Cloneable contrast across all biological length scales.

Area of Science:

• Biophysics
• Microscopy
• Nanotechnology

Background:

• Cloneable contrast agents like Green Fluorescent Protein (GFP) are vital for fluorescence microscopy.
• A lack of similar cloneable agents exists for biological electron microscopy.
• Existing methods struggle to provide contrast across all biological length scales.

Purpose of the Study:

• To report a novel cloneable Selenium NanoParticle (cSeNP) for molecular contrast in biological imaging.
• To demonstrate the utility of cSeNP across multiple imaging modalities, including electron microscopy, fluorescence microscopy, and X-ray computed tomography.
• To enable imaging from subcellular structures to whole organisms.

Main Methods:

• Development of a protein-conjugated, ~5 nm diameter Selenium Nanoparticle (cSeNP).
• Fusion of DNA encoding cSeNP protein with DNA encoding FtsZ, a bacterial tubulin analog.
• Expression of FtsZ-cSeNP fusion proteins in Escherichia coli and subsequent analysis via transmission electron tomography and fluorescence light microscopy.
• X-ray computed tomography was used to assess contrast in E. coli.

Main Results:

• cSeNPs exhibit high contrast in electron microscopy due to their electron density.
• Experiments confirmed cSeNP localization correlating with known FtsZ filament positions in E. coli.
• Less than 5% of cSeNPs were found in unexpected locations.
• X-ray imaging demonstrated distinguishable contrast from background noise.

Conclusions:

• The cSeNP serves as a versatile, cloneable imaging contrast agent.
• It facilitates the location and correlation of proteins across all biological length scales.
• This is particularly advantageous for electron microscopy, bridging the gap with larger-area imaging techniques like fluorescence microscopy.