あらゆる生物学的長さスケールにわたるクローン可能なコントラスト

Cloneable contrast in biological microscopy is exemplified by Green Fluorescent Protein (GFP) in fluorescence microscopy. There are no similarly useful cloneable contrast agents that function in biological electron microscopy. This paper reports a cloneable Selenium NanoParticle (cSeNP) that produces molecular contrast in imaging modalities including cellular electron microscopy, fluorescence microscopy, and X-ray computed tomography. This set of imaging modalities can image all biologically relevant length scales, from subcellular structure to whole organisms. The cSeNP is a ∼5 nm diameter Selenium nanoparticle that is made and conjugated by a protein. Because the cSeNP is electron dense compared to biological molecules, it has high contrast in biological electron microscopy. DNA encoding the cSeNP protein was concatenated to DNA encoding FtsZ, the procaryotic analog of tubulin. FtsZ is membrane associated throughout the cell cycle and localizes to the cleavage furrow of dividing cells. Escherichia coli cells expressing FtsZ-cSeNP fusion proteins were examined by transmission electron tomography and fluorescence light microscopy. These experiments show cSeNP decorated FtsZ filaments and/or cSeNPs in locations that correlate to known FtsZ locations, with less than 5% of cSeNPs in unexpected locations. X-ray imaging shows contrast attributable to cSeNPs is distinguishable from background in E. coli. The cSeNP, therefore, represents a cloneable imaging contrast agent that facilitates location and correlation of proteins-of-interest across all biological length scales. This is especially useful in biological electron microscopy, where larger-area imaging modalities such as fluorescence microscopy are employed to identify sub-areas containing a protein-of-interest to prepare for electron microscopy study.