Abstract

Confocal laser scanning fluorescence microscopy is demonstrated using a photonic crystal fiber-based excitation source. A 38 cm-long section of photonic crystal fiber is pumped with femtosecond pulses from a Ti:sapphire laser, and the resultant visible continuum is selectively filtered to provide the peak excitation wavelengths required for a range of fluorescently labeled biological tissue.

© 2004 Optical Society of America

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  1. J.B. PawleyHandbook of Biological Confocal Microscopy, 2nd (Plenum Press, New York, 1995).
    [CrossRef]
  2. J.M. Girkin, A.I. Ferguson, D.L. Wokosin, and A.M. Gurney, “Confocal microscopy using an InGaN violet laser diode at 406 nm,” Opt. Express. 7336–341 (2000); http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336
    [CrossRef] [PubMed]
  3. D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
    [CrossRef]
  4. W. Denk, J. Strickler, and W.W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 1073–75 (1990).
    [CrossRef]
  5. D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
    [CrossRef]
  6. J.C. Knight, T.A. Birks, P. St. J. Russell, and D.M. Atkin, “All-silica single-mode optical fiber with photonic crystal claddin,g” Opt. Lett. 211547–1549 (1996).
    [CrossRef] [PubMed]
  7. T.A. Birks, J.C. Knight, and P.St. J. Russell, “Endlessly single-mode photonic crystal fiber,” Opt. Lett. 22961–963 (1997).
    [CrossRef] [PubMed]
  8. J..K. Ranka, R.S. Windeler, and A.J. Stentz, “Visible continuum generation in air-silica microstructure optical fibers with anomalous dispersion at 800 nm,” Opt. Lett. 2525–27 (2000).
    [CrossRef]
  9. J.K. Ranka, R.S. Windeler, and A.J. Stentz, “Optical properties of high-delta air silica microstructure optical fibers,” Opt. Lett. 25796–798 (2000).
    [CrossRef]
  10. A.V. Husakou and J. Hermann, “Supercontinuum generation of higher-order solitons by fission in photonic crystal fibers,” Phys. Rev. Lett. 87203901–203904 (2001).
    [CrossRef] [PubMed]
  11. K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
    [CrossRef]
  12. G. McConnell and E. Riis, “Ultra-short pulse compression using photonic crystal fibre,” App. Phys. B. 78557–563 (2004).
    [CrossRef]
  13. K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
    [CrossRef]

2004 (1)

G. McConnell and E. Riis, “Ultra-short pulse compression using photonic crystal fibre,” App. Phys. B. 78557–563 (2004).
[CrossRef]

2003 (3)

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
[CrossRef]

2001 (1)

A.V. Husakou and J. Hermann, “Supercontinuum generation of higher-order solitons by fission in photonic crystal fibers,” Phys. Rev. Lett. 87203901–203904 (2001).
[CrossRef] [PubMed]

2000 (3)

1997 (1)

1996 (2)

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

J.C. Knight, T.A. Birks, P. St. J. Russell, and D.M. Atkin, “All-silica single-mode optical fiber with photonic crystal claddin,g” Opt. Lett. 211547–1549 (1996).
[CrossRef] [PubMed]

1990 (1)

W. Denk, J. Strickler, and W.W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 1073–75 (1990).
[CrossRef]

Armstrong, D.

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

Atkin, D.M.

Birks, T.A.

Centonze, V.

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

Coen, S.

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

Corwin, K.L.

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

Denk, W.

W. Denk, J. Strickler, and W.W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 1073–75 (1990).
[CrossRef]

Diddams, S.A.

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

Dudley, J.M.

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

Eliceiri, K.W.

D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
[CrossRef]

Ferguson, A.I.

J.M. Girkin, A.I. Ferguson, D.L. Wokosin, and A.M. Gurney, “Confocal microscopy using an InGaN violet laser diode at 406 nm,” Opt. Express. 7336–341 (2000); http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336
[CrossRef] [PubMed]

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

Girkin, J.M.

J.M. Girkin, A.I. Ferguson, D.L. Wokosin, and A.M. Gurney, “Confocal microscopy using an InGaN violet laser diode at 406 nm,” Opt. Express. 7336–341 (2000); http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336
[CrossRef] [PubMed]

Gurney, A.M.

J.M. Girkin, A.I. Ferguson, D.L. Wokosin, and A.M. Gurney, “Confocal microscopy using an InGaN violet laser diode at 406 nm,” Opt. Express. 7336–341 (2000); http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336
[CrossRef] [PubMed]

Hermann, J.

A.V. Husakou and J. Hermann, “Supercontinuum generation of higher-order solitons by fission in photonic crystal fibers,” Phys. Rev. Lett. 87203901–203904 (2001).
[CrossRef] [PubMed]

Hilligsøe, K.M.

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

Husakou, A.V.

A.V. Husakou and J. Hermann, “Supercontinuum generation of higher-order solitons by fission in photonic crystal fibers,” Phys. Rev. Lett. 87203901–203904 (2001).
[CrossRef] [PubMed]

Keiding, S. R.

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

Knight, J.C.

Larsen, J. J.

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

McConnell, G.

G. McConnell and E. Riis, “Ultra-short pulse compression using photonic crystal fibre,” App. Phys. B. 78557–563 (2004).
[CrossRef]

Newbury, N.R.

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

Paulsen, H.N.

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

Pawley, J.B.

J.B. PawleyHandbook of Biological Confocal Microscopy, 2nd (Plenum Press, New York, 1995).
[CrossRef]

Ranka, J..K.

Ranka, J.K.

Riis, E.

G. McConnell and E. Riis, “Ultra-short pulse compression using photonic crystal fibre,” App. Phys. B. 78557–563 (2004).
[CrossRef]

Robertson, G.

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

Russell, P. St. J.

Russell, P.St. J.

Squirrell, J.M.

D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
[CrossRef]

Stentz, A.J.

Strickler, J.

W. Denk, J. Strickler, and W.W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 1073–75 (1990).
[CrossRef]

Thøgersen, J.

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

Webb, W.W.

W. Denk, J. Strickler, and W.W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 1073–75 (1990).
[CrossRef]

Weber, K.

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

White, J.G.

D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
[CrossRef]

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

Windeler, R.S.

Wokosin, D.L.

D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
[CrossRef]

J.M. Girkin, A.I. Ferguson, D.L. Wokosin, and A.M. Gurney, “Confocal microscopy using an InGaN violet laser diode at 406 nm,” Opt. Express. 7336–341 (2000); http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336
[CrossRef] [PubMed]

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

App. Phys. B. (1)

G. McConnell and E. Riis, “Ultra-short pulse compression using photonic crystal fibre,” App. Phys. B. 78557–563 (2004).
[CrossRef]

IEEE J. Sel. Top. Quantum Electron. (1)

D.L. Wokosin, V. Centonze, J.G. White, D. Armstrong, G. Robertson, and A.I. Ferguson, “All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging,” IEEE J. Sel. Top. Quantum Electron. 21051–1065 (1996).
[CrossRef]

J. Opt. Soc. Am. B. (1)

K.M. Hilligsøe, H.N. Paulsen, J. Thøgersen, S. R. Keiding, and J. J. Larsen, “Initial steps of supercontinuum generation in photonic crystal fibers,” J. Opt. Soc. Am. B. 201887–1893 (2003).
[CrossRef]

Opt. Express. (1)

J.M. Girkin, A.I. Ferguson, D.L. Wokosin, and A.M. Gurney, “Confocal microscopy using an InGaN violet laser diode at 406 nm,” Opt. Express. 7336–341 (2000); http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336
[CrossRef] [PubMed]

Opt. Lett. (4)

Phys. Rev. Lett. (2)

K.L. Corwin, N.R. Newbury, J.M. Dudley, S. Coen, S.A. Diddams, K. Weber, and R.S. Windeler, “Fundamental noise limitations to supercontinuum generation in microstructure fiber,” Phys. Rev. Lett. 90113904-1-4 (2003).
[CrossRef]

A.V. Husakou and J. Hermann, “Supercontinuum generation of higher-order solitons by fission in photonic crystal fibers,” Phys. Rev. Lett. 87203901–203904 (2001).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

D.L. Wokosin, J.M. Squirrell, K.W. Eliceiri, and J.G. White, “Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities,” Rev. Sci. Instrum. 74193–201 (2003).
[CrossRef]

Science (1)

W. Denk, J. Strickler, and W.W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 1073–75 (1990).
[CrossRef]

Other (1)

J.B. PawleyHandbook of Biological Confocal Microscopy, 2nd (Plenum Press, New York, 1995).
[CrossRef]

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Figures (6)

Fig. 1. (a).
Fig. 1. (a).

Experimental set-up. The output of a mode-locked Ti:Sapphire laser is sent through a Faraday Isolator (F.I.) and coupled into a 38 cm section of anomalously dispersive photonic crystal fiber (PCF). The resultant visible continuum is filtered through a high quality bandpass filter (BP) and subsequently attenuated using a neutral density (ND) filter.

Fig. 1. (b).
Fig. 1. (b).

The filtered visible continuum was entered into a scan-head coupled to an inverted microscope. A 40x/1.4 NA microscope objective lens was used to focus the radiation onto the fluorescently stained sample.

Fig. 2.
Fig. 2.

Sample unfiltered spectral visible continuum transmitted through the PCF at a measured average output power of 51 mW, plotted on a linear scale.

Fig. 3.
Fig. 3.

(a) and 3(b). Fluorescence (3(a)) and transmission (3(b)) images of guinea pig detrusor labeled with anti-PGP 9.5 and Alexa 488, obtained under confocal excitation using the 1.26 mW of radiation at 488±5 nm from the filtered visible continuum. The fluorescence image was obtained at a depth of 41 µm within the sample.

Fig. 4. (a).
Fig. 4. (a).

Superimposed fluorescence and transmission image of guinea pig smooth muscle cell containing fluo-4 under excitation at λ=488±5 nm. A dead cell that also exhibits a fluorescent signal is observable to below the smooth muscle cell.

Fig. 4. (b).
Fig. 4. (b).

Mean fluorescence signal intensity from control (unloaded) and fluo-4 loaded smooth muscle cells from guinea pig bladder (n=10 samples) under excitation at λ=488±5 nm.

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