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Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 19, Iss. 9 — Apr. 25, 2011
  • pp: 8011–8018
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Imaging properties of supercritical angle fluorescence optics

Jörg Enderlein, Ingo Gregor, and Thomas Ruckstuhl  »View Author Affiliations


Optics Express, Vol. 19, Issue 9, pp. 8011-8018 (2011)
http://dx.doi.org/10.1364/OE.19.008011


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Abstract

In recent years, new optical systems have been developed with the ability to collect light at very high angles of emission, exceeding the critical angle of total internal reflection. Prominent examples are solid-immersion lenses and paraboloid collectors. These systems achieve high efficiencies in fluorescence detection which is an important issue for sensitive applications in analytical chemistry and biochemical assays. The exclusive collection of supercritical angle fluorescence (SAF) allows for the detection of evanescent modes and thus to confine the detection volume within one wavelength to an interface. For conventional optical systems with high numerical aperture a precise wave-optical theory of imaging was developed by Richards and Wolf in the fifties of the last century. However, their theory is not directly applicable to non-imaging, strongly aberratic light collection systems systems that collect a significant part of light above the critical angle. Here, we extend the theory to describe the optical properties of such systems.

© 2011 OSA

1. Introduction

A fundamental goal of physical optics is the characterization of the imaging properties of a given optical system. When dealing with imaging of non-coherent fluorescent sources, this is equivalent to calculating the image of single dipole emitters as a function of their position in sample space. For aplanatic optical systems with high numerical aperture, an exact wave-optical approach for calculating their imaging properties was laid down by Richards and Wolf in two seminal papers in the fifties [1

1. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. Roy. Soc. London A 253, 358–379 (1959). [CrossRef]

, 2

2. E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. Roy. Soc. London A 253349–357 (1959). [CrossRef]

]. Their approach employs the particular property of aplanatic imaging systems to obey Abbe’s sine condition [3

3. M. Born and E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, 1987).

]. Although their method can be also adapted for moderately aberratic optical systems, it becomes inapplicable for systems that are strongly violating Abbe’s sine condition (or any other simple relationship between propagation angles in sample and image space, such as Herschel’s condition [3

3. M. Born and E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, 1987).

]). Two such optical systems are of particular practical importance: light collection optics using parabolic mirrors [4

4. T. Ruckstuhl and S. Seeger, “Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens,” Appl. Opt. 42, 3277–3283 (2003). [CrossRef] [PubMed]

] or solid-immersion lenses [5

5. S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett. 57, 2615–2616 (1990). [CrossRef]

]. Such systems are far from being aplanatic, and collect a considerable amount of light above the critical angle of reflection (supercritical light detection), i.e. modes that are non-propagating in sample space but become propagating after tunneling into the glass of the optics (paraboloid glass segment, solid immersion lens). For this situation, there is no straightforward method available for computing the point spread function.

As far as we know, all previously described methods for calculating optical systems with high numerical aperture use the property that the imaging system is close to a perfect imaging system obeying Abbe’s sine condition. This allows to derive, in a rigorous way, the field distribution in image space when the field distribution in the focal region of the sample space is known, see for example Ref. [7

7. M. Foreman, R. Matthew, S. Sherif, P. R. T. Munro, and P. Török, “Inversion of the Debye–Wolf diffraction integral using an eigenfunction representation of the electric fields in the focal region,” Opt. Express 16, 4901–4917 (2008). [CrossRef] [PubMed]

]. However, this approach fails when the involved optics cannot be described by some simple relationship between plane wave modes in sample space and in image space. In that case, one usually takes recourse to ray tracing. Ray tracing is a general technique from geometrical optics that parses rays of light through an optical system as they interact with the optical interfaces of its elements. It can be generalized to include polarization effects by associating with each ray an electric-field polarization vector, and tracking its change during each interaction of the ray with an optical interface. Even more, ray tracing can be combined with wave-optics in the sense of Richards and Wolf by associating the rays impinging on the image plane with electromagnetic plane waves. The method can then be used to calculate point spread functions and imaging properties of arbitrary optical systems [8

8. P. Török, P. D. Higdon, and T. Wilson, “On the general properties of polarised light conventional and confocal microscopes,” Opt. Commun. 148, 300–315 (1998). [CrossRef]

10

10. M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16, 8519–8531 (2008). [CrossRef] [PubMed]

]. However, ray tracing in this simple form becomes problematic when trying to trace modes that are evanescent, because for an evanescent mode there is no obvious association with a light ray! However, evanescent modes are important for emitters that are located close to an optical interface of the imaging system. Then, a considerable amount of energy can be transferred into the optical system via evanescent modes, i.e. modes that are non-propagating within the medium of the emitter, but that transform into propagating modes above the critical angle of total internal reflection (TIR) within the optical system [11

11. W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface II. Radiation patterns of perpendicular oriented dipoles,” J. Opt. Soc. Am. 67, 1615–1619 (1977). [CrossRef]

, 12

12. W. Lukosz, “Light emission by magnetic and electric dipoles close to a plane interface III. Radiation patterns of dipoles with arbitrary orientation,” J. Opt. Soc. Am. 69, 1495–1503 (1979). [CrossRef]

]. For example, a fluorescing molecule in air or water located on or close to a glass surface emits a considerable part of its radiation as supercritical angle fluorescence (SAF) into the glass above the TIR angle [13

13. T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72, 2117–2123 (2000). [CrossRef] [PubMed]

, 17

17. T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12, 4246–4254 (2004). [CrossRef] [PubMed]

].

Thus, for an optical system which (i) does not allow for a simple relationship between modes in the front and in the back of the optics, such as Abbe’s sine condition, and which (ii) collects a significant part of light from evanescent modes of an emitter, there does not exist a straightforward method of calculating its point spread function. To derive and describe such a method is the purpose of the present manuscript. This will be done by a combined ray tracing and wave-optical treatment of evanescent emission of electric dipole emitters. The method allows for modeling the imaging and light collection properties of non-aplanatic optical systems with high numerical aperture and evanescent wave collection such as SAF. The method is exemplified on the example of a parabolic collection optics.

2. Theory

The theoretical treatment will be divided into several steps. Firstly, we briefly recall the angular distribution of radiation, the phase and the polarization properties of an oscillating electric dipole close to an optical interface. Secondly, we develop a method how to assign to that emission a bundle of rays with correct distribution of propagation directions, phases and polarizations. This is the core and original part of our work. Thirdly, we give a brief description of the ray tracing and image calculation procedure.

2.1. Angular distribution of electromagnetic field in glass half space

Consider a molecule in air deposited on a glass surface. The general geometry of imaging such a molecule is depicted in Fig. 1.

Fig. 1 Geometry of the emission of an electric dipole close to an interface: a single dipole emitter with an inclination angle β is placed at a distance h away from a glass surface. The angular distribution of radiation into the glass is depicted by the red solid line and is a function of angles θ and ϕ. Shown is a cross section of this function in a plane containing the optical axis (vertical) and the dipole axis. The critical TIR angle θcr between glass and air is also indicated by straight lines. It is important to realize that for plane wave modes above the TIR angle in the bottom medium, there are no corresponding propagating plane wave modes in the upper medium.

Omitting any constant factors, the angular distribution of the electric-field amplitude E(θ,ϕ) generated by an oscillating electric dipole with orientation angles θ, ϕ along direction κ = (sin(θ) cos(ϕ), sin(θ) sin(ϕ), −cos(θ)) per solid angle sin(θ)dθdϕ is given by [14

14. 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(θ,ϕ)=sinβ[tsEs(θ)sinϕ+tpEp(θ)cosϕ]+tpcosβEp(θ),
(1)
Ep(θ)=ngwgwmqnmTpexp(iwmh),
(2)
Ep(θ)=ngwgnmTpexp(iwmh),
(3)
Es(θ)=ngwgwmTsexp(iwmh),
(4)
where β is the polar angle of the molecule’s emission dipole orientation; h its vertical distance from the glass surface; t p and t s are orthogonal unit vectors perpendicular to the direction of emission κ,
ts=κ×ez|κ×ez|,tp=ts×κ,
(5)
whereby e z is a unit vector perpendicular to the surface; ng and nm denote the refractive index values of glass and air, respectively; and the Tp , s(θ) are Fresnel’s transmission coefficients for plane p- and s-waves through the air/glass interface traveling along direction κ into the glass, q is the in-plane component of the wave vectors, which is equal on both sides of the interface, and wg and wm are the z-components of the wave vectors in glass and air, respectively. Note that wm (but not wg) becomes purely imaginary for evanescent modes.

2.2. Virtual point of origin of light rays

As a function of θ, this expression describes the wave front of the expanding light wave. The core idea is that each patch of the wave front is associated with a light ray that is perpendicular to the wave front’s surface, i.e. perpendicular to the surface of constant phase [15

15. E. M. Lifshitz, L. D. Landau, and L. P. Pitaevskii, Electrodynamics of Continuous Media: 8 (Course of Theoretical Physics) (Butterworth Heinemann, 1984), Chap. X.

]. Applying the principle of stationary phase one thus finds
k0ng(zsinθ+ρcosθ)+ϕ(θ)=0,
(8)
where a prime denotes differentiation with respect to θ. Thus, the virtual point of origin on the optical axis ρ = 0 of the corresponding patch of the wave front is located at
z0(θ)=ϕ(θ)k0ngsinθ.
(9)

As an example, we consider the case of a vertically oriented dipole (dipole axis along optical axis) sitting in air directly on the glass surface. The refractive index of air is nm = 1, and that of glass is set to ng = 1.51. For this situation, Fig. 2 shows the virtual distance z 0 as a function of emission angle in glass. For the dipole sitting directly on the glass surface, the functions Φ(θ) and z 0(θ) are zero below the critical TIR angle.

Fig. 2 Function z 0(θ) for a parallel (red line) and a vertically (blue line) oriented dipole on an air/glass interface. Vertical green line indicates the position of the TIR angle, where z 0(θ) diverges towards infinity. Notice that even in the limit θ → 90°, z 0 does not approach zero: the dipole seems to hover over the surface at a finite distance. This shift is a similar effect as the virtual lateral shift of the reflection point of a light beam upon TIR, the so-called Goos-Hänchen effect [16]. It should be noted that this figure will not change when moving the dipole away from the surface: For evanescent modes, the exponents in Eqs. (2) through (4) become real-valued, and the phase Φ(θ), see Eq. (6), becomes independent on the dipole’s position above the surface.

2.3. Image formation

Fig. 3 Schematic of the parabolic light collection system.

The emission of a dipole into the glass segment is now considered to consist of bundles of light rays traveling into all possible directions, whereas a ray traveling along direction (sinθ cosϕ, sinθ sinϕ, cosθ) starts at position
r0=(z0tanθcosϕ+ρ0cosϕ0,z0tanθsinϕ+ρ0sinϕ0,0),
(10)
having an initial optical phase
ψ0=k0ngz0sin2θcosθ+ϕ(θ),
(11)
(see Eq. (7)) and an initial polarization equal to the polarization of E(θ,ϕ) in Eq. (1). For each ray, one performs a ray tracing of its subsequent propagation through the system (total internal reflection at paraboloid’s lateral surface, refraction through segment’s bottom surface, re-focusing by ideal lens towards the detector). During ray tracing, one records not only the position and direction of each ray, but also the change of the optical phase along its path and the re-orientation and attenuation of the associated electric field vector. This is done by handling all reflections and refractions as an interaction of a plane wave with a planar surface, thus taking into account also phase jumps upon TIR. In the image plane (position of the CCD detector), each light ray is finally associated with a plane wave traveling along the ray’s direction with corresponding optical phase and polarization. Finally, the intensity distribution in the image plane is calculated as the absolute square of the superposition of all plane waves.

3. Results and discussion

In what follows, all lengths are given in units of the vacuum wavelength. As a concrete example, we consider the imaging properties of the paraboloid light collection system as shown in Fig. 3 with the following parameters: radius of top surface of the paraboloid glass segment a = 104, axial position and focal length of the refocusing lens are zf = −5 · 105 and f = 106, respectively. This reflects fairly well the parameters of existing paraboloid light collection setups. The refractive index of the glass segment was assumed to be 1.51, that of air was set to 1. We calculated the image of a dipole emitter positioned directly on the glass surface and considered three different dipole orientations along x, y and z and six dipole positions along x between x 0 = 0 and x 0 = 5 in steps of one. We further considered two different imaging conditions: (i) only supercritical angle fluorescence is collected, and (ii) light is collected only below the critical angle down to a collection angle of ≈ 30°. The computational results are shown in Figs. 4 and 5.

Fig. 4 Images of a dipole emitter on glass for SAF collection. Shown are results for three dipole orientations along the horizontal figure axis (top row), vertical figure axis (middle row), and an orientation perpendicular to the figure plane (along the optical axis, bottom row). From left to right, the images show six different lateral dipole positions, from x 0 = 0 till x 0 = 5 in steps of one. Each of the 18 images has an edge length of 300.
Fig. 5 Same as Fig. 4 but for “classical” light collection below the TIR angle.

First at all, the results show how sensitive the images are against lateral displacement of the emitter from the optical axis. For the on-axis position, refocusing of the emission light is best for sub-critical angle light collection and less efficient for SAF light collection. However, sub-critical angle collection is extremely sensitive against lateral displacement, which is shown by the quick deterioration of the images with increasing displacement. It should be noted that the calculations are equivalent to calculating the point spread function (image of an isotropic emitter) of the system, which is obtained when adding the images for all three dipole orientations. When repeating the calculations for different axial dipole positions, one can obtain the three-dimensional molecule-detection function (MDF), which is of importance in Fluorescence Correlation Spectroscopy (FCS) [18

18. J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94, 221–229 (2008). [CrossRef]

]. Another potential application is defocused imaging of an emitter for determining its three-dimensional orientation in space [19

19. M. Bhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B 20, 554–559 (2003). [CrossRef]

]. As an example, we repeated the calculations of Figs. 4 and 5, but for an emitter on the optical axis and varying positions of the focal plane (by moving the image plane away from the focusing lens, see Fig. 3). The results for SAF as well as sub-critical light detection are shown in Fig. 7 and 6, respectively.

Fig. 6 Same as Fig. 7 but for ”classical” light collection below the TIR angle.
Fig. 7 Images of a dipole emitter on glass for SAF collection. Shown are results for two dipole orientations along the horizontal figure axis (top row), and for an orientation perpendicular to the figure plane (along the optical axis, bottom row). From left to right, the images show six different positions of the image plane away from the focal plane of the focusing lens, from z = 0 till z = −40 in steps of 8. As before, each of the 12 images has an edge length of 300.

4. Conclusion

We have presented a general approach for calculating the light collection/imaging properties for strongly aberratic systems which collect a significant part of light above the critical angle of TIR. One of the core results of our paper is given by Eq. (9), which determines the virtual origin of light pencils associated with a given patch of the wavefront in glass. We applied our general theoretical approach to imaging a dipole emitter with a parabolic light collection system.

Acknowledgment

J. Enderlein and I. Gregor thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (grant A11, SFB 937).

References and links

1.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. Roy. Soc. London A 253, 358–379 (1959). [CrossRef]

2.

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. Roy. Soc. London A 253349–357 (1959). [CrossRef]

3.

M. Born and E. Wolf, Principles of Optics, 6th ed. (Pergamon Press, 1987).

4.

T. Ruckstuhl and S. Seeger, “Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens,” Appl. Opt. 42, 3277–3283 (2003). [CrossRef] [PubMed]

5.

S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett. 57, 2615–2616 (1990). [CrossRef]

6.

P. Török, P. R. Munro, and E. E. Kriezis, “Rigorous near- to far-field transformation for vectorial diffraction calculations and its numerical implementation,” J. Opt. Soc. Am. A 23, 713–722 (2006). [CrossRef]

7.

M. Foreman, R. Matthew, S. Sherif, P. R. T. Munro, and P. Török, “Inversion of the Debye–Wolf diffraction integral using an eigenfunction representation of the electric fields in the focal region,” Opt. Express 16, 4901–4917 (2008). [CrossRef] [PubMed]

8.

P. Török, P. D. Higdon, and T. Wilson, “On the general properties of polarised light conventional and confocal microscopes,” Opt. Commun. 148, 300–315 (1998). [CrossRef]

9.

M. Leutenegger, R. Rao, R. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express 14, 11277–11291 (2006). [CrossRef] [PubMed]

10.

M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16, 8519–8531 (2008). [CrossRef] [PubMed]

11.

W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface II. Radiation patterns of perpendicular oriented dipoles,” J. Opt. Soc. Am. 67, 1615–1619 (1977). [CrossRef]

12.

W. Lukosz, “Light emission by magnetic and electric dipoles close to a plane interface III. Radiation patterns of dipoles with arbitrary orientation,” J. Opt. Soc. Am. 69, 1495–1503 (1979). [CrossRef]

13.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72, 2117–2123 (2000). [CrossRef] [PubMed]

14.

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

15.

E. M. Lifshitz, L. D. Landau, and L. P. Pitaevskii, Electrodynamics of Continuous Media: 8 (Course of Theoretical Physics) (Butterworth Heinemann, 1984), Chap. X.

16.

F. Goos and H. Hänchen, “Ein neuer und fundamentaler Versuch zur Totalreflexion,” Ann. Physik 436, 333–346 (1947). [CrossRef]

17.

T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12, 4246–4254 (2004). [CrossRef] [PubMed]

18.

J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94, 221–229 (2008). [CrossRef]

19.

M. Bhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B 20, 554–559 (2003). [CrossRef]

OCIS Codes
(260.2110) Physical optics : Electromagnetic optics
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence

ToC Category:
Spectroscopy

History
Original Manuscript: February 9, 2011
Revised Manuscript: February 10, 2011
Manuscript Accepted: March 24, 2011
Published: April 12, 2011

Virtual Issues
Vol. 6, Iss. 5 Virtual Journal for Biomedical Optics

Citation
Jörg Enderlein, Ingo Gregor, and Thomas Ruckstuhl, "Imaging properties of supercritical angle fluorescence optics," Opt. Express 19, 8011-8018 (2011)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-19-9-8011


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References

  1. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. Roy. Soc. London A 253, 358–379 (1959). [CrossRef]
  2. E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. Roy. Soc. London A 253349–357 (1959). [CrossRef]
  3. M. Born and E. Wolf, Principles of Optics , 6th ed. (Pergamon Press, 1987).
  4. T. Ruckstuhl and S. Seeger, “Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens,” Appl. Opt. 42, 3277–3283 (2003). [CrossRef] [PubMed]
  5. S. M. Mansfield and G. S. Kino, “Solid immersion microscope,” Appl. Phys. Lett. 57, 2615–2616 (1990). [CrossRef]
  6. P. Török, P. R. Munro, and E. E. Kriezis, “Rigorous near- to far-field transformation for vectorial diffraction calculations and its numerical implementation,” J. Opt. Soc. Am. A 23, 713–722 (2006). [CrossRef]
  7. M. Foreman, R. Matthew, S. Sherif, P. R. T. Munro, and P. Török, “Inversion of the Debye–Wolf diffraction integral using an eigenfunction representation of the electric fields in the focal region,” Opt. Express 16, 4901–4917 (2008). [CrossRef] [PubMed]
  8. P. Török, P. D. Higdon, and T. Wilson, “On the general properties of polarised light conventional and confocal microscopes,” Opt. Commun. 148, 300–315 (1998). [CrossRef]
  9. M. Leutenegger, R. Rao, R. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express 14, 11277–11291 (2006). [CrossRef] [PubMed]
  10. M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16, 8519–8531 (2008). [CrossRef] [PubMed]
  11. W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface II. Radiation patterns of perpendicular oriented dipoles,” J. Opt. Soc. Am. 67, 1615–1619 (1977). [CrossRef]
  12. W. Lukosz, “Light emission by magnetic and electric dipoles close to a plane interface III. Radiation patterns of dipoles with arbitrary orientation,” J. Opt. Soc. Am. 69, 1495–1503 (1979). [CrossRef]
  13. T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72, 2117–2123 (2000). [CrossRef] [PubMed]
  14. J. Enderlein and T. Ruckstuhl, “The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection,” Opt. Express 13, 8855–8865 (2005). [CrossRef] [PubMed]
  15. E. M. Lifshitz, L. D. Landau, and L. P. Pitaevskii, Electrodynamics of Continuous Media: 8 (Course of Theoretical Physics) (Butterworth Heinemann, 1984), Chap. X.
  16. F. Goos and H. Hänchen, “Ein neuer und fundamentaler Versuch zur Totalreflexion,” Ann. Physik 436, 333–346 (1947). [CrossRef]
  17. T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12, 4246–4254 (2004). [CrossRef] [PubMed]
  18. J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94, 221–229 (2008). [CrossRef]
  19. M. Bhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” J. Opt. Soc. Am. B 20, 554–559 (2003). [CrossRef]

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