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Confocal laser scanning fluorescence microscopy with a visible continuum source

Gail McConnell

Open Access Open Access

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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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.

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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.

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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.

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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.

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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.

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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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