Developing New Instruments for Fluorescent Research

Author: James Batey | Major: Chemistry and Physics | Semester: Spring 2023

My name is J. Ethan Batey, I am a Junior majoring in Chemistry and Physics. Since the Fall of 2021, I have been working with Dr. Bin Dong in the Department of Chemistry and Biochemistry. Our group has been engaged with developing new spectromicroscopic methodologies for the detection and analysis of nanoscale fluorogenic, catalytic, and single particle behavior in ensemble and lone environments. My projects in our group have consisted of single particle tracking and spectroscopy (SPT), fluorescent super-resolution and single molecule localization, as well as instrument/methodology development for spectromicroscopic analysis.

Fluorescent microscopy techniques are useful to virtually all areas of the physical and life sciences as well as in many areas of chemical, biological, and biomedical engineering. This type of microscopy works by observing fluorophores or fluorogenic behavior to gain understanding of a “bigger picture’. For example, these studies include tagging proteins with fluorophores to study mechanisms in cells and tissues or observing fluorophore synthesis to determine reaction rate with multiple catalysts.

These methods employ optical microscopes to gather data. Microscopes are optical systems that are diffraction limited, meaning the resolution is limited by the physics of diffraction through the individual optical components. This can result in spatial resolution that is not ideal.

Currently, I am working to construct an instrument capable of single molecule imaging with ultrahigh spatial resolution that can overcome the diffraction limit associated with traditional (epi-illumination) fluorescent techniques. This instrument works on a principle known as light-sheet fluorescent microscopy (LSFM). In LSFM, a laser is shaped into an oblique plane or thin sheet that excites a small number of fluorophores. These fluorophores blink rapidly as they are excited by the laser and then relax as the electrons return to a ground state. This process takes only nanoseconds so when imaging, the light sheet covers a small part of the sample for tens of milliseconds. This means those fluorophores have switched thousands of times! Recording the blinking of these fluorophores over multiple frames, moving the light sheet to excite new fluorophores, and repeating the process a large sample can be scanned. Then, by using stochastic reconstruction (STORM) to build a picture that shows all the excitation events from every active fluorophore, an image is obtained that shows the precise location of each fluorophore. This allows smaller features to be resolved than what could be obtained if all the fluorophores were excited at the same time. Thus, breaking the diffraction limit!

When selecting this project and finding my mentor, I began by digging into the literature of the faculty members. I found many interesting subjects, but I was truly enticed by Dr. Dong’s work. Chemical imaging was not something I had heard of before, and it piqued my interest. When I joined the Dong lab, I was fascinated by the optical systems that had been and were being constructed. I hoped to one day build one of these myself. I was ecstatic the day the Dr. Dong had an idea for me to construct one of my own.

From being a member of the Dong lab, I have learned many things about the chemistry here, as well as many things about myself. From being in the lab, I have learned everything from how to make figures and use a microscope, to how to read scientific literature and give proper scientific presentations. There truly are too many things to count, the experience and knowledge that I have gained here is invaluable to my career and day-to-day life. The most important lesson that doing this research has taught me is how to approach and solve problems that are daunting or seem impossible. Whenever difficult problems arose Dr. Dong has always been available to help. Not to mention the indispensable advice that has come from other faculty members such as Dr. Martin Edwards, Dr. Julie Stenken, Dr. Ingrid Fritsch, Dr. Mya Norman, and more. These faculty members as well as graduate students Meek Yang, Elric Pott, Megan Magness, Nick Rathke, and countless others are an inspiration and great contributors to my success. By doing this research I have met these individuals who are not only my mentors and advisors, but also some of my greatest friends.

The work I have been a part of here has solidified my plans of going to graduate school to pursue a PhD. I hope to focus in physical chemistry and continue working with optics throughout my graduate school career. I hope to work as a postdoc and then continue to a life in academia. I aspire to one day be a great professor that can produce research with great impact and to someday inspire my own students to find a love for research.