Enhanced live cell membrane imaging using surface plasmon-enhanced total internal reflection fluorescence microscopy

Experimental evidence suggests that the molecular interactions which occur on or near the surfaces of cell membranes (typically within 50 nm) have different properties from those which occur in the bulk solution. To visualize these events within the cell-substrate contact region more clearly, it is desirable to use observation techniques which suppress the interference arising from background fluorescence. In total internal reflection fluorescence microscopy (TIRFM), an incident light ray with an incident angle greater than the critical angle is used to generate an evanescent field to excite and image fluorophores on or very near (i.e. within 100 nm) the liquid/ solid interface. Fluorophores located within or close to the plasma membrane are excited by the shallow evanescent field, resulting in images with very low background fluorescence and no out-of-focus fluorescence. TIRFM is particularly suited to the investigation of live cells since it causes minimal photodamage or photobleaching. Furthermore, since TIRFM typically illuminates a wide-field thinner vertical slice compared to confocal scanning systems, the signal-to-noise ratio (SNR) and attainable frame rate are significantly improved. Although confocal microscopes can generate deep three-dimensional images, TIRFM provides a complementary approach which can be readily combined with other microscopy techniques. Therefore, TIRFM has been widely used in studies of cell-substrate contact regions, protein dynamics, endocytosis or exocytosis, and membrane-associated photosensitizers.

Experimental evidence suggests that the molecular interactions which occur on or near the surfaces of cell membranes (typically within 50 nm) have different properties from those which occur in the bulk solution.To visualize these events within the cell-substrate contact region more clearly, it is desirable to use observation techniques which suppress the interference arising from background fluorescence.In total internal reflection fluorescence microscopy (TIRFM), an incident light ray with an incident angle greater than the critical angle is used to generate an evanescent field to excite and image fluorophores on or very near (i.e.within 100 nm) the liquid/ solid interface.Fluorophores located within or close to the plasma membrane are excited by the shallow evanescent field, resulting in images with very low background fluorescence and no out-of-focus fluorescence.TIRFM is particularly suited to the investigation of live cells since it causes minimal photodamage or photobleaching.Furthermore, since TIRFM typically illuminates a wide-field thinner vertical slice compared to confocal scanning systems, the signal-to-noise ratio (SNR) and attainable frame rate are significantly improved.
Although confocal microscopes can generate deep three-dimensional images, TIRFM provides a complementary approach which can be readily combined with other microscopy techniques.Therefore, TIRFM has been widely used in studies of cell-substrate contact regions, protein dynamics, endocytosis or exocytosis, and membrane-associated photosensitizers.
Recently, some improvements in TIRFM have been made regarding its experimental configuration and image processing techniques.However, the fluorescence signal in live cell imaging still needs enhancement, if dynamic images of the molecular interactions which take place on or near the surface of the cell membrane are to be acquired.As of this point, even if given a readily detectable fluorescence signal of single molecules, reduced detection limits are still required.In recent investigations, metallic surfaces or particles has been exploited to enhance the fluorescence of the fluorophores in order to develop more efficient fluorescence immunoassays.In these techniques, surface plasmons (SPs) or localized SPs on the metallic surfaces or nanoparticles enhance the local electro-magnetic (EM) field around the fluorophore and therefore increase the intensity of the detected fluorescence signal.Through the moderate modification of a metal film or particles on a substrate and the metal-fluorophore distance, the plasmonic effect is the observed increase in the quantum yield and photostability, the reduction in the fluorescence lifetime, and the specific orientation in the typically isotropic emission.These effects are not due to the reflection of the emitted photons, but rather as the result of the fluorophore dipole interacting with free electrons in the metal.Therefore, this study proposes a surface plasmon-enhanced fluorescence measurement technique capable of imaging the molecular interactions between a cell membrane and a biosurface in real-time.Figure 1(a) shows the optical configuration of the proposed prism-coupled surface plasmon-enhanced TIRFM, in which the attenuated-total-reflection (ATR) method is used to excite the SPs in order to enhance the local EM field and to increase the SNR of the fluorescence.In this configuration, a thin silver film via a sputtering deposition process, a chemical self-assembly monolayer (SAM), and a biomolecular layer are sequentially deposited on an SF-11 slide in accordance with an optimal design which is known to enhance the intensity of the fluorescence and therefore to increase the attainable frame rate.As shown in Fig. 1(a), a light beam from a diode-pumped solid-state laser (20 mW, λ = 473 nm) is passed initially through two polarizers, which enable the intensity and plane of polarization of the laser beam to be adjusted such that the light beam which exits the polarizers contains a higher ratio of ppolarization wave than s-polarization wave.The light beam is then focused by a convergence lens (f = 175 mm), passes through a coupled SF-11 hemispherical prism, a layer of index-matching oil, and the SF-11 slide, and then incidents directly upon the interface between the slide and the thin silver film.Fluorescence from the cell membrane excited by the SPs or the evanescent wave is collected and imaged from the reverse side of the prism by an immersion water objective (100×, N.A. = 1.0,Olympus) dipped into the window of the flow cell, and is then passed through a band-pass filter (BPF, λ = 500~535 nm, Semrock) into a high-speed frame rate CCD camera (iXon DV885, Andor).
The cells were then re-suspended in a small volume of fresh Dulbecco's Modified Eagle Medium (DMEM).In order to compare the fluorescence intensities of a conventional total internal reflection (TIR) sensing chip with that of the proposed enhanced surface plasmon resonance (SPR) chip, the required number of cells (approximately 10 6 cells/cm 2 ) were added to collagens immobilized by chemical SAMs on a naked SF-11 slide and on a silver thin film slide containing pre-warmed medium, respectively.Figure 1(b) shows a cell cultured on a collagen-coated slide modified with a silane SAM.In fabricating this conventional TIR chip, the naked SF-11 slide was immersed in 20% (3-aminoproply) triethoxysilane solution in order 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-(3dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride, 2 mM] and NHS(N-hydroxysuccinimide, 5 mM) for 6 hours.In Fig. 1(c), the cell is cultured on a collagen-coated silver thin film modified with a thiol SAM.In developing this enhanced SPR chip, the metal film was immersed in 1 mM 2aminoethanethiol hydrochloride solution to form a dense SAM on its surface.As with the TIR chip, covalent activation was then performed by immersing the chip in a solution containing EDC[N-(3dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride, 2 mM] and NHS(N-hydroxysuccinimide, 5 mM) for 6 hours to immobilize the protein collagen.
The local field enhancement of the enhanced SPR chip (75.61) is theoretically 20.87 times higher than that of the conventional TIR chip (3.62).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 over a short metal-fluorophore distance is happened, and hence the fluorescence quenching effect is counted.Taking the competing phenomena into account, the enhancement in the fluorescence signal obtained from the proposed SPR chip is more likely to be no more than 20.87 times that of the TIR chip.To compensate for the two competing effects, a spacer is inserted (a thiol SAM and a collagen layer in this study) to increase the distance between the live cell membrane and the metal film such that the experimental results demonstrate a capability of producing 10-times brighter fluorescent live cell imaging via the SPs.

Fig. 1 .
Fig. 1.(a) Schematic illustration of experimental configuration employed for live cell imaging using conventional and surface plasmon-enhanced TIRFM techniques.(b) Conventional TIR chip: cell is cultured on collagen-coated slide modified with silane.(c) Enhanced SPR chip, cell is cultured on collagen-coated silver thin film modified with thiol.

Fig. 2 .
Fig. 2. (a) TIRFM live cell membrane image and (b) surface plasmon-enhanced TIRFM image both exposed at 0.5 seconds; (c) Distribution of fluorescence intensity along x-axis of central crosscut lines of (a) conventional TIRFM live cell membrane image (dashed line) and (b) surface plasmon-enhanced TIRFM image (solid line).

Figure 2
Figure 2(a) presents a conventional TIRFM image of a melanoma-green fluorescent protein (GFP)tagged thrombomodulin (TM) cell (measuring approximately 30 × 30 μm 2 ) cultured on collagen-coated slides modified with silane.The exposure time of the image in Fig. 2(a) is 0.5 seconds, and the