Increased resolution of in situ hybridization signal by electron microscopy: A comparison with fluorescence microscopy

Cytogenetic studies by in situ hybridization (ISH) have proven to be valuab le for gene mapping on banded chromosomes when combined with fluorescence m icroscopy (FISH). However, even under the best conditions, FISH technology has a resolving power inherent to light of just 0.2 m. Its utilization is f urther limited by the diffusion of light coming from the fluorescent signal which covers an area considerably larger than the target DNA sequence. The development of new ISH protocols applied to electron microscopy (EMISH) sh ould increase the resolution for cytogenetic mapping and fine chromosomal s tructure studies. Despite these advances, few attempts have been made which exploit this increased resolution. Here we present a detailed analysis of ISH signals obtained by fluorescence and electron microscopy methodologies to demonstrate and define the higher sensitivity obtainable by electron mic roscopy. This comparative study was conducted with probes of different orig ins: telomeric, classical satellite, alpha satellite, and single-copy DNA s equences, which provide a good reference point for later studies. We were a lso able to map a 200-bp cDNA probe by EMISH.This study assesses the nature of the resolution and the better definition of the EMISH signal, which con firms the greater resolution of electron microscopyas compared with that ac hieved with light microscopy. It also indicates that better delineation of two closely linked sequences is achieved at the electron microscopy level.