The excision of introns from nascent eukaryotic transcripts is catalyzed by the 
spliceosome, a highly complex and dynamic macromolecular machine composed of RNA 
and protein. Because of its complexity, biochemical analysis of the spliceosome 
has been previously limited to bulk assays in largely unfractionated cell 
extracts. We now report development of methodologies for studying the splicing 
of isolated single pre-mRNA molecules in real time. In this system, a 
fluorescently tagged pre-mRNA is tethered to a glass surface via its 3'-end. 
Splicing can be observed in Saccharomyces cerevisiae whole cell extract by 
monitoring loss of intron-specific fluorescence with a multi-wavelength total 
internal reflection fluorescence (TIRF) microscope. To prolong fluorophore 
lifetime, two enzyme-based O2 scavenging systems compatible with splicing were 
also developed. This work provides a powerful new approach for elucidating the 
mechanisms of spliceosome function and demonstrates the feasibility of utilizing 
TIRF microscopy for biochemical studies of single molecules in highly complex 
environments.