This study utilized a developed surface plasmon polariton (SPP) phase microscopy to observe cell-biosubstrate contacts. The developed SPP phase microscopy is highly sensitive to cell membrane contact with biosubstrates and also provides long-term phase stability to achieve time-lapse living cell observation. As such, an SPP intensity and phase sensitivity comparison demonstrates that the sensitivity of the phase measurement can be 100-fold greater than that of the intensity measurement. Also, a more than 2-hour cell apoptosis observation via the SPP phase microscopy is presented. To implement the incident angle from 70° to 78°, cell-biosubstrate contact images corresponding to the surface plasmon resonance (SPR) angles are obtained by utilizing the SPP phase measurement. According to the information of the corresponding SPR angle image and a multilayer simulation, the contact distances between a living melanoma cell and a bovine serum albumin substrate at four different locations have been estimated.
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The study of cell adhesion, migration, and apoptosis are very important in biomedical science such as wound healing, tissue remodeling, and metastasis of tumor cells [1,2]. Molecular interactions occur on or near surfaces of cell membranes that are expected to have different properties from those occurring in bulk solution. By utilizing the optical near field approach, such as an evanescence wave, measurement of the molecular interaction within cell-substrate contact regions within 100 nm depth is possible . A total-internal-reflection fluorescence microscope (TIRFM) can induce an evanescent field from an incident light with an incident angle greater than the critical angle to selectively excite fluorescent molecules on or near a surface in an aqueous or cellular environment [4,5]. TIRFM has been used to observe living cell-substrate contact regions and to study protein dynamics, endocytosis, or exocytosis, as well as acquire detailed information about membrane-associated photosensitizers [6–9]. However, the TIRFM needs fluorescence labeling and the contact distances between the cell membrane and the substrate are not acquired easily from the TIRFM. Herein, we use surface plasmon polariton (SPP) microscopy as an alternative. The SPP microscopy also uses an evanescence wave to excite the SPP at the metal and dielectric interface . It offers high sensitivity, real-time, and label-free advantages, and has been widely applied to biomolecular interaction analysis such as DNA and protein interactions and live cell membrane imaging [11–13].
Conventionally, SPP imaging systems apply a parallel monochromatic light beam incident on a gold film through prism-coupled attenuated total reflection (ATR) or grating-coupled methods. When the incident angle is close to the surface plasmon resonance (SPR) angle, the SPP reflectivity patterns are captured by a CCD camera [14–16]. Giebel et al demonstrated an SPP intensity microscope with the prism-coupled ATR method to study cell-substrate contact . Peterson et al also used an SPP intensity microscope to monitor live cell interaction with an extracellular matrix (ECM) . However, they have two main drawbacks: image quality and sensitivity. Most SPP image systems adopt the prism-coupled ATR method, but the image aberrations are affected by the prisms, especially in microimaging applications. Furthermore, the spatial resolution is proportional to the numerical aperture (NA) of the objective. An SPP microscope in tandem with the high NA objective-coupled ATR method was used to correct image distortions  and observe living cell images . In addition, the sensitivity is too low to detect biomolecules of low molecular weight and low concentration by the ATR SPP intensity measurement. Comparing SPR sensing at various configurations, an ATR SPP phase measurement can provide the best detection limit . Hence, we have developed an SPP phase imaging system for high-throughput real-time dynamic measurement of biomolecular interaction causing slight variation in the dielectric constant or thickness of biomolecular material . However, this system, like other SPP phase imaging systems, cannot match the strict demand of real-time kinetic study for biomolecular interaction analysis because it lacks long-term stability [21,22]. Therefore, an SPP phase imaging system with the advantages of the common-path phase-shift interferometry (PSI) technique can overcome the phase drifts due to environmental change, mechanical vibration, and light source fluctuation to provide long-term stability and high resolution . Furthermore, an SPP phase microscopy and SPP-enhanced fluorescence microscopy via a high NA objective coupling was developed to image living cell membranes on the surface of a biosubstrate .
In this study, we utilized an SPP phase microscope based on a high NA objective to observe cell-biosubstrate contacts with high-quality imaging, long-term stability, and high sensitivity. This SPP phase microscope is first utilized to time-lapse observe the contact between a living B16F10 melanoma cell and a bovine serum albumin (BSA) substrate during cell apoptosis. Additionally, by scanning the incident angle from 70° to 78°, the cell-biosubstrate contact image corresponding to the SPR angles, which are estimated by the SPP phase measurement, is obtained. Finally, according to the information of the corresponding SPR angle image and a multilayer simulation, the contact distances between the living melanoma cell and the BSA substrate were estimated.
2. Principle and experimental setup
2.1. Surface plasmon excitation
Excitation of the SPR occurs when the parallel component of the wave vectors of the incident light, klx, and the wave vector, ksp, of the SPP, satisfy the following matching condition by using the objective-coupled ATR coupling method:
where θ is the incident angle of the light, and ε 0, ε 1 and ε 2 are the wavelength-dependent complex dielectric constants of the objective, the dielectric sample, and the metal, respectively. When the matching condition is satisfied, most of the incident light energy is transferred to the SPP. With the multilayer modeling simulation, it can be shown that the reflectivity of the p-wave has an absorption dip and the phase is changed abruptly at the resonance angle while the s-wave does not excite the SPP and therefore does not demonstrate an absorption dip and drastic phase variation, i.e. a phase jump, in its reflection [10,11,25,26].
2.2. Optical setup
Figure 1(a) shows the oil-immersion objective based SPP microscopy. The light source is a He-Ne laser (5mW, Melles Griot) with a wavelength of 632.8 nm. A linear polarizer positioned at an adjustable angle between the optical axes controls the p-wave and s-wave components. The incident beam then passes through a nematic liquid-crystal phase retarder (LVR-200, Meadowlark Optics) while the slow-axial is positioned along the s-wave. The s-wave phase delay can be applied by driving appropriate voltages for the PSI . The light passes a beam expander with a spatial filter to form a smooth collimating beam, and is then focused onto the back focus plane (BFP) of a high NA oil-immersion micro-objective (60X, NA = 1.49, Nikon,) through a focusing lens (f = 400 mm). The output of the light from the BFP is an approximate plane wave while the incident angle θ can be controlled through changing the focal position d on the BFP as a function of sine condition :
where f is the focal length of the objective (shown in Fig. 1(b)). A 0.17 mm thin cover slide (BK7) coated with a 46 nm Au thin film is placed on top of the oil-immersion objective, and refraction index matching oil is filled up. The SPP are excited at near the SPR angle, and the reflected light passes through a beam splitter, a linear polarizer, and an imaging lens. The interference patterns between the p-wave and s-wave components are then captured by a CCD camera (TM-1320-15CL, PULNiX). The linear polarizer is used to induce the p-wave and s-wave interference. The coupling angle can be adjusted from 0 to 79.5° in this configuration.
The nematic liquid crystal phase retarder is applied to produce PSI in the common optical path. Applying the voltages calibrated to produce the optical axis at these five-step phase shifts, θ 0, θ 0 + π/2, θ 0 + π, θ 0 + 3π/2, and θ0 + 2π, where θ 0 is an initial phase, five interference images of different phase shifts can be displayed through the linear polarization and captured by a regular CCD camera as digital image data (I 1, I 2, I 3, I 4, I 5, respectively). Applying a five-step phase shift reconstruction algorithm, a 2D wrapped phase distribution ϕ(x,y) can be obtained with high resolution as follows :
Then, the unwrapped phase from ϕ(x,y) will be completely reconstructed .
2.3. Surface coating and modification
A BK7 cover slip was used as sample substrate and washed by typical cleaning procedure. The metal film was typically Au or Ag for SPP excitation in the visible light region. Since Au is rather stable in biochemical procedures and has a shorter wave propagation length, it was chosen as the material for the metallic thin film. A sacrificial layer of 3 nm thick Cr was pre-coated on the cover slip to increase Au adhesion on the sample substrate, and then a 46 nm thick Au film was coated on it via a sputtering deposition process. The slide was immersed in a 1 mM thiol solution (HS(CH2)15COOH, Sigma) in alcohol over 12 hours to form a self-assembled monolayer (SAM), and then washed by distilled water. Subsequently, it was placed in a solution with 0.2 M EDC hydrochloride (N-Ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride, Fluka) and 0.5 M NHS (N-hydroxysuccinimide, Fluka) in distilled water for a further 6 hours for surface activation, and then washed by distilled water. In order to form a biocompatible monolayer on the surface, the slide was then immersed in a BSA (Sigma) solution with a concentration of 10 mg/ml in phosphate buffered saline (PBS) for 1 hour to form a BSA SAM, and finally rinsed with PBS.
2.4. Cell line and culture
B16F10 murine melanoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.292 g/liter of L-glutamine, 2% sodium bicarbonate, 1% sodium pyruvate, 1% penicillin, and 10% fetal bovine serum at 37°C under a humid atmosphere and 5% CO2. For the SPP imaging experiments, the cells were cultured on the BSA-coated Au thin film modified with the thiol.
3. Experimental results
3.1. Lateral spatial resolution test
In order to examine the lateral spatial resolution of the SPP imaging, some patterns are designed for clarification. A 3 nm Cr and 46 nm Au film was coated on the BK7 substrate, and then a 30 nm PMMA film was deposited on the Au film by spin coating. Finally, a square-hole pattern was completed by E-beam lithography. Figure 2(a) shows the SEM image of the pattern. The sizes of the square holes are 1 × 1 µm2, 3 × 3 µm2, 5 × 5 µm2, and 7 × 7 µm2, and the distance between the square holes is 5 µm. The pattern substrate was then placed in the air. The incident angle was adjusted to excite the SPR condition for the PMMA/metal interface, but not for the air/metal interface. Herein, the incident direction (x direction) of excitation light is from left to right. The SPP intensity and phase images are shown in Figs. 2(b) & 2(c). According to the feature discrimination of the smallest square holes and the minimal coupling effect between the holes, the spatial resolution can achieve around 1 µm in the y direction and 3 µm in the x direction for both the SPP intensity and phase images. The difference in the resolution is due to the SPP propagation on the surface in the x direction.
The x-direction lateral resolution of SPP microscopy is limited by the propagation length of the SPP. The SPP field decays exponentially in the z direction while the SPP travels along a dielectric/metal interface with a propagation length given by the imaginary part of the ksp in Eq. (1) . In theory, the propagation length of the SPP is 3.5 µm at the air/metal interface at a 632.8 nm wavelength. The experimental result is comparable to the theoretical value of both the intensity and phase images.
3.2. Long-term live cell membrane imaging
The SPP microscope can be used for real-time observation of various cell behaviors such as adhesion, migration, and apoptosis to obtain information of interaction and distance between cell membrane and substrate. Here we first utilize the microscope to observe the apoptosis of melanoma cells. The cells were placed on the Au-coated and BSA-modified biocompatible substrate described in Sec. 2. After a 5-hour cell incubation period, the substrate with the cells was observed under the SPP microscope. Figure 3 shows the images of the B16F10 cell apoptosis with time-lapse observation. The left picture of Fig. 3(a) is an epi-illuminated image of the living cell adhered on the biosubstrate after 50 min of real-time observation. By changing the phase delay of the s-wave via the liquid crystal, the five-step PSI SPP phase images of the cell membrane on the biosubstrate were taken and are shown in Fig. 3(b). The complete SPP phase image was obtained by the phase reconstructed algorithm in Eq. (3) and our developed multichannel phase wrapping algorithm , as shown in the first image of Fig. 3(c). Also, the cell detaching from the biosubstrate was observed after 50 min of seeding. The remaining images of Fig. 3(c) show the complete SPP phase images of the cell apoptosis at the 70th, 90th, 110th, and 130th minute with a 20 min time interval. There still exists slightly residual cell-biosubstrate contact, as shown in the last image of Fig. 3(c). An epi-illuminated image of the cell death is shown in the right picture of Fig. 3(a).
3.3. Cell-biosubstrate contact distance
The cell-substrate contact distance information can be measured by both the SPP intensity and phase microscopy. Herein, the intensity and phase curves of the reflected light can be obtained by modulating the incident angle, and then the SPR angle can be found based on the intensity and phase curves with polynomial curve fitting. Figure 4(a) shows the bright-field epi-illuminated image of a living melanoma cell. Three spots, A, B, and C, are marked as the locations of the background as no cell contact, the inside of cell-biosubstrate contact, and the edge of cell-biosubstrate contact, respectively. In order to get the p-wave reflectivity, the linear polarizer before the imaging lens is adjusted along the p-wave direction. The incidence angle is scanned from 62° to 78° to estimate the SPR angles for different cell-biosubstrate contact areas. The SPP intensity images are demonstrated at the incident angles of 70.75°, 71.45°, 72.19°, 72.96°, 73.75°, 74.59°, 75.48°, and 76.42° in Fig. 4(b). The background images are altered from bright to dark. Also, the intensity of cell location is greater than that of the background at the SPR angle of around 73.75°, as shown in the fifth image of Fig. 4(b). When the incident angle is further increased to approach the SPR angle of the cell location at around 75.48°, the background becomes bright while the cell location darkens, as shown in the seventh image of Fig. 4(b). Figure 4(c) shows the reflectivity of the three different locations and their corresponding SPR angles. The SPR angle of the background (A) is the smallest while the cell locations such as B and C have greater SPR angles. The greater the SPR angle, the closer the cell is to the biosubstrate.
The SPP phase images can be also obtained by the PSI and phase reconstruction algorithm. The incidence angle is also scanned from 62° to 78° to estimate the SPR angles for different cell-biosubstrate contact areas. Figure 5(a) shows the SPP phase images at the incident angles of 70.75°, 71.45°, 72.19°, 72.96°, 73.75°, 74.59°, 75.48°, and 76.42°, respectively. Figure 5(b) shows that the phase jumps of the A, B, and C locations appear at their corresponding SPR angles of around 73.75°, 75.48°, and 74.59°, respectively. Obviously, the phase measurement has a much higher sensitivity than the intensity measurement. The slope of the intensity measurement is about 75 a.u. (grey level) per incident angle of degree (a.u./deg) and the slope of the phase measurement can reach 0.938 π/deg. The resolution of the intensity measurement is 13 grey-level units based on the 5% intensity stability in a 256 grey-level CCD camera. The resolution of the phase measurement resolution based on the SPP phase system can be 10−3 π . If the same phase resolution can be achieved in the SPP phase imaging system, the sensitivity of the phase measurement can be about 160 times (0.938 π/deg /10−3π/deg/75 a.u./ 13 a.u.) higher than that of the intensity measurement. The 160-fold sensitivity is similar to the value in SPR biosensing [12,23]. The phase measurement can achieve a very high sensitivity at a fixed incident angle near the SPR angle, but its linear dynamic range is fairly narrow.
Cell composition is quite complex. It is covered with a plasma membrane and contains cytoplasm and many organelles such as the nucleus, mitochondrion, endoplasmic reticulum, etc. Therefore, the optical property, geometry, and structure of cells are difficult to individually analyze by a single imaging technique. Cells can be simplified as a homogeneous and transparent material in the study of cell-biosubstrate contacts. The refractive index (n) and thickness (d) is defined as the following: BK7 cover slip (n = 1.515), Cr (n = 3.135 + 3.31j, d = 3 nm), Au (n = 0.223 + 3.294j, d = 46 nm), thiol SAM (n = 1.464, d = 1.49 nm), BSA SAM (n = 1.4, d = 4.0 nm), PBS solution (n = 1.338), and cell (n = 1.36) at the incident wavelength of 632.8 nm. The multilayer configuration consists of BK7/Cr/Au/thiol/BSA/PBS/cell/PBS. The unknown parameters are the thickness of cell and cell-substrate distance. Cell thickness is assumed to be greater than 300 nm, so we can simplify the calculation to estimate the cell-substrate distance. It can be inferred by fitting the SPR angle with the phase measurement. Figure 6(a) shows the 3D image of the corresponding SPR angles at different locations. The image is converted by estimating the SPR angles at different locations through the SPP phase images at the incident angles from 70° to 78°. Its 2D image is shown in Fig. 6(b).
Four locations are chosen for further discussion as shown in Fig. 6(b). First, location A on the background surface is filled by only the PBS solution above the biosubstrate. The simulation result with the configuration of BK7/Cr/Au/thiol/BSA/PBS approaches the corresponding SPR angle of 73.75° from the SPP phase measurement at location A. Locations B and D are situated inside the main cell and contain a lot of organelles with a typical cell thickness about 10 to 100 µm, which is much thicker than the depth of the SPP evanescent field. The upper layer is assumed to be the cell, and hence a configuration of BK7/Cr/Au/thiol/BSA/PBS/cell is adopted. The cell-biosubstrate distances, i.e. the PBS gaps, are estimated as 10 nm and 65 nm for B and D, respectively. The biggest SPR angle position locates on B where there is only about a 10 nm space, which means that the cell acts as a focal contact on the biosubstrate. Location C looks like a lamellipodium with the SPR angle of 74.59°. The lamellipodium is very thin compared to the depth of the SPP evanescent field, so a configuration of BK7/Cr/Au/thiol/BSA/PBS/cell/PBS is considered reasonable for use. If we assume that the lamellipodium thickness is 40 to 50 nm, then the cell-biosubstrate distance could be estimated as 15 to 30 nm. The experiment results roughly shows the cell adhered on the biosubstrate with various contact distances, but the exact values cannot be obtained due to inaccuracies such as the optical property, the topography of the cell, and the measurement error. Despite the inaccuracies, this study has demonstrated that the results are reasonable for the actuality in the TIRFM [6–9].
In this paper, the adhesion of melanoma living cells on a BSA biosubstrate has been studied by the developed SPP phase microscopy. The developed SPP phase microscopy has a spatial resolution of 3 µm in the SPP propagation direction, possesses a sensitivity more than 100-fold greater compared to SPP intensity microscopy, and provides long-term phase stability for time-lapse cell observation. With the help of an incident angle scanning mechanism, the corresponding SPR angle image based on the SPP phase measurement is obtained. According to the corresponding SPR angle image and a BK7/Cr/Au/thiol/BSA/PBS/cell/PBS multilayer configuration simulation, cell-biosubstrate contact distances at different locations can be approximately estimated.
This work was supported by the National Research Program for Genomic Medicine (NRPGM) of the National Science Council (NSC) in Taiwan (NSC 97-3112-B-006-013), NSC 97-3111-B-006-004, and Advanced Optoelectronic Technology Center of National Cheng Kung University.
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