Nanosphere Lithography of Zero Mode Waveguides for Single Molecule Fluorescence Microscopy

Example of single molecule fluorescence microscopy with an insert of Kevin and a word bubble saying "wow cool"

Funding Source

Students

2018
College

Faculty

Project Summary

This summer, I worked in Dr. Yale E Goldman’s single molecule biophysics lab, which I joined my freshman spring semester. Since then, I have been fabricating devices called zero mode waveguides for single molecule fluorescence microscopy. Single molecule fluorescence microscopy allows biophysicists like me to observe individual proteins moving and interacting with other molecules in real time. This is a big step from looking at test tube reactions where millions of molecules are interacting and one just measures the bulk average value of an interesting parameter. For me, I want to look at the reactions that form proteins in our cells. This reaction, otherwise known as translation, is facilitated by a large macromolecular machine called a ribosome, which is a complex of multiple of proteins and RNA molecules that catalyze the protein synthesis reactions. However, to observe translation at the single molecule level, experimentalists usually dilute the concentrations of fluorescently labeled ligands, such as the transfer RNA molecules that bring individual pieces of the protein to the ribosome, to non-physiological concentrations. This slows down the reaction. A good analogy of why this is important is Eadweard Muybridge’s photographic study of a running horse’s motion. If Muybridge had only looked at walking horses, he would have never known that all four of a horse’s feet are actually off the ground at the same time when it runs! Thus, I want to look at a ribosome making proteins at physiological rates with physiological concentrations of ligands, which is what the zero mode waveguides allow me to do.  

I focused this summer on chemically passivating the metal surfaces of the waveguides so that the molecules I want to look at do not stick to the metal walls. Metal can quench the fluorescence from a fluorophore, so I want my fluorescently labeled molecules to only adhere to the glass surface at the bottom of the waveguides. To do this, I used a chemical passivation technique using PVPA (poly-vinyl phosphonic acid) that blocks nonspecific interactions of molecules with the aluminum walls of the waveguides. This took a lot of troubleshooting and optimization. I am now close to finishing the passivation and cannot wait to see individual eukaryotic ribosomes making proteins in my waveguides.  

In the meantime, I have been purifying and labeling transfer RNA molecules for my single molecules fluorescence experiments, which has taught me a lot of biochemistry I didn’t know before. I have done preliminary fluorescence experiments with individual ribosomes adhered to a plain glass surface, and I am proud to say that I can now obtain my own single molecule fluorescence data of translating ribosomes, which is very exciting for me. It was no easy feat. Although the method is pretty well developed in our lab, single molecule experiments are still quite finicky, and I am amazed every time it works.

Overall, engineering and passivating the waveguides helped me appreciate science as an interdisciplinary endeavor, as I learned nanofabrication techniques from the semiconductor industry and numerical simulation methods from physics to design and make the waveguides. I also experienced both the frustrations and excitement of method development. The ability to now answer new questions about translation with the waveguides excites me and bolsters my passion for research. I am very grateful to CURF and my lab mentors for all their amazing support and advice. I am especially grateful to Dr. Ann Vernon Grey for her healthy sass and mentorship. As she once wisely said, “Kevin Chen takes a village.” Although she was just cracking another joke at me, I have learned from my research that all important things, whether it is understanding protein synthesis or picking out the right suit for myself, takes teamwork and collaboration.