Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope

doi:10.1364/OE.14.007878

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Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope

J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski »View Author Affiliations

Optics Express, Vol. 14, Issue 17, pp. 7878-7888 (2006)
http://dx.doi.org/10.1364/OE.14.007878

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▸ Article Outline
– Abstract
1. Introduction
2. Material & methods
3. Results
4. Discussion
– References and links

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Abstract

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.

1. Introduction

Molecular dynamics is conveniently studied by Fluorescence Correlation Spectroscopy (FCS) [1

R. Rigler and E. L. Elson, “Fluorescence Correlation Spectroscopy: Theory and Applications,” (Berlin Springer, 2001).

]. In particular, the study of dynamics of attachment of single molecules to surfaces [2

M. K. Auer, J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, “Fluorescence correlation spectroscopy: lead discovery by miniaturized HT,” Drug Discovery Today 3, 457–465 (1998). [CrossRef]

] 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 [2

] and to study their conformational changes [3

L Edman, U. Mets, and R. Rigler, “Conformational transitions monitored for single molecules in solution,” Proc Natl. Acad. Sci. U S A. 93, 6710–6715 (1996). [CrossRef] [PubMed]

]. 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 [4

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys J. 33, 435–454 (1981). [CrossRef] [PubMed]

]. 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 [5

A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys J. 85, 3294–302 (2003). [CrossRef] [PubMed]

] and surface binding of biomolecules to silica surfaces [6

R. L. Hansen and J. M. Harris, “Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy,” Anal. Chem. 70, 4247–4256 (1998). [CrossRef]

]. Recently, by combining TIRF with confocal detection [7

T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29, 569–571 (2004). [CrossRef] [PubMed]

], the sensitivity of TIRF-FCS has been significantly improved [8

K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys J. 88, L01–L03 (Epub 2004). [CrossRef] [PubMed]

While TIRF-FCS is a significant improvement over confocal-FCS [9

P. Schwille, “TIR-FCS: staying on the surface can sometimes be better,” Biophys J. 85, 2783–2784 (2003). [CrossRef] [PubMed]

], 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

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, “Surface plasmon-coupled emission with gold films,” J. Phys. Chem. 108, 12568–12574 (2004). [CrossRef]

, 11

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowicz, N. Calander, and Z. Gryczynski, “Two-photon induced surface plasmon-coupled emission,” Thin Solid Films 491, 173–176 (2005). [CrossRef]

]. Recently, we have described a method to implement it in the microscope [12–14

Z. Gryczynski, J. Borejdo, E. Matveeva, N. Calander, R. Grygorczyk, J. Harper, and I. Gryczynski, ldquo;Minimization of detection volume by Surface Plasmon-Coupled Emission,” in Ultrasensitive and Single-Molecule Detection Technologies, J. Enderlein, and Z. K. Gryczynski, eds., Proc. SPIE S1–S10 (2006).

] (a related method was described by Burghardt and his collaborators [15

T. P. Burghardt, J. E. Charlesworth, M. F. Halsetad, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film enhanced total internal reflection microscopy,” Biophys J. 90, 4662–4671 (2006). [CrossRef] [PubMed]

]). 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 [16

J. Enderlein and T. Ruckstuhl, “The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection,” Opt. Express 13, 8855–8865 (2005). [CrossRef] [PubMed]

], 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
E. Matveeva, Z. Gryczynski, I. Gryczynski, J. Malicka, and J. R. Lakowicz, “Myoglobin immunoassay utilizing directional surface plasmon-coupled emission,” Anal. Chem. 76, 6287–6292 (2004). [CrossRef] [PubMed]
].
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 [20
V. Kiessling, B. Muller, and P. Fromherz, “Extracellular resistance in cells adhesion measured with a transistor probe,” Langmuir 16, 3517–3521 (2000). [CrossRef]
].

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

Fig. 1. Prismless confocal SPCE-FCS microscope. Not to scale.

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 [21

D. Axelrod, “Total internal reflection fluorescence microscopy,” Methods Cell Biol. 30, 245–270 (1989). [CrossRef] [PubMed]

]. 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.

2.4. Calculations

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. [22

TFC-Calc, “Optical Coating Design Software” Software Spectra, Inc.: Portland, OR 97229 (2005).

3. Results

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.

Fig. 2. The intensity fluctuations (minus background) caused by diffusion of 100 nm spheres through detection volume in SPCE experiment on gold substrate (A) and the corresponding autocorrelation function (B).

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).

Fig. 3. The autocorrelation functions of diffusion of 100 nm spheres in SPCE experiment on gold substrate (red) and silver (gray).

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.

Fig. 4. Comparison of TIRF (green) and SPCE on silver (gray) autocorrelation functions. The samples and conditions were identical for two experiments.

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

Fig 5. Excitation (blue) and emission (red) intensities near the surfaces. a) A schematics of a random movement of fluorophore near the surface. b) The excitation decays exponentially away from the surface. c) The emission that enters the objective is exponential in TIRF and d) biphasic in SPCE.

It may be approximated by the sum of two exponentials as shown in Fig. 6. Hassler et al. [8

] calsulated autocorrelation function for a single exponential as:

G (τ) = 1 + \frac{1}{2 N} {(1 + \frac{τ}{τ_{xy}})}^{- 1} [(1 - \frac{τ}{{2 τ}_{z}}) w (i \sqrt{\frac{τ}{{4 τ}_{z}}}) + \sqrt{\frac{τ}{{πτ}_{z}}}]

Fig. 6. Left: A sum of a two exponentials approximates the emission at SPCE; Right: The detection intensity versus distance to metal approximated by bi-exponentials. Note the quenching near the surface.

w (x) = \exp (- x^{2}) erfc (- ix)

τ_{z} = \frac{d^{2}}{4 D}

τ_{xy} = \frac{ω_{xy}^{2}}{4 D}

where N is the average number of molecules in the effective volume defined by V_eff = π

ω_{xy}^{2}

d. ω_xy 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 [23

T. E. Starr and N. L. Thompson, “Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion,” Biophys J. 80, 1575–84 (2001). [CrossRef] [PubMed]

], 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:

G (t) = 1 + \frac{1}{2 N} \frac{\sum_{nm} \frac{d_{n} d_{m}}{d_{n} + d_{m}} A_{n} A_{m} R_{nm} (t)}{\sum_{nm} \frac{d_{n} d_{m}}{d_{n} + d_{m}} A_{n} A_{m}}

where

R_{nn} (t) = {(1 + \frac{Dt}{σ^{2}})}^{- 1} ((1 - \frac{2 Dt}{d_{n}^{2}}) erfc (\sqrt{\frac{Dt}{d_{n}^{2}}}) \exp (\frac{Dt}{d_{n}^{2}}) + \sqrt{\frac{4 Dt}{{πd}_{n}^{2}}})

R_{nm} (t) = {(1 + \frac{Dt}{σ^{2}})}^{- 1} (\frac{d_{m}}{d_{m} - d_{n}} erfc (\sqrt{\frac{Dt}{d_{m}^{2}}}) \exp (\frac{Dt}{d_{m}^{2}}) + \frac{d_{n}}{d_{n} - d_{m}} erfc (\sqrt{\frac{Dt}{d_{n}^{2}}}) \exp (\frac{Dt}{d_{n}^{2}}))

and ω_xy = 2σ

In case of bi-exponential:

A_{0} = 1

(zero at metal surface)

A_{1} = - 1

G (t) = 1 + \frac{1}{2 N} \frac{d_{0} R_{00} (t) - 4 \frac{d_{0} d_{1}}{d_{0} + d_{1}} R_{01} (t) + d_{1} R_{11} (t)}{d_{0} - 4 \frac{d_{0} d_{1}}{d_{0} + d_{1}} + d_{1}}

The fit to the above equation is shown in Fig. 7. The diffusion coefficient was taken as 4.12×10^-12 m²/s [24

C. Tanford, “Physical Chemistry of Macromolecules” (John Wiley & Sons New York, 1963).

Fig. 7. Fit of theoretical FCS curves to experimental data. Single-exponential for TIRF and bi-exponential for SPCE. Note that the y-axis is linear.

The fitted parameters are (ω_xy fixed at 0.5 μm):

For TIRF:

d = 65 nm

N = 5

For SPCE with gold:

d_{0} = 35 nm

d_{1} = 35 nm

d = \sqrt{d_{0}^{2} + d_{1}^{2}} = 36.4 nm (RMS value)

N = 8.33

For SPCE with silver:

d_{0} = 50 nm

d_{1} = 15 nm

d = \sqrt{d_{0}^{2} + d_{1}^{2}} = 52.2 nm (RMS value)

N = 6.25

There is a significant tail for the longer times for silver, which doesn’t fit into the theory of Hassler et. [8

]. 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).

Fig. 8. Fit of theoretical FCS curves to experimental data. Single-exponential for TIRF and bi-exponential for SPCE. Note that the y-scale is log minus one.

4. 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 [20

V. Kiessling, B. Muller, and P. Fromherz, “Extracellular resistance in cells adhesion measured with a transistor probe,” Langmuir 16, 3517–3521 (2000). [CrossRef]

] (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

D. Braun and P. Fromherz, “Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye,” Biophys J. 87, 1351–9 (2004). [CrossRef] [PubMed]

, 26

Y. Iwanaga, D. Braun, and P. Fromherz, “No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil,” Eur Biophys J. 30, 17–26 (2001). [CrossRef] [PubMed]

]), 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 ω_xy 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 ω_xy 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 ω_xy, because it is determined by the correlation function for small times:

N = {\frac{1}{2}}_{τ \to 0} lim \frac{1}{G (τ) - 1}

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.

Acknowledgments

Supported by NIH RO1 AR048622 & NCI-CA114460.

References and links

1.	R. Rigler and E. L. Elson, “Fluorescence Correlation Spectroscopy: Theory and Applications,” (Berlin Springer, 2001).
2.	M. K. Auer, J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, “Fluorescence correlation spectroscopy: lead discovery by miniaturized HT,” Drug Discovery Today 3, 457–465 (1998). [CrossRef]
3.	L Edman, U. Mets, and R. Rigler, “Conformational transitions monitored for single molecules in solution,” Proc Natl. Acad. Sci. U S A. 93, 6710–6715 (1996). [CrossRef] [PubMed]
4.	N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys J. 33, 435–454 (1981). [CrossRef] [PubMed]
5.	A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys J. 85, 3294–302 (2003). [CrossRef] [PubMed]
6.	R. L. Hansen and J. M. Harris, “Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy,” Anal. Chem. 70, 4247–4256 (1998). [CrossRef]
7.	T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29, 569–571 (2004). [CrossRef] [PubMed]
8.	K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys J. 88, L01–L03 (Epub 2004). [CrossRef] [PubMed]
9.	P. Schwille, “TIR-FCS: staying on the surface can sometimes be better,” Biophys J. 85, 2783–2784 (2003). [CrossRef] [PubMed]
10.	I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, “Surface plasmon-coupled emission with gold films,” J. Phys. Chem. 108, 12568–12574 (2004). [CrossRef]
11.	I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowicz, N. Calander, and Z. Gryczynski, “Two-photon induced surface plasmon-coupled emission,” Thin Solid Films 491, 173–176 (2005). [CrossRef]
12.	Z. Gryczynski, J. Borejdo, E. Matveeva, N. Calander, R. Grygorczyk, J. Harper, and I. Gryczynski, ldquo;Minimization of detection volume by Surface Plasmon-Coupled Emission,” in Ultrasensitive and Single-Molecule Detection Technologies, J. Enderlein, and Z. K. Gryczynski, eds., Proc. SPIE S1–S10 (2006).
13.	Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, “Minimization of detection volume by surface plasmon-coupled emission,” Anal. Biochem. , in press (2006). [CrossRef] [PubMed]
14.	J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of Surface Plasmon Coupled Emission to study of muscle” Biophys. J. , in press (2006). [CrossRef] [PubMed]
15.	T. P. Burghardt, J. E. Charlesworth, M. F. Halsetad, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film enhanced total internal reflection microscopy,” Biophys J. 90, 4662–4671 (2006). [CrossRef] [PubMed]
16.	J. Enderlein and T. Ruckstuhl, “The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection,” Opt. Express 13, 8855–8865 (2005). [CrossRef] [PubMed]
17.	E. Matveeva, Z. Gryczynski, I. Gryczynski, J. Malicka, and J. R. Lakowicz, “Myoglobin immunoassay utilizing directional surface plasmon-coupled emission,” Anal. Chem. 76, 6287–6292 (2004). [CrossRef] [PubMed]
18.	J. Malicka, I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, “Surface plasmon-coupled ultraviolet emission of 2,5-diphenyl-1,3,4-oxadiazole,” J. Phys. Chem. B. 108, 19114–19118 (2004). [CrossRef] [PubMed]
19.	I. Gryczynski, J. Malicka, W. Jiang, H. Fischer, W.C. W. Chan, Z. Gryczynski, W. Grudzinski, and J. R. Lakowicz, “Surface plasmon-coupled emission of quantum dots,” J. Phys. Chem. B. 109, 1088–1093 (2005). [CrossRef]
20.	V. Kiessling, B. Muller, and P. Fromherz, “Extracellular resistance in cells adhesion measured with a transistor probe,” Langmuir 16, 3517–3521 (2000). [CrossRef]
21.	D. Axelrod, “Total internal reflection fluorescence microscopy,” Methods Cell Biol. 30, 245–270 (1989). [CrossRef] [PubMed]
22.	TFC-Calc, “Optical Coating Design Software” Software Spectra, Inc.: Portland, OR 97229 (2005).
23.	T. E. Starr and N. L. Thompson, “Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion,” Biophys J. 80, 1575–84 (2001). [CrossRef] [PubMed]
24.	C. Tanford, “Physical Chemistry of Macromolecules” (John Wiley & Sons New York, 1963).
25.	D. Braun and P. Fromherz, “Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye,” Biophys J. 87, 1351–9 (2004). [CrossRef] [PubMed]
26.	Y. Iwanaga, D. Braun, and P. Fromherz, “No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil,” Eur Biophys J. 30, 17–26 (2001). [CrossRef] [PubMed]

OCIS Codes
(110.0180) Imaging systems : Microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Microscopy

History
Original Manuscript: June 22, 2006
Revised Manuscript: July 31, 2006
Manuscript Accepted: August 1, 2006
Published: August 21, 2006

Virtual Issues
Vol. 1, Iss. 9 Virtual Journal for Biomedical Optics

Citation
J. Borejdo, N. Calander, Z. Gryczynski, and I. Gryczynski, "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope," Opt. Express 14, 7878-7888 (2006)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-17-7878

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References

R. Rigler, and E. L. Elson, "Fluorescence Correlation Spectroscopy: Theory and Applications," (Berlin Springer, 2001).
M. K. Auer, J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, "Fluorescence correlation spectroscopy: lead discovery by miniaturized HT," Drug Discovery Today 3, 457-465 (1998). [CrossRef]
L Edman, U. Mets, and R. Rigler, "Conformational transitions monitored for single molecules in solution,"Proc Natl. Acad. Sci. U S A. 93, 6710-6715 (1996). [CrossRef] [PubMed]
N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981). [CrossRef] [PubMed]
A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,"Biophys J. 85, 3294-302 (2003). [CrossRef] [PubMed]
R. L. Hansen, and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998). [CrossRef]
T. Ruckstuhl, and S. Seeger, "Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy," Opt. Lett. 29, 569-571 (2004). [CrossRef] [PubMed]
K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-L03 (Epub 2004). [CrossRef] [PubMed]
P. Schwille, "TIR-FCS: staying on the surface can sometimes be better," Biophys J. 85, 2783-2784 (2003). [CrossRef] [PubMed]
I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface plasmon-coupled emission with gold films," J. Phys. Chem. 108,12568-12574 (2004). [CrossRef]
I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowicz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005). [CrossRef]
Z. Gryczynski, J. Borejdo, E. Matveeva, N. Calander, R. Grygorczyk, J. Harper, and I. Gryczynski, "Minimization of detection volume by Surface Plasmon-Coupled Emission," in Ultrasensitive and Single-Molecule Detection Technologies, J. Enderlein, and Z. K. Gryczynski, eds., Proc. SPIE S1-S10 (2006).
Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of detection volume by surface plasmon-coupled emission," Anal. Biochem., in press (2006). [CrossRef] [PubMed]
J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, "Application of Surface Plasmon Coupled Emission to study of muscle" Biophys. J., in press (2006). [CrossRef] [PubMed]
T. P. Burghardt, J. E. Charlesworth, M. F. Halsetad, J. E. Tarara, and K. Ajtai, "In situ fluorescent protein imaging with metal film enhanced total internal reflection microscopy," Biophys J. 90, 4662-4671 (2006). [CrossRef] [PubMed]
J. Enderlein, and T. Ruckstuhl, "The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection," Opt. Express 13, 8855-8865 (2005). [CrossRef] [PubMed]
E. Matveeva, Z. Gryczynski, I. Gryczynski, J. Malicka, and J. R. Lakowicz, "Myoglobin immunoassay utilizing directional surface plasmon-coupled emission," Anal. Chem. 76, 6287-6292 (2004). [CrossRef] [PubMed]
J. Malicka, I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, "Surface plasmon-coupled ultraviolet emission of 2,5-diphenyl-1,3,4-oxadiazole,"J. Phys. Chem. B. 108,19114-19118 (2004). [CrossRef] [PubMed]
I. Gryczynski, J. Malicka, W. Jiang, H. Fischer, W.C. W. Chan, Z. Gryczynski, W. Grudzinski, and J. R. Lakowicz, "Surface plasmon-coupled emission of quantum dots,"J. Phys. Chem. B. 109,1088-1093 (2005). [CrossRef]
V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000). [CrossRef]
D. Axelrod, "Total internal reflection fluorescence microscopy," Methods Cell Biol. 30, 245-270 (1989). [CrossRef] [PubMed]
TFC-Calc, "Optical Coating Design Software," Software Spectra, Inc., Portland, OR 97229 (2005).
T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion," Biophys J. 80,1575-84 (2001). [CrossRef] [PubMed]
C. Tanford, "Physical Chemistry of Macromolecules" (John Wiley & Sons New York, 1963).
D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87,1351-9 (2004). [CrossRef] [PubMed]
Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30,17-26 (2001). [CrossRef] [PubMed]

Ajtai, K.
Auer, M. K.
Axelrod, D.
Borejdo, J.
Braun, D.
Burghardt, T. P.
Calander, N.
Chan, W.C. W.
Charlesworth, J. E.
Cush, R. C.
Edman, L
Enderlein, J.
Fischer, H.
Fromherz, P.
Goldys, E. M.
Grudzinski, W.
Gryczynski, I.
Gryczynski, Z.
Guenther, R.
Halsetad, M. F.
Hansen, R. L.
Harris, J. M.
Iwanaga, Y.
Jiang, W.
Kiessling, V.
Lakowicz, J. R.
Lieto, A. M.
Malicka, J.
Matveeva, E.
Matveeva, E. G.
Mets, U.
Meyer-Almes, F. J.
Moore, J.
Muller, B.
Muthu, P.
Pope, A. J.
Rigler, R.
Ruckstuhl, T.
Schwille, P.
Seeger, S.
Starr, T. E.
Stoeckli, K. A.
Tarara, J. E.
Thompson, N. L.

Anal. Biochem.

Z. Gryczynski, J. Borejdo, N. Calander, E. G. Matveeva, and I. Gryczynski, "Minimization of detection volume by surface plasmon-coupled emission," Anal. Biochem., in press (2006). [CrossRef] [PubMed]

Anal. Chem.

R. L. Hansen, and J. M. Harris, "Measuring reversible adsorption kinetics of small molecules at solid/liquid interfaces by total internal reflection fluorescence correlation spectroscopy," Anal. Chem. 70, 4247-4256 (1998). [CrossRef]
E. Matveeva, Z. Gryczynski, I. Gryczynski, J. Malicka, and J. R. Lakowicz, "Myoglobin immunoassay utilizing directional surface plasmon-coupled emission," Anal. Chem. 76, 6287-6292 (2004). [CrossRef] [PubMed]

Biophys J.

N. L. Thompson, T. P. Burghardt, and D. Axelrod, "Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy," Biophys J. 33, 435-454 (1981). [CrossRef] [PubMed]
A. M. Lieto, R. C. Cush, and N. L. Thompson, "Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,"Biophys J. 85, 3294-302 (2003). [CrossRef] [PubMed]
P. Schwille, "TIR-FCS: staying on the surface can sometimes be better," Biophys J. 85, 2783-2784 (2003). [CrossRef] [PubMed]
T. P. Burghardt, J. E. Charlesworth, M. F. Halsetad, J. E. Tarara, and K. Ajtai, "In situ fluorescent protein imaging with metal film enhanced total internal reflection microscopy," Biophys J. 90, 4662-4671 (2006). [CrossRef] [PubMed]
T. E. Starr and N. L. Thompson, "Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion," Biophys J. 80,1575-84 (2001). [CrossRef] [PubMed]
D. Braun and P. Fromherz, "Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye," Biophys J. 87,1351-9 (2004). [CrossRef] [PubMed]

Biophys. J.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, "Application of Surface Plasmon Coupled Emission to study of muscle" Biophys. J., in press (2006). [CrossRef] [PubMed]

Drug Discovery Today

M. K. Auer, J. Moore, F. J. Meyer-Almes, R. Guenther, A. J. Pope, and K. A. Stoeckli, "Fluorescence correlation spectroscopy: lead discovery by miniaturized HT," Drug Discovery Today 3, 457-465 (1998). [CrossRef]

Eur Biophys J.

Y. Iwanaga, D. Braun, and P. Fromherz, "No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil," Eur Biophys J. 30,17-26 (2001). [CrossRef] [PubMed]

J. Phys. Chem.

I. Gryczynski, J. Malicka, Z. Gryczynski, and J. R. Lakowicz, "Surface plasmon-coupled emission with gold films," J. Phys. Chem. 108,12568-12574 (2004). [CrossRef]

J. Phys. Chem. B.

J. Malicka, I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, "Surface plasmon-coupled ultraviolet emission of 2,5-diphenyl-1,3,4-oxadiazole,"J. Phys. Chem. B. 108,19114-19118 (2004). [CrossRef] [PubMed]
I. Gryczynski, J. Malicka, W. Jiang, H. Fischer, W.C. W. Chan, Z. Gryczynski, W. Grudzinski, and J. R. Lakowicz, "Surface plasmon-coupled emission of quantum dots,"J. Phys. Chem. B. 109,1088-1093 (2005). [CrossRef]

Langmuir

V. Kiessling, B. Muller, and P. Fromherz, "Extracellular resistance in cells adhesion measured with a transistor probe," Langmuir 16, 3517-3521 (2000). [CrossRef]

Methods Cell Biol.

D. Axelrod, "Total internal reflection fluorescence microscopy," Methods Cell Biol. 30, 245-270 (1989). [CrossRef] [PubMed]

Opt. Express

J. Enderlein, and T. Ruckstuhl, "The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection," Opt. Express 13, 8855-8865 (2005). [CrossRef] [PubMed]

Opt. Lett.

T. Ruckstuhl, and S. Seeger, "Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy," Opt. Lett. 29, 569-571 (2004). [CrossRef] [PubMed]

Proc Natl. Acad. Sci. U S A.

L Edman, U. Mets, and R. Rigler, "Conformational transitions monitored for single molecules in solution,"Proc Natl. Acad. Sci. U S A. 93, 6710-6715 (1996). [CrossRef] [PubMed]

Thin Solid Films

I. Gryczynski, J. Malicka, E. M. Goldys, J. R. Lakowicz, N. Calander, and Z. Gryczynski, "Two-photon induced surface plasmon-coupled emission," Thin Solid Films 491, 173-176 (2005). [CrossRef]

Other

Z. Gryczynski, J. Borejdo, E. Matveeva, N. Calander, R. Grygorczyk, J. Harper, and I. Gryczynski, "Minimization of detection volume by Surface Plasmon-Coupled Emission," in Ultrasensitive and Single-Molecule Detection Technologies, J. Enderlein, and Z. K. Gryczynski, eds., Proc. SPIE S1-S10 (2006).
K. Hassler, T. Anhut, R. Rigler, M. Gosch, and T. Lasser, "High count rates with total internal reflection fluorescence correlation spectroscopy," Biophys J. 88, L01-L03 (Epub 2004). [CrossRef] [PubMed]
TFC-Calc, "Optical Coating Design Software," Software Spectra, Inc., Portland, OR 97229 (2005).
C. Tanford, "Physical Chemistry of Macromolecules" (John Wiley & Sons New York, 1963).
R. Rigler, and E. L. Elson, "Fluorescence Correlation Spectroscopy: Theory and Applications," (Berlin Springer, 2001).

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