This paper demonstrates the first combination for wide-field surface plasmon (SP) phase microscopy and SP-enhanced fluorescence microscopy to image living cells’ contacts on the surface of a bio-substrate simultaneously. The phase microscopy with a phase-shift interferometry and common-path optical setup can provide high-sensitivity phase information in long-term stability. Simultaneously, the fluorescence microscopy with the enhancement of a local electromagnetic field can supply bright fluorescent images. The combined microscope imposes a high numerical aperture objective upon the excitation of surface plasmon through a silver film with a thickness of 30 nm. The developed SP microscope is successfully applied to the real-time bright observation of the transfected fluorescence of living cells localized near the cell membrane on the bio-substrate and the high-sensitivity phase image of the cell-substrate contacts at the same time.
©2010 Optical Society of America
Numerous biochemical functions of proteins are mediated on cell membranes. Therefore, how to develop a microscope that can image the cell-substrate contact region more efficiently is a crucial research topic for concluding the role of signaling proteins in cell spreading and migration. By inducing the evanescent field from incident light with an incident angle greater than the critical angle to selectively excite fluorescent molecules on or near a surface in a cellular environment only, total internal reflection fluorescence microscopy (TIRFM) has been utilized to observe the cell-substrate contact region  and to study dynamics of proteins , endocytosis and exocytosis  near the cell membrane [1,4]. Nevertheless, fluorescent molecules are needed to dye or transfect into observed cells in the TIRFM. Intensity microscopy based on the enhancement of surface plasmons (SPs) has been utilized to observe cell-substrate contact localities without any addition of dye labels .
SPs are oscillations of the free electrons located on the surface of a metal film and can be usually excited by incident light based on the prism-coupled attenuated total reflection (ATR) or grating-coupled diffraction methods . Conveniently, the prism-coupled excitation can be substituted via the excitation of a highly oblique collimating beam that is generated by focusing a light beam to the outer regions of the back focal plane (BFP) of a high numerical aperture (NA) objective . When the wave vector of an incident evanescent transverse magnetic (TM) light matches that of the SPs, the so-called surface plasmon resonance (SPR) phenomenon occurs and the SPR associated with the EM field is greatly enhanced . The SPs induce a quantum of the photon-photon transverse wave field with a skin depth of less than a few hundred nanometers, which is suitable for the biomolecular scale. The two main features of SPs in bio-sensing and imaging are: 1) very high sensitivity to the interface change to detect tiny biomolecular interactions on the metal surface based on the intensity and phase variations of reflection light; and 2) to enhance the local electro-magnetic field to improve fluorescence and Raman signals .
According to the first feature, intensity-based SPR microscopes with high NA objectives have been utilized to image the cell surface interface in fluid at improved lateral resolution. Additionally, they can contrast  and provide the quantitative data of a refractive index map regarding interactions between cell membrane and extracellular matrix in the cell-substrate contact region . To enhance the sensitivity for detecting the tiny changes of biomolecular interaction, we have integrated the SPR and common-path phase-shift interferometry (PSI) techniques to develop a phase microscope for imaging the two-dimensional (2D) spatial phase variation caused by biomolecular interactions on a sensing chip without the need for additional labeling [10,11]. The common-path PSI technique has the advantage of long-term stability, even when subjected to external disturbances. The SP phase microscope has demonstrated a phase shift stability of 2.5 × 10−4π over four hours and a resolution of 2 × 10−7 refraction index difference. However, either the SPR intensity or phase microscopes cannot provide fluorescence signals, which are sometimes regarded as the information for molecular interactions near the bio-surface and to identify the specific protein binding areas . Therefore, to apply the second feature, the prism-based SP-enhanced fluorescence image can be enhanced at least 10 fold compared to that of the TIRFM [12,13]. Although prism-based SPR images of cell-substrate interactions have previously been elucidated with the corresponding fluorescence images, the cells were fixed by paraformaldehyde after SPR experiments . Furthermore, the physical restraint of the prism confines the NA and magnification of the imaging system. Hence, a simultaneous and real-time observation of objective-based SP-enhanced fluorescence and phase image will make the biochemical responses of living cells near the cell-substrate contacts to be clearly identified according to the brighter fluorescence and more sensitive phase information under the high NA imaging system.
This paper reports the first combination of high NA oil-immersion objective-based wide-field SP-enhanced fluorescence microscopy and SP phase microscopy. To diminish the need of a very high incident angle associated with SP images in buffer solution, we adopt a reduction in the thickness of thin metal film and do not attempt to find the highest NA objective. According to theoretical investigations, the decrease of thin silver film thickness not only decreases the propagation length of SPs, so that more detailed intensity and phase features can be recognized owing to improved lateral resolution, but also decreases the SPR angle so as to resolve the restriction associated with the maximum angle imposed by finite NA value of the objective [6,15,16]. In addition, the sensitivity in the index of refraction is not seriously compromised for cell-substrate contacts detection. The SP-enhanced fluorescence imaging of living cells is also able to be achieved by making the use of backward oil-immersion objective (collecting the fluorescence from the bottom of the slide) as well as forward water-immersion objective (from the upper side of the slide). The employment of the silver film as an SP excitation candidate for imaging cells transfected with enhanced green fluorescent protein (eGFP) can enhance the fluorescence via SPs excited by a blue laser . Through a moderate spacer to adjust the metal-fluorophore distance, the SPs obviously contribute to the increase in the quantum yield for achieving brighter a fluorescent signal and a reduction in the fluorescence lifetime for attaining better photostability [17,18]. A candidate tumor suppressor WW domain-containing oxidoreductase, known as marine WOX1 or WWOX, and monkey kidney COS7 fibroblasts are used to demonstrate the imaging capabilities of the developed microscopy . WOX1 proteins play an important role in the regulation of a wide variety of cellular functions such as protein degradation, transcription, and RNA splicing .
2. Methods and materials
2.1 Surface plasmons excited by light and simulation
SPR is an optical phenomenon in which an incident TM wave excites SPs, i.e. a surface charge density oscillation, at the interface between a thin metal film and a dielectric sample medium. SPR occurs when the parallel component of the wave vector of the incident TM wave, , matches the wave vector of the SP traveling over a semi-infinite structure, , i.e.
In order to unravel the constraint relevant to the maximum incident angle imposed by a finite NA value of the objective in the objective-based SP microscope, the SPR angle based on the three-layer configuration (substrate (BK7)/metal (Ag)/dielectric (H2O)) can be diminished from 83.9° to 78.3° by decreasing the thickness of the thin silver film from 50 nm to 30 nm at the wavelength of 473 nm. The angle associated with the maximum fluorescence enhancement, i.e. 77.5°, is the incident angle of the excitation which slightly lower than the SPR angle at 78.3°. An incident angle smaller than the SPR angle is usually employed to optimize the contrast of reflectivity . Also, in the visible light and near infrared ranges, the longer the incident wavelength used, the smaller the SPR angle is. Therefore, the constraints regarding the range of available angles can be solved with the use of the 30 nm silver film excited by 473 nm or longer wavelength light. However, a shorter SP excitation wavelength will broaden the SPR reflectivity spectrum, which would then clearly reduce the contrast of the SPR image . Also, a thinner silver film would reduce the sensitivity of the SPR image; meanwhile, a shorter incident wavelength could decrease the penetration depth into cell samples.
As a result of the higher sensitivity attained by means of phase measurement rather than intensity detection, in this study the common-path PSI is adopted to obtain the phase information of living cell observations . Also, a 473 nm blue laser with a shorter incident wavelength is utilized due to the lateral resolution issue and to excite the eGFP simultaneously.
Figure 1(a) illustrates the angle-resolved phase reflectivity spectrum based on the substrate (BK7)/metal (Ag/30nm)/dielectric (H2O) at the wavelength of 473 nm. In this simulation for the SP phase microscopy, the green line and red line represent the simulated phase reflectivity spectrum of the SP chip with and without the cell adhered on the top of bio-surface, respectively, where the phase difference between the two lines is 0.38 π at the excitation angle. Figure 1(b) shows that the distribution of the enhancement factor of the electric field intensity is perpendicular to the interface of the SP chip at 77.5° with two additional layers of thiol and collagen. The thicknesses of the collagen and thiol layers are approximately 5.5 nm and 1.1 nm, respectively, and the corresponding refractive indexes are 1.46 and 1.49 . The enhancement factor at the interface between the collagen and the buffer has a value around 16.5, as shown in Fig. 1(b). Based on the simulation for the TIRFM chip, the maximum value of the enhancement factor is 1.1. From the calculations with the 30 nm silver film, an approximately 15 times higher local field enhancement of the SP chip, compared to that of the conventional TIRFM chip, could be estimated. Local field enhancement leads to an increased excitation rate, and is therefore expected to enhance the fluorescence intensity. However, the interaction of fluorophores with SPs under a short metal-fluorophore distance occurred, and hence the fluorescence quenching effect was counted . Taking the competing phenomena into account, the enhancement in the fluorescence signal obtained from the proposed SP chip is more likely to be no more than 5 times that of the TIRFM chip. To compensate for the competing effect and the thinner silver film issue, a spacer is inserted (a thiol SAM and a collagen layer) to increase the distance between live cell membranes and the 30 nm silver film such that the experimental results demonstrate a capability of producing an approximately 5 times brighter fluorescent live cell image via the excitation of SPs, as discussed in Section 3B.
2.2 Overall system
Figure 2 shows the system configuration of the wide-field oil-immersion objective-based SP microscopy. The SP phase and fluorescence images are mainly obtained by utilizing a diode-pumped solid-state laser light source (20 mW, λ = 473 nm) to excite SPs on the thin silver film. A 632.8 nm He-Ne laser which can be switched by a flipper mirror is available as well. The laser beam passes through two linear polarizers to control the polarization and intensity. The liquid crystal adjusts the phase delay between the TM wave and transverse electric (TE) wave for the PSI which is mentioned above. Then, the light is focused onto the BFP of a high NA oil-immersion objective (60 ×, NA = 1.49, Nikon) through an objective (40 ×, Nikon) and a relay lens pair, finally, emerging from the objective as a parallel beam.
The incident angle is controlled by means of adjusting the focusing spot position on the BFP through a linear translation stage. A 0.17 mm cover slide (BK7) coated with metal film via a RF sputtering deposition process is coupled into the oil-immersion objective by adding index-matching oil. The range of available excitation angles of the setup is up to 79.5°. The SPs are excited and the reflection from the metal film comes into the objective, and then goes through a beam splitter, a linear polarizer, and an imaging lens; finally, they are imaged on to a regular CCD camera. The linear polarizer is adjusted at a suitable angle between its optical axis and the incident plane in order to make the TM wave and TE wave interference with better contrast . In comparison with the 50 nm thin silver film, a coating with 30 nm is an adequate thickness for simultaneously achieving the SP phase imaging and fluorescence imaging with the NA 1.49 oil-immersion objective. At the same time, the SP fluorescence imaging of living cells is also able to be achieved by using the bottom oil-immersion objective and an upper water-immersion objective (100 × , NA = 1.0, Olympus) as well. The eGFP fluorescence excited via SPs from the cell-substrate contact region is collected by the oil-immersion objective and the water-immersion objective, and individually imaged onto two high-speed frame rate EMCCD cameras (Luca and iXon DV885, Andor) by passing through band-pass filters (BPF, λ = 500 - 535 nm, Semrock) and imaging lenses.
As aforementioned, a nematic liquid crystal phase shifter is applied to produce PSI in the common optical path. It is a positive crystal of single optical axis. As a linear polarization beam with two polarization components in orthogonal directions enters the liquid crystal, they experience different optical paths resulting in different phase shifts. If the incident angle is fixed, the direction of the optical axis of the liquid crystal can be changed by altering the voltage applied to the liquid crystal. Shifts of the modulated phase between the fast axis and slow axis are thus created. Applying the voltages calibrated to produce the optical axis at these five-step phase shifts δ, δ + π/2, δ + π, δ + 3π/2, and δ + 2π, where δ is an initial phase, five interference images of different phase shifts can be displayed through the linear polarization and be captured by the regular CCD camera as digital image data (I 1, I 2, I 3, I 4, I 5 in order). Applying a five-step phase shift reconstruction algorithm , the 2D wrapped phase distribution can be obtained with high resolution as follows:
Finally, the unwrapped phase will be completely reconstructed by using our developed multichannel phase wrapping algorithm .
2.3 Cell culture protocol
In this study, cultured monkey kidney COS-7 fibroblasts were suspended in a serum-free culture medium, containing 2 mg/ml bovine albumin, and electroporated with EGFP-WOX1 construct (BTX ECM 830 Electroporator, Genetronics; 5 μg DNA/3 × 106 cells, 220 volt and 50 msec). Albumin enhances both the transfection efficiency and gene expression 3-5 fold . The cells were cultured 24-48 hrs, and then observed by the proposed wide-field oil-immersion objective-based SP microscope.
In order to observe the molecular interactions between cell membranes and extracellular matrix with the SP microscope, the cells were added to collagen immobilized by chemical self-assembly monolayers (SAMs) on a thin silver film BK7 slide containing a pre-warmed medium. In developing this SP chip, the metal film was immersed in 1 mM 2-aminoethanethiol hydrochloride solution to form a dense SAM on its surface. To immobilize the protein collagen, covalent activation was conducted by immersing the chip in a solution containing EDC[N-(3-dimethylaminopropyl)-N’-ethylcarbodimide hydrochloride, 2 mM] and NHS(N-hydroxysuccinimide, 5 mM) for 6 hrs. One prior study for detecting fluorescent erythrocytes on metal surfaces to induce the quenching effect with a similar cell culture protocol can be referred to in .
3. Experimental results and discussions
3.1 Considerations of enhancement and lateral resolution
The complex dielectric constants of gold and silver have large negative real numbers in the visible light region. Therefore, they are good candidates as the medium of SP excitation. In this study, a 473 nm blue laser is adopted as a light source for the excitation of eGFP via SPs, the SP intensity and phase images. On account of preceding studies of SP chips with thin silver film coatings, it has been shown that not only the fluorescence intensity can be enhanced by the excitation of SPs, but also the photostability of fluorophores can be improved . Compared with thin gold films, superior fluorescence enhancement is able to be obtained by the excitation of thin silver films with the blue laser beam.
The lateral resolution of SP microscopy is usually limited by the propagation length of SPs. The SP field decays exponentially while traveling along a dielectric/metal interface with the propagation length determined by the imaginary part of the SP wave vector . Usually, the propagation length of gold is shorter than that of silver. Also, the propagation length excited by a shorter wavelength is shorter than that by a longer wavelength. Furthermore, the propagation length excited under an optimal thickness is longer than that under others. For example, an around 45 nm thin gold film excited by a 632.8 nm laser in air is an optimal thickness for the excitation of the SPR. A 30 nm silver film excited by the 473 nm laser in air is not. Figure 3(a) shows E-beam lithographical PMMA removed square patterns at different sizes (1 × 1 μm2, 3 × 3 μm2, 5 × 5 μm2, & 7 × 7 μm2) on a BK7 substrate coated with a metal film. The space between the square holes is 5 μm and the thickness of the PMMA is controlled at 30 nm. The patterned SP images arise due to the refractive index difference between the PMMA and air.
The incident angle is modulated to excite the SPR condition for the PMMA/metal interface, not for the air/metal interface. Figures 3(b) and 3(c) indicate that the SP intensity and phase images excited by the 632.8 nm He-Ne laser are based on an optimal SPR condition of 45 nm gold film, while Figs. 3(d) and 3(e) show a 30 nm silver film excited by the 473 nm laser at the incident angle of 77.5°. The phase images are inverted due to the phase reconstruction. Also, the incident light is from left to right. The compromise based on the above issues is that the propagation length of SPs at the 30 nm silver film excitation condition could be shorter than that of the 45 nm gold film excitation condition. As shown in Figs. 3(b)-3(e), the experimental results of the patterned SP intensity and phase images demonstrate that the spatial resolution of the 30 nm thin silver film condition is better than that of the 45 nm gold film condition according to the feature discrimination of the smallest square holes and the minimal coupling effect between the holes. The lateral resolution of the SP phase image based on the setup approaches to 1 μm, as checked in Fig. 3(e).
As with any microscopy, lateral spatial resolution problems in SP microscopy cannot be discussed without addressing the question of contrast. From Figs. 3(b)-3(e), the superior contrasted images with higher maximum-to-minimum ratios are found in the gold film condition due to the optimal thickness at a longer excitation wavelength. From the experimental results and prior studies, a non-absorbing dielectric spacer (a thiol SAM and a collagen layer) that is 6.1 nm thick not only reduces the effect of metallic surface-induced fluorophore quenching at very short distances in SP-enhanced fluorescence microscopy, but also promotes high contrast SP images without loss of lateral resolution [12,21,27]. Therefore, associated with the thinner silver film setup, the SP phase image shows an acceptable lateral resolution, while an SP-enhanced fluorescence image can also be enhanced significantly.
3.2 Appropriate setup for surface plasmon-enhanced fluorescence excitation
Recent investigations have supported the contention that thrombomodulin, an integral membrane glycoprotein, plays an important role in extravascular activities  and thrombomodulin-mediated cell adhesion . As indicated in Figs. 4(a) and 4(b), the SP-enhanced fluorescence imaging of a living melanoma-GFP-tagged thrombomodulin cell can be realized at an incident angle of 77.5° associated with the maximum fluorescence enhancement by making utilization of the bottom oil-immersion objective through the thinner silver film as well as the upper water-immersion objective. The fluorescence signal collected from the bottom objective is quenched when a thicker sliver film is adopted. A five times brighter fluorescent live cell image via the SPs can be observed by the bottom oil-immersion objective based on the appropriate microscopy setup. Compared to the observation of melanoma-GFP-tagged thrombomodulin cells through the culture medium with the water-immersion objective, better collection efficiency and spatial resolution of SP-enhanced fluorescence images can be obtained by the use of the oil-immersion objective with a higher NA value, as shown in Figs. 4(a) and 4(b). The decrease in thickness of the silver film solves the two problems regarding imaging in fluid via the 1.49 NA oil-immersion objective and improvement in the lateral resolution. Moreover, a simultaneous observation of phase image can be made with this setup.
3.3 Simultaneous fluorescence and phase imaging
Figures 5(a) , 5(b) and 5(c) demonstrates the simultaneous fluorescence and phase imaging of a single living COS7 fibroblast transiently transfected with the eGFP-WOX1 construct cultured on the collagen-coated SP chip based on the developed SP-enhanced fluorescence and phase microscopy. In contrast to the flat adhesion of the melanoma-GFP-tagged thrombomodulin cell illustrated in Fig. 4, the expression of WOX1 proteins that possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence, and a C-terminal short-chain alcohol dehydrogenase/reductase domain (which contains a mitochondria-targeting sequence) tagged with eGFP is imaged with the punctuated adhesion of the COS7 fibroblast near its plasma membrane area, as shown in Fig. 5(a) . Also, it presents the localization to the perinuclear region and mitochondria of the WOX1-containing clusters. Simultaneously, Figs. 5(b) and 5(c) show the acquired 2D and three-dimensional (3D) SP phase images of the same cell, while the phase difference between the areas with and without the cell is seen to be approximately 0.35 π. The regions with large phase differences indicate the localities that are directly in contact with the bio-surface and the areas of greater density which previous research has explained reflect where the contraction of microfilaments in the cytoskeleton have sustained the adhesion of the cell . Contrary to the dense perinuclear localization of the WOX1-containing clusters, the SP phase image corresponding to the cell’s footprint exhibited greater phase difference resulting from shorter cell-substrate distance and greater density in the cell cytoplasm close to the membrane. Further instrumental developments and theoretical modeling are planned to employ both the SP phase microscopy and SP-enhanced fluorescence microscopy to allow quantitative measurements of cell-substrate distances at the same time.
This study has developed the first combination of SP phase microscopy and SP-enhanced fluorescence microscopy with a 1.49 NA oil-immersion objective by decreasing the thickness of a silver film to 30 nm. The distributions of WOX1-containing clusters and cell-substrate distance can be qualitatively illustrated at the same time through a comparison of the SP-enhanced fluorescence and phase images. The experimental results, consistent with the simulation, have shown that a 5-fold enhancement of the SP-enhanced fluorescence image and a better than 3 μm lateral resolution of the SP phase image could be achieved by the simultaneous observation of a living COS7 fibroblast transfected with the eGFP-WOX1 construct on the cell-substrate contact region.
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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