SOURCES FOR ILLUMINATION: Single-mode fibers deliver beneficial illumination
The beauty of a fiber-optic laser delivery system is the flexibility it offers the instrument designer when routing the laser beam.
The beauty of a fiber-optic laser delivery system is the flexibility it offers the instrument designer when routing the laser beam. It allows the laser source to be separated from the instrument front end and removes the need for traditionally bulky optomechanics. As a result, it offers a smaller, neater instrument design in, for instance, DNA sequencing, protein crystallography, and fluorescence microscopy (see Fig. 1).
FIGURE 1. Fiber-optic laser delivery in instrumentation can provide light that is almost Gaussian in profile, can function as a cold light source, and can dramatically reduce jitter.
When used with a diode laser, fiber-optic delivery actually improves the quality of the beam by suppressing the higher-order transverse modes and transmitting a single traverse mode of light that is almost Gaussian in profile, removing the need for additional filters.
When used with a gas laser, such as a helium-neon or argon-ion laser, fiber-optic delivery protects the instrument from the heat and vibration generated by the laser. The single-mode fiber functions as a cold light source: heat and vibration at the input are not transmitted to the output.
All types of lasers exhibit transverse jitter, or beam-pointing instability, caused by thermal and optomechanical instabilities in the laser cavity-typically in the region of 30 µrad/°C. Use of a single-mode fiber for delivery dramatically reduces this instability-the best designs offer a beam-pointing stability of better than 1 µrad/°C regardless of laser technology.
Dispelling the myths
The core diameter of a single-mode fiber operating within the visible wavelength range is typically about 3 to 5 µm. Because of this, the fiber system is sometimes thought to be inefficient, difficult to use, and potentially unstable. But, in fact, significant technology developments over the last 15 years have overcome these objections. Careful design of the input optics to mode-match the laser-beam parameter to the fiber achieves excellent results-typically 70% coupling efficiency or better (see Fig. 2).
FIGURE 2. Optimal design of the fiber input coupler in an illumination application using a 488 nm laser can produce repeatable high coupling efficiencies (distribution shown for 1000 units of the same design).
The latest fiber-optic delivery systems are exceptionally easy to use and highly repeatable. The Point Source system, for example, takes about two minutes to align and suffers less than 0.5% variation in coupling efficiency over 100 repeat insertions. This efficiency has been achieved through a combination of detailed kinematic design of the laser-to-fiber interface and submicron factory optical alignment that guarantees beam position and beam angle of less than 100 µm and 200 µrad, respectively (see Fig. 3).
A further objection to single-mode fiber is that, because it has a circular cross section, it degrades the polarization of the laser because the stress-optic coefficient of the glass induces birefringence when the fiber is bent or coiled. The outcome is two modes of propagation, which recombine with an unpredictable phase relationship at the output, resulting in polarization drifting and fading. Modern systems avoid this problem with the use of polarization-preserving fiber, which is not optically symmetrical and has strong internal birefringence caused by stress-applying sectors. The internal birefringence is significantly higher than normal bend-induced levels; therefore, as long as the laser is correctly aligned to either of the two birefringent axes of the fiber, polarization is preserved.
Point Source has worked with many OEM customers to integrate single-mode-fiber technology solutions into specialist microscopy instrumentation. This technology is now used in more than 50% of all confocal microscopes worldwide, and we are working with instrumentation builders involved in total-internal-reflection-fluorescence (TIRF) microscopy and related areas such as fluorescence recovery after photobleaching (FRAP). The unique illumination benefits of fiber-optic beam delivery are also achieving significant performance benefits in specialized measurement instrumentation for DNA sequencing and protein crystallography.
The speed of development in DNA-sequencing technology is astonishing and has facilitated the first-ever large-scale biological project. The Human Genome and related projects spanned many continents in a cooperative effort to generate the DNA sequences of many genomes. The focus is now shifting to resequencing in an immense effort to establish a link between genotypic variation and phenotype. Next-generation sequencing technologies will make it even cheaper and quicker to sequence a genome, allowing complete genome sequences to be determined from many different individuals of the same species. These technologies will provide a better understanding of aspects of human genetic diversity.
To make sequencing a viable technology, instruments have been developed that can run more than one sample at a time. These new platforms are based on parallel sequencing of millions of DNA fragments. High-sensitivity fluorescence detection is achieved using laser excitation and total-internal-reflection optics. Laser lifetime-even of the latest solid-state sources-is still a consideration in the design of instruments, to minimize downtime and cost of ownership. Thus, the critical optical alignment of the laser beam to the high-density microarrays is a significant overhead consideration during manufacture and in field replacement.
Fiber-optic beam-delivery systems used in the design of DNA sequencing systems eliminate the need for expensive line-of-sight optics. Remote siting of the laser head and power supply decouples sources of heat and vibration from the instrument and allows a smaller footprint for the instrument head. Optomechanical alignment considerations of the laser beam to the application are simplified and no optical realignment within the instrument head is required during laser replacement, resulting in minimal downtime.
The low beam-pointing error inherent in the single-mode fiber solution also reduces possible fluctuations in the signal-to-noise ratio from Rayleigh and Raman scattering for nonconfocal detection architectures (see Fig. 4).
Macromolecular crystallography is a powerful tool in revealing the structures and interactions of protein molecules. It provides valuable information that can be used to develop effective pharmaceutical compounds more rapidly. Recent advances have established protein crystallography as the lead instrument in structure-based drug design. However, until now, the actual crystal growth has been the largest bottleneck in this area of proteomics.
In the dynamically controlled protein-crystal growth (DCPCG) experiment conducted onboard NASA Space Shuttle mission STS-95 in 1998, fiber-coupled laser diodes were used in the study of protein-crystal growth in space (see Fig. 5). When detrimental gravitational effects such as convection and sedimentation are eliminated, protein-crystal growing conditions are optimized. Ultimately, this can yield larger and more perfect crystals, achieving crystallization levels unobtainable on Earth.
The laser light-scattering subsystem used in the experiment detects an aggregation event earlier than standard video-analysis methods, thus enabling more precise control and optimization of the crystal growth process.
Specifically, the onset of aggregation leads to an increase in scatter of a collimated laser beam directed through the sample cell. This scatter is detected at 90° by a photodiode shielded by a small pinhole aperture. There is a separate laser light-scattering system-each with its own laser-for each of the 10 growth-cell blocks in the experiment, as well as for the control cell. Because the system activation is determined by scattered light, if the beam moves relative to the detector pinhole, the change in background scatter signal could be misinterpreted as the onset of aggregation.
Apart from obvious benefits brought by diode-based laser systems, such as small size and low power consumption, the key to the success of the crystallography system is the low beam-pointing error enabled by fiber coupling of the laser diode. The stability of direction and point in space means confident detection of the onset of crystal aggregation. The angular pointing error of the beam is less than 1 µrad/°C. The experiment was housed in a thermally controlled environment, with the fiber-optic cables providing the ability to locate the lasers remotely and therefore removing any possibility of thermal perturbation to the crystallization experiments.
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