MICROSCOPY: Supercontinuum white-light source enhances confocal microscope

Confocal laser-scanning microscopy (CLSM), in which optics collect the scattered light from a sample-sensing focused laser spot, has become a standard investigation technique for the life sciences.

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Confocal laser-scanning microscopy (CLSM), in which optics collect the scattered light from a sample-sensing focused laser spot, has become a standard investigation technique for the life sciences. Biological objects are typically stained with appropriate fluorescing dyes. High-contrast and high-resolution scanning can be performed layer by layer (tomographically), producing 3-D representations of the sample. Diode lasers have partially replaced gas and frequency-doubled solid-state lasers for CLSM; however, diode lasers emit at fixed wavelengths, restricting the choice of dyes that show sufficient absorption at the laser wavelengths. Furthermore, to cover a broader spectral range, several lasers must be kept on hand.

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FIGURE 1. A confocal laser-scanning microscope is equipped with a supercontinuum white-light source for fluorescence excitation. Light from a 200-fs modelocked Ti:sapphire laser at a 76-MHz pulse rate with a center wavelength of 803 nm is coupled into a tapered fiber where the continuum is generated. Spectrally split at beamsplitter C into its near-IR (1300-700 nm) and visible (700-430 nm) parts, the radiation is either fed directly (near-IR) or acousto-optically wavelength-selected (visible spectrum; inset) to the scanning unit of the microscope. The fluorescence passes the AOBS undeflected.
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This situation is changing, now that a laser-like supercontinuum white-light source has been introduced that only requires a single short-pulse laser for excitation. A group of German researchers at the universities of Bonn and Leipzig have used a tapered fiber to transform 200-fs narrowband laser radiation into a continuum spectrum that spans a wide spectral range in the visible and near-IR spectral region, and applied it for CLSM.1

Boosting peak intensities

The tapered fiber was produced by pulling a standard single-mode 125-µm-diameter telecommunications fiber into a 2.1 µm waist about 90 mm long with tapered regions approximately 15 mm long on each end.2 The small core diameter leads to peak pulse intensities on the order of several hundred GW/cm2, with self-phase modulation and other nonlinear effects leading to spectral broadening. An average laser power of 650 mW yielded 280-mW output with a perfect TEM00 mode profile and a pulse duration of a few picoseconds.

An acousto-optical beamsplitter (AOBS) was used to single out a spectral band that would best excite the chosen dye (see Fig. 1). Spectral bands with wavelengths between 450 and 633 nm and bandwidths of a few nanometers or less could be selected.

The measured power of about 60 µW in the selected spectral band was sufficient for fluorescent excitation. The total IR portion of the supercontinuum spectrum produced a power of about 11.6 mW at the sample; a focal spot of less than 1‑µm diameter resulted in a peak power of 1.7 GW/cm2, sufficient for multiphoton excitation.

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FIGURE 2. A neuron-like cell fixed and stained for actin (red) and microtubules (green) was imaged by fluorescence excited by 488-nm and 543-nm selected from the supercontinuum. The scale bar is 10 µm. A two-photon-excitation fluorescence image of two cells was recorded using IR light (bottom). The scale bar is 20 µm.
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Using single-photon fluorescence, actin and microtubules of a neuron-like cell were imaged with diffraction-limited resolution; two-photon-excitation images using the entire IR portion of the supercontinuum source were also obtained (see Fig. 2).

The use of the white-light source in fluorescence confocal microscopy allows the wavelength for optimal excitation to be readily selected. In addition, by applying the IR part of the spectrum two-photon excitation is possible. Furthermore, continuous tuning offers the possibility of scanning samples by sensing the molecular absorption, without staining.

Uwe Brinkmann

REFERENCES

1. T. Betz et al., J. Biomedical Optics 10(5), 054009 (2005).

2. J. Teipel et al., Appl. Phys. B77, 245 (2003).

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