Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein-DNA complexes

Atomic force microscopy (AFM) and fluorescencemicroscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents forbothAFMtopography andfluorescenceimaging, facilitatesthecombinationofbothimagingtechniques,andwiththe addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes.We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA.