Advances in the automated detection of metaphase chromosomes labeled with fluorescence dyes.

Applications of fluorescence in situ hybridization (FISH) for translocation studies and biological dosimetry would benefit substantially from reliable and efficient automatic detection of metaphase chromosomes labeled with fluorescent dyes. We replicated and evaluated a fluorescence metaphase finder previously developed at the Medical Research Council (MRC), Human Genetics Unit (Scotland) and at Lawrence Berkeley Laboratory (LBL; California). The MRC/LBL system seemed to detect nearly all of the metaphases on the test slides, but it presented an unacceptable number of false positives (about five false positives per one true positive). Furthermore, we determined that the system actually overcalled true detections by counting certain metaphase spreads twice (duplicates). Through modifications of the MRC/LBL system, we developed the Lawrence Livermore National Laboratory (LLNL) system, which minimizes the detection of duplicates, incorporates new detection features, uses a binary decision tree (BDT) for classification, and provides functionalities to improve scanning accuracy and improve the post-detection review. To test the new system, DAPI-stained preparations of metaphase chromosomes from blood lymphocytes of four unrelated donors were placed on slides in drops ranging from 7 mm to 20 mm in diameter. Drops contained between 5 and 200 scorable metaphases each. The LLNL system achieved approximately 90% detection of non-duplicated metaphases as verified by an expert cytogeneticist, with typically less than one false positive per every one true positive detected.

The effect of nonablative laser energy on the ultrastructure of joint capsular collagen.

This study was designed to evaluate the effect of laser energy at nonablative levels on the ultrastructure of joint capsular collagen. The femoropatellar joint capsules of six mature New Zealand white rabbits were harvested immediately after death. Specimens were divided into three treatment groups (5, 10, and 15 watts) and one control group. Laser energy was applied using a holmium: YAG laser. Transmission electron microscopy showed significant ultrastructural alterations in collagenous architecture for all laser treatment groups, with increased fibril cross-sectional diameter for each of the treated groups. The fibrils began to lose their distinct edges and their periodical cross-striations at subsequently higher energy densities. A morphometric analysis showed that each subsequently higher laser energy caused a significant increase in collagen fibril diameter. Ultrastructural alteration of collagen fibril architecture caused by the thermal effect of laser energy is probably the dominant mechanism of laser-induced tissue shrinkage.

Semiautomated DNA probe mapping using digital imaging microscopy: I. System development.

Algorithms have been developed to help automate the mapping of DNA sequences along metaphase chromosomes using fluorescence in situ hybridization (FISH). Custom algorithms computationally define chromosome boundaries and compute chromosomal medial axes. A dynamic regional thresholding (DRT) algorithm is described that allows reliable detection of hybridization domains, even when they differ substantially in size and intensity. Chromosomal locations are calculated by determining the fractional location of each hybridization probe along the medial axis of a metaphase chromosome relative to the short arm (FLpter). These algorithms were tested on simulated data and by analysis of the location of probes that had been previously mapped by other techniques. These algorithms allow probes to be mapped rapidly along human chromosomes with a precision of 2-3 Mb.