphate buffered saline (DPBS; Invitrogen, Carlsbad, CA) for 1 h to 2 h. For the SPRI kinetics experiment, an alkanethiol patterned gold slide was mounted into an environmental cell culture/viewing chamber (FCS2; Bioptechs, Butler, PA), rinsed by flowing through DPBS, and placed onto the SPRI device. A solution of 25 μg/ml fibronectin in DPBS was flowed through the chamber at 1 mL/min, stopped, and the kinetics of deposition monitored by SPRI for 1 h at room temperature.
Cell culture, fixing, and staining
The rat aortic vascular smooth muscle cell line (vSMC) (A10; ATCC, Manassas, VA) was maintained in Dulbecco's Modified Eagles Medium (DMEM; Mediatech, Herndon, VA) supplemented with nonessential amino acids, glutamine, penicillin (100 units/mL), streptomycin (100 μg/mL), 10% by volume FBS (Invitrogen, Carlsbad, CA) and 25 mM HEPES, and maintained in a humidified 5% CO2 balanced-air atmosphere at 37°C. Cells were removed from tissue culture polystyrene flasks by trypsinization, and were seeded in culture medium onto the fibronectin patterned substrates at a density of 2000 cells/cm2. After 24 h incubation, cells on the substrates were washed with warm Hanks Balanced Salt Solution (HBSS; ICN Biomedicals, Costa Mesa, CA), fixed in 1% (v/v) paraformaldehyde in DPBS (30 min) at room temperature, quenched in 0.25% (m/v) NH4Cl in DPBS (15 min) and rinsed with DPBS. Cells were permeabilized and stained for 1 h with Texas Red-C2-Maleimide as described previously  (10 mg/mL in DMF stock, dissolved in DPBS (1 mg/mL) containing 0.1% (v/v) Triton X-100). After rinsing with DPBS, the fixed cell substrates were mounted into an environmental chamber (FCS2, Bioptechs) and kept under DPBS for all microscopy measurements. For the SPRI kinetics experiments with live cells, cells were seeded by gravity flow onto the fibronectin patterned substrate that was already mounted in the environmental chamber, and kept at 37°C for the duration of the experiment.
A SPR imaging instrument was built to provide a horizontal imaging surface for ease of compatibility with custom and commercially available cell chamber devices (Figure 1). An adjustable constant current power supply (Wavelength Electronics, location, Bozeman, MT) was used to power high candela LEDs (THC3; LSDiodes, Lake Oswego, Oregon) of wavelength 470 nm +/- 5 nm (blue) or 630 nm +/- 10 nm (red). The incoherent light was collimated, spatially filtered through a pin hole, recollimated, passed through a linear polarizer (Newport, Irvine, CA), and directed to the prism imaging surface at the incident angle of 56° by two broadband dielectric mirrors (Thorlabs, Newton, NJ). The environmental flow chamber was assembled with a gold-coated SF-10 glass slide; this slide serves as the substrate which was prepared as described above. The cell culture chamber was mounted on top of a SF-10 rhomboid prism (Optical Fabrication Shop, NIST, Gaithersburg, MD). An 'optical stand-off' (10 mm × 10 mm × 1 mm SF-10 glass slide) was designed to bridge the gap (~1 mm) between the bottom of the FCS2 Bioptechs chamber window with the top of the SPR prism. SF-10 index matching fluid, n = 1.725 (Cargille Laboratories, Cedar Grove, NJ), was used to optically couple each interface. The refractive index of the SF-10 glass is greater than the refractive index of the medium above it, and the geometry is configured for total internal reflection. The incident light couples into the plasmons of the gold film, resulting in a fraction of the p-polarized light being absorbed and fraction of the light being reflected as a function of the refractive index of the medium, the angle of incidence and the wavelength of light . The reflected SPR image was collected by two achromatic lenses (Thorlabs) arranged in the configuration of a 10× Lister objective  with a working distance of 30 mm and a calculated NA = 0.38. According to theory, this imaging objective is estimated to resolve <1 μm features. The image was reflected off a mirror onto a 12-bit (2048 pixel × 2048 pixel) Retiga 4000 CCD camera (Qimaging, Surrey, Canada). Images were collected with Qimaging software.
SPRI collection and analysis
For each SPR image, both p- and s-polarized light images were taken by manually rotating the linear polarizer 90°. Only p-polarized light interacts with the surface plasmons while s-polarized light remains proportional to the incident light. Dividing p- by s-polarized light provides a normalized value for reflectivity, and also normalizes for spatial inhomogeneity in incident light. For each static image a 630 nm and 470 nm wavelength excitation SPR image was obtained. For the kinetics of fibronectin deposition, a 630 nm p- and s-polarized image was taken at the beginning and end of the experiment while only 630 nm p-polarized light was used for the kinetics. Regions of interest (ROI) were selected on the hexadecanethiol and PEG-thiol areas (Figure 3) and the kinetics of deposition were monitored by 'difference imaging' in which the change in reflectivity is measured by subtracting the first time point image from all subsequent images.
For live cell time-lapse images, a 470 nm p- and s-polarized image was taken at the beginning and end of the experiment while only 470 nm p-polarized light was used for the time-lapse imaging. Live-cell time-lapse images presented in Figure 6 are displayed as reflectivity intensity values as indicated by the intensity scalebar.
SPR imaging suffers from two types of image distortion that is rarely discussed in the SPR imaging literature  but is addressed in the Brewster angle microscopy literature [40, 41] which also is dependent on imaging based on an oblique angle of the sample relative to the collection optics. The first is image skew in one axis of the x-y plane of the image; the second is out of focus regions in the image because the object plane is not parallel to the image plane. We use image processing to correct for image skew by dividing the y-direction of the image (the direction of lateral plasmon propagation and image illumination) by 1/cos(θ) where θ = 56° for the angle of incident illumination. We limit analysis to the center portion of the image that is in focus.
A previous report details the conversion of SPRI reflectivity changes into mass/area coverage . In that study, a calibration factor for the SPR system is necessary. Here, we use the relation, θ = sΔR, where mass/area (in ng/cm2) (θ) is directly proportional to the change in reflectivity (ΔR) and multiplied by a system calibration constant (s). The calibration constant is determined from the fibronectin deposition experiment assuming that the effective thickness of a fibronectin layer deposited onto a hexadecanethiol monolayer is 3 nm  and the partial specific volume of fibronectin  is 0.73 cm3/g. All subsequent coverage values reported (in ng/cm2), in the case of serum proteins adsorbed or putative cell deposited proteins, assume a partial specific volume 0.73 cm3/g. This value is in good agreement to values for many globular proteins [26, 27], and fibrillar proteins such as collagen .
All image analysis was performed using custom and stock code written in MATLAB and MATLAB Image processing toolbox (Mathworks, Natick, MA).
Image analysis routine for protein deposition at cell periphery
Fluorescence and phase contrast microscopy
Phase contrast and fluorescence microscopy images were acquired using a 10×/0.3 NA objective on a Zeiss Axiovert 200 inverted microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) and a CoolSNAP HQ CCD camera (Roper Scientific, Tucson, AZ). Flatfield images generated with a 1 μL droplet of a 50% fluorescein solution in 0.1 mM NaHCO3 between a microscope slide and coverslip  were used to correct for inhomogeneity of the fluorescence illumination. Because of the larger field of view afforded by the SPRI setup, images from two adjacent fields of phase contrast and fluorescence microscopy were stitched together to be compared to the corresponding SPR image (Fig, 4A). The SPR image was registered to the phase image using 6 fiduciary marks. The SPR incident wavelength, 470 nm, and fluorescence excitation wavelength (555 nm) for Texas Red staining were chosen so as not to have an overlapping excitation.
Certain commercial products are identified in order to adequately specify the experimental procedure; this does not imply endorsement or recommendation by NIST.
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