#microtwjc session 3 – 17th Feb 2015 8 PM

I have chosen a paper based on a technique I saw being exploited at a recent conference I attended. The paper can be found using the link below and I hope it really is open access:

Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules

This is a bit different from our usual papers but there are aspects of this technique that make one think of what we can now do! However the usual rules apply:

Discussion points:

1. Is the paper well written and concise?

2. Are the experiments well designed?

3. Anything you would have done differently?

Abstract: Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, 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 electroporation. 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 desirable properties of organic fluorophores to perform single-molecule Förster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeastSaccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.