We propose to develop new techniques for specimen preparation for the assessment of chromosome breakage in human cells by fluorescent in situ hybridization. The methods are applicable to automated image analysis at high resolution so that very low levels of chromosome damage will be detectable. We will develop new methods for depositing lymphocytes on slides at high density, using centrifugal cytology. Methods are proposed to-decrease the error rate for FISH detection of target sequences, so that infrequent breakage events such as translocation can be quantitated. A new technique for fluorescent counterstaining of nuclei is to be developed, employing narrow-band emission reporters such as europium chelates and cyanine dyes. This project will contribute to our goal of labeling of chromosomes 1,2,3 of the human genome with three-color fluorescence painting probes to detect chromosome damage of these three chromosomes with automated instrumentation.
