Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (FCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. FCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ~35 nm.
©2006 Optical Society of America
Molecular dynamics is conveniently studied by Fluorescence Correlation Spectroscopy (FCS) . In particular, the study of dynamics of attachment of single molecules to surfaces  requires that the thickness of detection volume be minimized, that the background be low, and that the fluorescence change associated with alteration in axial position of a molecule be maximized. Conventional confocal detection scheme goes a long way towards achieving these goals. It has made possible to investigate dynamical processes of biomolecules attached to a surface  and to study their conformational changes . However, in confocal detection the axial dimension of the detection volume is of the order of few microns, large compared to the axial extent of the attached biomolecule. Further, the change in axial position of a molecule leads to only a small change in signal. Finally, in confocal detection, a significant background is contributed by the reflection of the excitation light from the surface.
Total Internal Reflection Fluorescence (TIRF) correlation spectroscopy (TIRF-FCS) partially resolves these problems . In this method, a sample is excited by an evanescent field, generated by total internal reflection of a laser beam from the coverslip on which a sample is placed. The intensity of the evanescent field decays exponentially within the solution with increasing distance from the interface. It typically drops to 1/e after ~200 nm. This is substantially smaller than in confocal FCS. The method has been applied successfully to investigate ligand-receptor binding on membranes  and surface binding of biomolecules to silica surfaces . Recently, by combining TIRF with confocal detection , the sensitivity of TIRF-FCS has been significantly improved .
While TIRF-FCS is a significant improvement over confocal-FCS , it is possible to further minimize the thickness of a detection volume, reduce the background and to maximize change of fluorescent signal upon axial movement of a molecule by the application Surface Plasmon Coupled Emission [10, 11]. Recently, we have described a method to implement it in the microscope [12–14] (a related method was described by Burghardt and his collaborators ). In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle. At this angle of incidence, the laser beam excites surface plasmons in the metal film. Excitation light produces an evanescent field on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent field penetration depth and distance-dependent fluorescence coupling with surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to the surface. The fluorescent light is emitted through the metal only at an SPCE angle (on a surface of a cone in 3D) and efficiently collected by a high numerical aperture objective (APO NA=1.65, 100x, focal length=150 μm, Olympus). A confocal aperture inserted in the conjugate image plane of the objective reduces lateral dimensions of the detection volume to 0.5 μm. We show here that on gold surfaces, the detection volume is ~35 nm thick.
In summary: SPCE offers no significant advantages over TIRF as far as fluorescence collection efficiency and brightness are concerned , but SPCE-FCS has five advantages over TIRF-FCS:
- The detection volume is thinner than in TIRF. This assures that the major part of fluctuations is provided by one dimensional diffusion in the axial direction. It is important to note that plasmon coupling preserves spectral properties of fluorophores [17–19].
- The background is greatly reduced. Only the SPCE emission is able to penetrate the metal film. All other emission is reflected by the metal film.
- Changes of fluorescence upon axial movement of a molecule are caused not only by gradient of evanescent wave intensity as in TIRF, but are enhanced by quenching of fluorescence near the surface of a metal.
- The photobleaching is reduced. Coupling to surface plasmons enhances excitation field and allows less excitation power to be used. In addition, fluorescence lifetime is partially reduced.
- The major part of fluorescence signal is contributed by fluorophores at least 10 nm away from the surface. Since this is equal to the approximate thickness of cell membrane, the method is particularly well suited for studying events inside a cell, without contribution by molecules whose diffusion is slowed down by the surface. This is only true in systems in which the distance between a cell and a substrate is small (~10 nm), such as erythrocyte ghosts on polylysine .
In this paper we show that that high refractive index coverslips (n=1.77) coated with metal together with high numerical aperture objective (NA=1.65) combine to make microscopic SPCE-FCS an efficient way to study molecular diffusion.
2. Material & methods
2.1. Chemicals and solutions
Rhodamine-labeled microspheres (100 nm diameter) were from Molecular Probes (Eugene, OR). All the other solvents were from Sigma (St Louis, MO).
2.2. Preparation of coverslips
High refractive index coverglasses from Olympus were coated by vapor deposition by EMF Corp. (Ithaca, NY). A 52 nm thick layer of silver and 48 nm layer of gold were deposited on the coverslips. 5 nm of silica was deposited on top of silver to protect it from oxidation. 2 nm chromium undercoat was used as an adhesive background. For TIRF experiments, custom made sapphire coverslides (MPA Crystal Co, San Francisco, CA) were used.
2.3. Microscopic measurements.
The schematic of the microscope is shown in Fig. 1. Excitation light from an expanded Diode Pumped Solid State laser beam (Compass 215M, Coherent, Santa Clara, CA) enters the epi-illumination port of the inverted microscope (Olympus IX51). The expanded laser beam, focused at the back focal plane of the objective (Olympus Apo 100x, 1.65 NA), is directed by the movable optical fiber adapter to its periphery. It then
propagates towards the high-refractive-index glass-metal/buffer interface. When the incidence angle is equal to the SPR angle, the light is able to penetrate the metal and illuminate a cell. Excitation light produces an evanescent wave on the aqueous side of the interface . The detection volume is a product of evanescent wave penetration depth and distance-dependent coupling with surface plasmons. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The fluorescent light, emitted at SPCE angle, is collected by the objective. The sample rests on a moveable piezo stage (Nano-H100, Mad City Labs, Madison, WI) controlled by a Nano-Drive. The fluorescent light is collected through the same objective and projected onto a tube lens, which focuses it at the conjugate image plane. A 50 μm confocal aperture or an optical fiber (whose core acts as a confocal aperture) is inserted at this plane. A pair of Avalanche Photodiodes (APD, Perkin-Elmer SPCM-AQR-15-FC) collects light emerging from the aperture. The TTL signal from the diodes is fed to correlator (Flex02-08D, Correlator Inc, Bridgewater, NJ) and autocorrelation function is displayed on a PC.
It is assumed that the exciting field is continuous in time and weak enough such that the time between excitations is much longer than the time between excitation and emission of the fluorophore. This means that the average number of photons emitted per unit time and therefore the average total emitted power does not depend on the lifetime; they only depends on the average time between excitations. The refractive indices of the metals used in the calculations are interpolated from Ref. .
3.1. SPCE autocorrelation
Suspension of 0.1 μm diameter microspheres (Molecular Probes, Eugene, OR) was diluted 100x to 3.6 × 108 spheres/mL. The spheres were placed on a coverslip coated with gold. The intensities were measured in 160 μs intervals for 30 sec. Figure 2(A) shows a typical trace of intensity fluctuations. The fluctuations are caused by spheres entering and leaving the detection volume. Figure 2(B) shows corresponding autocorrelation function.
3.2. Comparison of SPCE on gold and silver
Samples were placed on coverslips coated with gold and silver. The changes in intensity were measured in 160 μs intervals for 30 sec. Figure 3 compares the autocorrelation functions of spheres diffusing on gold (red) and silver substrates (gray). Autocorrelation on silver is slightly larger and has slowly decaying tail (see below).
3.3. Comparison of SPCE and TIRF
The comparison is shown in Fig. 4. The autocorrelation function of SPCE on silver has slowly decaying tail. The TIRF signal is noisy in comparison with SPCE signal, both at short and long times.
3.4. Data fitting
Panel (a) in Fig. 5 shows schematically fluorophore moving randomly near the surface. Panel (b) shows that the rate of excitation decays exponentially away from the surface. The light entering the objective is a single exponential in TIRF (c) and biphasic in SPCE (d) because of quenching near the surface [12–14].
where N is the average number of molecules in the effective volume defined by Veff = πd. ωxy is the width of the lateral Gaussian, d is the thickness of the evanescent field and D is the diffusion coefficient. This result is based on description by Starr and Thompson , but takes into account the influence of transversal diffusion, which is necessary since the observation volume was laterally restricted by introducing a pinhole. Hassler et al. used a Gaussian function to model the lateral intensity distribution and an exponential decay in axial direction. Further, their model contained a term accounting for triplet state kinetics.
Extending this to multi-exponential emission, as shown in Fig. 6, gives the correlation function:
and ωxy = 2σ
In case of bi-exponential:
The fitted parameters are (ωxy fixed at 0.5 μm):
For SPCE with gold:
For SPCE with silver:
There is a significant tail for the longer times for silver, which doesn’t fit into the theory of Hassler et. . The tail is better seen in the log-log plot (Fig. 8). We think it is due to adsorption of spheres to the metal surface (see Discussion).
The fact that in SPCE-FCS the axial dimension is ~14 times smaller then the lateral dimension (for gold) assures that the major part of fluctuations is provided by one dimensional diffusion in the axial direction - important consideration in applications where ligand-receptor kinetics or adsorption kinetics of small molecules at solid/liquid interfaces is measured [5, 6]. Thin detection volume also increases change of fluorescence signal upon axial movement of a molecule. The thickness of detection volume is reduced not only by evanescent excitation, but also by distance-dependent emission coupling and by quenching of fluorescence emitted from fluorophores near the metal surface. Changes of fluorescence associated with axial movement of a molecule are further enhanced by quenching of fluorescence near the surface of a metal. We note that the fluorophores are not point-like. Spheres may not be able to penetrate so well into the quenched region near the metal surface. If the fluorophores were point-like, the detection volume thickness d would have been much smaller for SPCE than for TIRF.
An important advantage of SPCE is a significant reduction of the background fluorescence. Only SPCE emission couples through the metal film and is collected by the objective. All other fluorescence is reflected by metal film.
Another advantage of SPCE-FCS is that fluorescence signal is contributed by fluorophores at least 10 nm away from the surface. Since 10 nm is a typical thickness of cell membrane, the method allows looking at diffusion of fluorophores inside a cell and avoiding monitoring events occurring at a cell membrane. This is only true in systems in which the distance between a cell and a substrate is small (~10 nm), such as erythrocyte ghosts on polylysine  (in cells where the distance of the major part of the membrane is separated by >50 nm from the surface (e.g. kidney cells, rat neurons or fibroblasts [25, 26]), the SPCE quenching offers no advantage).
In SPCE photobleaching is reduced because coupling to surface plasmons enhances excitation field and allows less excitation power to be used.
Since we used very high NA (1.65) objective, all the light emitted in the lower half space and not reflected is collected by the high NA objective giving high photon counts per molecule compared to a prism-based setup. It is important to emphasize here that it is impossible to obtain SPCE image of samples in aqueous solutions using conventional TIRF (PlanApo NA=1.45) objective.
The long tail of autocorrelation function (Fig. 8) is most likely due to the adsorption of spheres to the metal surface. If ωxy could have been confirmed to be larger than 0.5 μm (TIRF value), than the effect of surface plasmon travel may have been noticed. However, the analysis showed that autocorrelation function was rather insensitive to ωxy suggesting that the tail is most likely due to adsorption of the beads to the metal surface. The effect was most prominent on silver. There was no tail in TIRF suggesting that the beads stick to metal but not to the glass.
The average number of molecules in the detection volume, N, is a parameter fitted independent of d and ωxy, because it is determined by the correlation function for small times:
We note that the method can also be applied in bulk (in a spectrofluorometer, not in a microscope), where directional and highly polarized character of SPCE signal enables better suppression of background noise.
Supported by NIH RO1 AR048622 & NCI-CA114460.
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