Experimental setup for the study of immobilized single nucleosomes using total internal reflection fluorescence

Abstract

An experimental setup for study of immobilized molecules and their complexes by fluorescence microscopy with sensitivity at a single fluorophore level was developed. The installation records fluorescence images of immobilized molecules in two spectral ranges simultaneously allowing analysis based on the Förster resonance energy transfer effect. The fluorescence excitation is caused by evanescent light wave formed by the total internal reflection effect. Registration of signal is conducted with a highly sensitive detection system that allows measurements with a temporal resolution of approximately 100 ms. Glass surface modification protocol was developed for immobilization of nucleosomes via high-affinity streptavidin-biotin interactions. To ensure immobilization, one of the ends of the fluorescently labeled nucleosomal DNA was biotinylated. The algorithm of image processing for analysis of structural rearrangements at a single nucleosome level was developed. Fluorescence microscopy of single immobilized molecules and their complexes allows the analysis of nucleosome structural dynamics during transcription, and its interactions with various nuclear proteins.