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31 Aug 2007
Many biomedical applications are now benefiting from the unique optical properties of supercontinuum fibre lasers. John Clowes of Fianium tracks recent developments.
Supercontinuum fibre laser sources have advanced significantly in the last two years to a stage where they are now a mature technology that is being used within a wide range of applications. In the bio-medical sector, flow cytometry, confocal microscopy and optical coherence tomography (OCT) are all exploiting the unique properties of this white-light source.
Back in 1999–2000, the first groups to generate a supercontinuum used highly nonlinear optical fibres such as photonic crystal fibres (PCFs) and tapered fibres to produce a continuous spectrum from 400 nm through to almost 2 µm.
These early demonstrations of fibre supercontinuum generation involved launching high-intensity femtosecond pulses from a Ti:sapphire laser or nanosecond pulses from a Q-switched microchip laser across free-space into the fibre. While providing a useful white-light laser source for use in the laboratory, the end-launching of high-intensity pulses into waveguides as small as 3 µm in diameter had inherent problems with instability and reliability, making it unsuitable for commercial applications.
In 2005, we introduced a super-continuum fibre laser covering the 450 nm to 2 µm spectrum that delivered an average power of 2 W. Based on an all-fibre architecture, the product incorporated a picosecond mode-locked fibre laser, high-power fibre amplifier and highly nonlinear optical fibre. Relying on fusion splicing of optical fibres rather than end-launching into PCF, this fibre supercontinuum source did not suffer from the reliability and stability issues associated with solid-state laser generation of a supercontinuum.
The supercontinuum fibre laser has been a commercial product for almost three years and has undergone many improvements. We believe that our SC450-2 was pushing the limit in terms of wavelength range and output power in 2005. Improvements over the past two years have focused on enhanced brightness and generation of shorter wavelengths, essential for many imaging applications. Table 1 compares the specifications of our sources today with those of 2005.
Perhaps the most important improvement, however, has been in the reliability of the products. This is thanks to increased use in the field within applications as diverse as OCT to fluorescence imaging and as a versatile high-brightness illumination source within numerous areas of fundamental research.
Fluorescence imaging
Flow cytometers (or fluorescence-activated cell sorters) are critical tools for biomedical research. These complex instruments measure the properties of individual cells, often by detecting fluorescent molecules (fluorophores) attached to their surface. These fluorescent molecules act as probes for analysing the identity of cells, studying the immune system, identifying cancer cells and diagnosing disease.
Flow cytometers rely almost exclusively on lasers to excite fluorophores. While a laser's coherence and output power makes it ideal for illuminating individual cells, its discrete wavelength limits the types of fluorescent probes that can be analysed.
Although solid-state laser technology has increased the variety of discrete laser wavelengths available, there are still significant gaps in the excitation capabilities that put limitations on the fluorescent probes used for biomedical analysis.
Confocal fluorescence microscopy has similar laser source requirements to those of flow cytometry and is one of the most powerful techniques for probing a wide range of phenomena within biological sciences.
Almost all commercially available confocal microscope systems use a combination of lasers to excite fluorescence at a few discrete wavelengths within the visible region of the spectrum. As with flow cytometry, only a small number of excitation wavelengths are available by using fixed wavelength lasers. Many fluorophores are therefore unusable because of this limitation.
A recent development, which further aids fluorophore excitation, is the combination of multichannel acousto-optic tunable filters (AOTFs) with the supercontinuum fibre laser. An SC400 supercontinuum laser in conjunction with an eight-channel AOTF delivers up to eight laser lines within the visible region of the spectrum. Each line is individually tunable across the entire spectrum and is emitted as a diffraction-limited, collinear beam.
The supercontinuum-AOTF combination provides the required flexibility for optimal excitation and detection of a wide range of fluorophores within flow cytometers or fluorescence confocal microscopes. The laser can be tuned precisely to the excitation peak of the fluorescent probes needed for analysis.
A supercontinuum also improves performance and is cost-effective. For example, a high-specification flow cytometer or confocal microscope might include up to eight lasers, ranging from an expensive frequency tripled Nd:YVO4 laser at 355 nm to multiple DPSS sources at 488, 532 and 561 nm; diode lasers at 405 and 440 nm; and HeNe lines at 633 or 594 nm (figure 2).
With the exception of the ultraviolet (UV) source, a single SC400–AOTF can replace all of these sources, along with all beam combination and steering optics. As supercontinuum fibre laser technology continues to advance, it is only a matter of time before the spectrum is extended further down into the UV enabling improvements to the highest specification imaging systems.
While commercial vendors of confocal microscopes will soon incorporate supercontinuum fibre lasers within their imaging systems, it is often research groups who are first to investigate the technology. Clemens Kaminski and his team at the Laser Analytics Group in Cambridge, UK, have recently incorporated a supercontinuum fibre laser within an Olympus Fluoview microscope scanning unit to demonstrate 2D, 3D and live-cell imaging.
Figure 3 shows high-resolution 2D fluorescent confocal images of plants taken using a combined SC450–AOTF system. A Convallaria majalis (Lily of the Valley) specimen was stained with safranin and fast green dyes that have peak excitation wavelengths of 530 and 620 nm, respectively.
Scanning the excitation wavelength highlights different regions of the sample as the dyes have affinities to separate regions. Fast green stains the cellulosic cell walls present over the whole sample (620 and 640 nm images). The safranin dye stains lignin, present within the endodermis and xylem, highlighted within figure 3 at 540 to 560 nm excitation wavelengths.
Optical coherence tomography
OCT is an interferometric, non-invasive imaging technique offering millimetre penetration (up to 3 mm in tissue) with micrometre-scale axial resolution.
Superluminescent diodes used within conventional OCT systems typically suffer from limited bandwidths resulting in axial resolutions of 10–15 µm. Ultrahigh-resolution (UHR–OCT) systems typically use cost-extensive and complex femtosecond solid-state lasers and have demonstrated sub-micron axial resolution at 725 nm.
A supercontinuum fibre laser source achieves even higher spectral bandwidths, as well as increased optical power and tunability over the spectrum. Felix Spöler and colleagues from the Institute of Semiconductor Electronics at RWTH Aachen, Germany, have recently used the high brightness and broad bandwidth properties of a supercontinuum fibre laser to demonstrate UHR–OCT. Their approach was to filter two spectral bands centred on 840 and 1230 nm where each band had more than 100 mW of average power over a spectral width of approximately 200&thinp;nm.
Dual wavelength OCT at 840 and 1230 nm exploits the high axial resolution of the short wavelength illumination as well as the high penetration depths achievable at longer wavelengths. The team achieved a free-space resolution of 1.8 and 3.8 µm at 840 and 1230 nm, respectively, using this technique. Using both wavelengths simultaneously also enables frequency compounding of the OCT images at each wavelength resulting in reduced speckle.
The image contrast can also be enhanced by employing differential spectral imaging. Here, back-scattered light from the sample is analysed at each wavelength band and provides another level of information about the sample's structure. Figure 4 shows an in vivo differential spectral image of a human nail fold showing the various layers and blood vessels within the region. The difference in scattering intensity between the applied wavelength bands is encoded by the colour.
An even brighter future Huge improvements in supercontinuum laser technology have been made over the past two years, not least a three-fold increase in brightness and the development of sub-400 nm supercontinuum sources as off-the-shelf turnkey products. The technology and range of applications enabled by this unique source will no doubt continue to grow. Over the next two years we can expect to see UV supercontinuum sources and even further scaling in brightness.
• This article originally appeared in the September 2007 issue of Optics & Laser Europe magazine.
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