Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH.

The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.

Molecular classification of cutaneous malignant melanoma by gene expression profiling.

The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.

Long-term stability of two diode-laser-pumped nonplanar ring lasers independently stabilized to two Fabry-Perot interferometers.

We have locked two 1064-nm diode-laser-pumped Nd:YAG lasers to two ultralow-expansion glass-ceramic Fabry-Perot interferometers by using the Pound-Drever discriminator technique. The interferometers have finesses of approximately 200,000 and 5-kHz linewidths and are housed in separate temperature-stabilized vacuum vessels. Allan variance measurements of the beat note between the two lasers are as low as 10(-14) for delay times between 0.5 and 2 s and increase with the time interval for times longer than 2 s, an improvement of 4 orders of magnitude over the free-running performance. Daily variations of the beat-note frequency are less than 1 MHz, which corresponds to a relative temperature change of approximately 100 mK between the interferometers.

Stabilization of laser beam alignment to an optical resonator by heterodyne detection of off-axis modes.

We demonstrate a method for real time alignment of a Gaussian beam to an optical resonator. While thefrequency of a source laser is stabilized to a fundamental cavity mode resonance, phase modulation sidebands are applied at the off-axis mode frequencies. Asymmetrical transmission of the sideband at the frequency of each off-axis mode produces amplitude modulated optical signals and indicates the extent of the misalignments. Phase sensitive detection of these optical signals provides the error signals which are minimized by a control system that steers the input beam. In this way, optimum coupling of an injected source beam can be maintained to the fundamental mode of the resonator. This active alignment technique has demonstrated a sensitivity to tilts of 0.1 nrad/ radicalHz and to lateral beam displacements of 0.08 nm/ radicalHz in the ~1-Hz-1-kHz frequency range. These values correspond to 2 parts in 10(7) radicalHz for both the far-field divergence angle and the beam waist size. Such performance is within a factor of 2 of the shot noise limitation of the error signal measurement for a detected power of 160 microW.