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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References

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App. Phys. B. (1)

G. McConnell and E. Riis, ???Ultra-short pulse compression using photonic crystal fibre,??? App. Phys. B. 78 557-563 (2004).
[CrossRef]

Handbook of Biological Confocal Microsco (1)

J.B. Pawley Handbook of Biological Confocal Microscopy, 2nd (Plenum Press, New York, 1995).
[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. 2 1051-1065 (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. 20 1887-1893 (2003).
[CrossRef]

Opt. Express (1)

Opt. Lett. (4)

Phys. Rev. Lett. (2)

A.V. Husakou and J. Hermann, ???Supercontinuum generation of higher-order solitons by fission in photonic crystal fibers,??? Phys. Rev. Lett. 87 203901-203904 (2001).
[CrossRef] [PubMed]

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. 90 113904-1-4 (2003).
[CrossRef]

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. 74 193-201 (2003).
[CrossRef]

Science (1)

W. Denk, J. Strickler and W.W. Webb, ???Two-photon laser scanning fluorescence microscopy,??? Science 10 73-75 (1990).
[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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