Abstract

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.

© 2008 Optical Society of America

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  1. S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994).
    [CrossRef] [PubMed]
  2. T. A. Klar and S. W. Hell, Opt. Lett. 24, 954 (1999).
    [CrossRef]
  3. G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lurmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
    [CrossRef] [PubMed]
  4. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, Nature 440, 935 (2006).
    [CrossRef] [PubMed]
  5. B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, J. Cell Biol. 174, 153 (2006).
    [CrossRef] [PubMed]
  6. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 97, 8206 (2000).
    [CrossRef] [PubMed]
  7. V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, Appl. Phys. Lett. 82, 3125 (2003).
    [CrossRef]
  8. C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, J. Phys. D 37, 3296 (2004).
    [CrossRef]
  9. M. A. A. Neil, T. Wilson, and R. Juskaitis, J. Microsc. 197, 219 (2000).
    [CrossRef] [PubMed]
  10. P. Torok and P. R. T. Munro, Opt. Express 12, 3605 (2004).
    [CrossRef] [PubMed]
  11. B. R. Boruah and M. A. A. Neil, Opt. Express 14, 10377 (2006).
    [CrossRef] [PubMed]
  12. A. Kudlinski, A. K. George, J. C. Knight, J. C. Travers, A. B. Rulkov, S. V. Popov, and J. R. Taylor, Opt. Express 14, 5715 (2006).
    [CrossRef] [PubMed]

2006 (5)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lurmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, Nature 440, 935 (2006).
[CrossRef] [PubMed]

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, J. Cell Biol. 174, 153 (2006).
[CrossRef] [PubMed]

A. Kudlinski, A. K. George, J. C. Knight, J. C. Travers, A. B. Rulkov, S. V. Popov, and J. R. Taylor, Opt. Express 14, 5715 (2006).
[CrossRef] [PubMed]

B. R. Boruah and M. A. A. Neil, Opt. Express 14, 10377 (2006).
[CrossRef] [PubMed]

2004 (2)

P. Torok and P. R. T. Munro, Opt. Express 12, 3605 (2004).
[CrossRef] [PubMed]

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, J. Phys. D 37, 3296 (2004).
[CrossRef]

2003 (1)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, Appl. Phys. Lett. 82, 3125 (2003).
[CrossRef]

2000 (2)

M. A. A. Neil, T. Wilson, and R. Juskaitis, J. Microsc. 197, 219 (2000).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 97, 8206 (2000).
[CrossRef] [PubMed]

1999 (1)

1994 (1)

Appl. Phys. Lett. (1)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, Appl. Phys. Lett. 82, 3125 (2003).
[CrossRef]

J. Cell Biol. (1)

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, J. Cell Biol. 174, 153 (2006).
[CrossRef] [PubMed]

J. Microsc. (1)

M. A. A. Neil, T. Wilson, and R. Juskaitis, J. Microsc. 197, 219 (2000).
[CrossRef] [PubMed]

J. Phys. D (1)

C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Onfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, J. Phys. D 37, 3296 (2004).
[CrossRef]

Nature (1)

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, Nature 440, 935 (2006).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

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

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lurmann, R. Jahn, C. Eggeling, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 103, 11440 (2006).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, Proc. Natl. Acad. Sci. U.S.A. 97, 8206 (2000).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

STED microscopy setup: FR, Faraday rotator; λ 2 , achromatic half-wave plate; λ 4 , achromatic quarter-wave plate; PBS, polarizing beam splitter; GB, SF57 Glass block; RRC, retroreflector corner cube; MOF, microstructured optical fiber; SMF, single mode fiber; F1, bandpass filter ( 628 40 nm ) ; F2, combination of short-pass filter ( 750 nm ) and bandpass filter ( 692 40 nm ) ; SLM, spatial light modulator; DC, (infrared port) dichroic; BS, 30 70 reflection/transmission beam splitter; XY, x y galvanometer scanner; Z, galvanometer driven specimen stage; PMT, photomultiplier (TCSPC detector); PH1, pinhole; PH2, confocal pinhole. Inset, blazed holograms written to the SLM to produce the STED beams in the + 1 diffracted order: type I, helical phase distribution, type II, π phase-shifted central disk.

Fig. 2
Fig. 2

PSFs of STED beams measured with 200 nm gold beads. (a), (b) Type I hologram and (c) type II hologram, with aberrations corrected. (d) Line profile through the middle of the doughnut (a) along the y direction.

Fig. 3
Fig. 3

(a), (b) Lateral and (d), (e) axial fluorescence images of 200 nm beads with (a), (d) confocal acquisition; (b), (e) acquisition in STED mode [(b) with type I hologram, (e) with type II hologram]. (c) Normalized fluorescence intensity line profiles of lateral images and (f) axial images with FWHM specified for corresponding curves.

Fig. 4
Fig. 4

Intensity merged fluorescence lifetime images ( x y plane) obtained from the same data set as shown in Fig. 3, recorded in (a) confocal mode and (b) STED mode with the doughnut-shaped STED beam. (c) Spatially integrated fluorescence decay curves obtained from confocal and STED images.

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