Development of single-molecule tracking confocal microscope combined with force spectroscopy for gene-expression analysis

Abstract

We have constructed a confocal fluorescence micro-scope combined with force measurements. Our method allows for simultaneous measurements of fluorescence anisotropy, energy transfer and correlation. The metho-dology and the sensitivity of the set-up using enhanced green fluorescent protein are described. We present results on (a) the detection of mRNA polymerization during in vitro transcription using fluorescence corre-lation spectroscopy and anisotropy, (b) detection of in vivo protein-DNA interactions using fluorescence ani-sotropy and (c) nanomanipulation of polytene chromosomes using the micropippete force sensor. Such a combined method allows for probing novel structure–function relationship underlying gene-expression.