Abstract

STED microscopes are commonly built using separate optical paths for the excitation and the STED beam. As a result, the beams must be co-aligned and can be subject to mechanical drift. Here, we present a single-path STED microscope whose beams are aligned by design and hence is insensitive to mechanical drift. The design of a phase plate is described which selectively modulates the STED beam but leaves the excitation beam unaffected. The performance of the single-beam setup is on par with previous dual-beam designs.

© 2009 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [CrossRef] [PubMed]
  2. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
    [CrossRef] [PubMed]
  3. V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903–143904 (2005).
    [CrossRef] [PubMed]
  4. G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
    [CrossRef] [PubMed]
  5. S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
    [CrossRef] [PubMed]
  6. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
    [CrossRef] [PubMed]
  7. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
    [CrossRef] [PubMed]
  8. D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
    [CrossRef] [PubMed]
  9. H.-Y. Tsai, H. I. Smith, and R. Menon, “Reduction of focal-spot size using dichromats in absorbance modulation,” Opt. Lett. 33(24), 2916–2918 (2008).
    [CrossRef] [PubMed]
  10. R. Menon, P. Rogge, and H.-Y. Tsai, “Design of diffractive lenses that generate optical nulls without phase singularities,” J. Opt. Soc. Am. A 26(2), 297–304 (2009).
    [CrossRef]
  11. L. Kastrup, and V. Westphal, “Wavelength or polarisation sensitive optical assembly and use thereof,” German Patent DE102007025688A1, 2007/06/01.
  12. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
    [CrossRef] [PubMed]
  13. V. V. Kotlyar, A. A. Almazov, S. N. Khonina, V. A. Soifer, H. Elfstrom, and J. Turunen, “Generation of phase singularity through diffracting a plane or Gaussian beam by a spiral phase plate,” J. Opt. Soc. Am. A 22(5), 849–861 (2005).
    [CrossRef]
  14. G.-H. Kim, J.-H. Jeon, K.-H. Ko, H.-J. Moon, J.-H. Lee, and J.-S. Chang, “Optical vortices produced with a nonspiral phase plate,” Appl. Opt. 36(33), 8614–8621 (1997).
    [CrossRef]
  15. V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
    [CrossRef]
  16. X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
    [CrossRef]
  17. I. Gregor and J. Enderlein, “Focusing astigmatic Gaussian beams through optical systems with a high numerical aperture,” Opt. Lett. 30(19), 2527–2529 (2005).
    [CrossRef] [PubMed]

2009 (2)

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
[CrossRef] [PubMed]

R. Menon, P. Rogge, and H.-Y. Tsai, “Design of diffractive lenses that generate optical nulls without phase singularities,” J. Opt. Soc. Am. A 26(2), 297–304 (2009).
[CrossRef]

2008 (3)

2007 (3)

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

2006 (1)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

2005 (3)

2002 (1)

V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
[CrossRef]

2000 (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

1997 (1)

1994 (1)

Ahluwalia, B. P. S.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Alekseev, A. N.

V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
[CrossRef]

Almazov, A. A.

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Bu, J.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Burge, R. E.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Chang, J.-S.

Cheong, W. C.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Donnert, G.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Eggeling, C.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Egner, A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Elfstrom, H.

Enderlein, J.

Gregor, I.

Harke, B.

Hell, S. W.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903–143904 (2005).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[CrossRef] [PubMed]

Izdebskaya, Y. V.

V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
[CrossRef]

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Jakobs, S.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Jeon, J.-H.

Kastrup, L.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

Keller, J.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Khonina, S. N.

Kim, G.-H.

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Ko, K.-H.

Kotlyar, V. V.

Lee, J.-H.

Lin, J.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Medda, R.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Menon, R.

Moon, H.-J.

Rittweger, E.

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Rogge, P.

Schönle, A.

Shvedov, V. G.

V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
[CrossRef]

Smith, H. I.

Soifer, V. A.

Tao, S. H.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Tsai, H.-Y.

Turunen, J.

Ullal, C. K.

Volyar, A. V.

V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
[CrossRef]

Westphal, V.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903–143904 (2005).
[CrossRef] [PubMed]

Wichmann, J.

Wildanger, D.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

Yuan, X.-C.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Zhang, L. S.

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

Appl. Opt. (1)

Appl. Phys. B (1)

X.-C. Yuan, B. P. S. Ahluwalia, S. H. Tao, W. C. Cheong, L. S. Zhang, J. Lin, J. Bu, and R. E. Burge, “Wavelength-scalable micro-fabricated wedge for generation of optical vortex beam in optical manipulation,” Appl. Phys. B 86(2), 209–213 (2007).
[CrossRef]

J. Microsc. (1)

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. (2009), doi:.
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (2)

Opt. Express (3)

Opt. Lett. (3)

Phys. Rev. Lett. (1)

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903–143904 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (2)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Science (1)

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

Tech. Phys. Lett. (1)

V. G. Shvedov, Y. V. Izdebskaya, A. N. Alekseev, and A. V. Volyar, “The formation of optical vortices in the course of light diffraction on a dielectric wedge,” Tech. Phys. Lett. 28(3), 256–259 (2002).
[CrossRef]

Other (1)

L. Kastrup, and V. Westphal, “Wavelength or polarisation sensitive optical assembly and use thereof,” German Patent DE102007025688A1, 2007/06/01.

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (8)

Fig. 1
Fig. 1

Setup of a single-beam path STED microscope. The excitation and the STED beams are combined with a dichroic beamsplitter (DC) and are jointly coupled into a polarization preserving single-mode optical fiber (PMF). The combined beams are collimated with a lens (L), travel through a phase filter (PF) and a quarter-wave plate (QWP) and are focused by the objective lens. The collected fluorescence is separated from the laser beams with a second dichroic (DC), is focused through a pinhole (PH) and detected with an avalanche photodiode (APD). When used with a broadband or a multiline laser source the beam combination before the fiber coupling may become obsolete.

Fig. 2
Fig. 2

Concept of a wavelength-selective phase filter based on dispersion. The refractive index of both materials A and B is matched at the excitation wavelength λexc but is notably different at λSTED. As a result, the transmitted wavefront only of the STED beam is modified. The phase shift ϕ depends on the thickness d, the index difference Δn and the wavelength λSTED.

Fig. 3
Fig. 3

Dispersion curves of the optical glasses N-KZFS4 (blue) and N-SK4 (green) at 21 °C. The refractive indices are identical at a wavelength of 630.5 nm but differ by Δn = 1.24×10−3 at the STED design wavelength (750 nm). This combination is suitable for use with e. g. the ATTO 633 fluorescent dye.

Fig. 4
Fig. 4

Comparison of a continuous vortex phase filter (a), a discretized vortex (a, inset) and a dual ramp phase filter (b). The plots show intensity profiles along the x and y axes of the PSFs generated by the vortex (gray) and the dual ramp (red, dotted). All three phase filters generate similar PSFs; only the intensity along the rim of the torus is slightly modulated in the case of the dual ramp filter. The linear ramp filter is significantly easier to manufacture from two solid materials.

Fig. 5
Fig. 5

Design of a dual-wedge phase plate. (a) Two identical glass wedges are attached to each other with opposite slopes (left) and are cemented together (right). (b) The wedge angle α is chosen such that the phase gradient runs from 0 to π in the front part of the phase filter and from π to 2π in the back part.

Fig. 6
Fig. 6

(a) Point-spread functions due to phase plate #6 from Tab. 1. Lateral (xy) and axial (yz) sections (top) through the excitation (green) and the STED PSF (red) and corresponding intensity profiles (bottom) along the lines indicated in the images above. For the z plot, the intensity was integrated along y. (b) Photo of the phase plate #3 (top) and xy (middle) and yz sections (bottom) of the PSF generated by this filter. Scale bars: 200 nm.

Fig. 7
Fig. 7

Thermal drift measurements. (a) The reference beam (blue) was initially co-aligned with the combined excitation (green) and STED beams (red). (b) After heating the setup to 45 °C the excitation and STED beams remain aligned with respect to each other while the reference beam is displaced by 140 nm. (c) After the setup has been cooled to 6 °C the reference beam is displaced by 233 nm. However, the excitation and STED beams of the STED microscope remain largely co-aligned, thus proving the ruggedness of the self-aligned single beam STED setup with respect to drastic changes of environmental temperature.

Fig. 8
Fig. 8

Images of the microtubular network of PtK2 cells imaged with a confocal (a) and a single-beam STED microscope (b). The confocal image shows much less detail than the STED image which clearly discerns single fibers. Even an inhomogeneous staining is revealed in the STED image as evidenced by the dot-like structures (c). The smallest distance between well-resolved features is 80 nm (d). Scale bar: 1 µm.

Tables (1)

Tables Icon

Tab. 1. Glass combinations for the fabrication of wavelength-selective phase plates to be used in STED microscopy with various dyes. Materials were chosen such that n Aexc) = n Bexc) but ΔnSTED) > 5×10−4. α is the wedge angle calculated for a beam diameter of d = 5.6 mm; the design temperature is 21.0 °C. The phase plates shown in bold were fabricated and experimentally tested.

Equations (5)

Equations on this page are rendered with MathJax. Learn more.

dλ/(2NA1+I/Is).
φ=2πdΔnλSTED.
n2(λ)=1+B1λ2λ2C1+B2λ2λ2C2+B3λ2λ2C3.
h=λφ2πΔn
α=tan1(h2d),

Metrics