Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One power- ful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; how- ever, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing elec- troporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desir- able properties of organic fluorophores to perform single-molecule Forster resonance energy transfer measurements in the cyto- plasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions address- able in microbes by single-molecule fluorescence.