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It was a sweltering Tuesday in June, my second day volunteering in the lab. To my surprise, after observing my PI meticulously sacrifice a mouse, isolate its cardiothoracic cavity, and collect blood samples, he handed me a pair of forceps and told me to isolate the rest of the aorta myself. With shaky hands and only a two-dimensional understanding of anatomy from textbook diagrams, I began the delicate process. What started as a nerve-wracking challenge soon became one of my favorite skills to practice in the lab. Yet, the experience also underscored the ethical complexities of science. Even now, it’s difficult to witness a mouse being sacrificed, despite understanding its necessity for the advancement of medicine. This duality—awe at the skills and techniques I’ve learned and the ethical weight of the work—continues to shape my perspective on the responsibility that comes with scientific research.

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My first dissection/isolation of a TRPC6 KO mouse aorta

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My first dissection/isolation of a TRPC6 KO mouse aorta

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The general focus of Dr. Obukhov's lab is on understanding vascular and metabolic dysfunction, with a primary emphasis on the role of TRPC channels in metabolic syndrome-associated atherosclerosis. Additionally, the lab investigates broader mechanisms of cellular and vascular dysfunction, including uterine contractility, endothelial dysfunction, diabetic neuropathy, traumatic brain injury, catecholamine secretion, and the function of ion transporters in the corneal endothelium.

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This semester, our work primarily focused on completing data analysis for a long-standing project on atherosclerosis and TRPC6 channel physiology. Shifting from the hands-on wet lab and microscopy techniques I had previously learned, I gained a deeper appreciation for the analytical side of research. It was an invaluable experience to develop new math and data analysis skills, which gave me a more holistic understanding of the research process. One aspect I particularly enjoyed was using smart segmentation in image analysis, which allowed me to approach data interpretation with precision and efficiency. This opportunity to engage with different facets of research has broadened my skill set and deepened my appreciation for the multifaceted nature of scientific inquiry.

We also were on the initial stages of developing a new project involving immunocytochemistry, a technique used to detect specific proteins within cells by employing antibodies that bind to the target protein and fluorescent dyes for visualization. Much of our time was spent refining the protocol through repeated tests, adjusting variables to optimize the process. These early trials were a valuable learning experience, as I had the chance to explore the basics of fluorescence imaging to observe cellular activity. In particular, we used the Fura protein, a ratiometric calcium indicator dye that emits fluorescence upon binding to calcium ions, allowing us to track calcium dynamics within the cells. This process not only deepened my understanding of fluorescence imaging but also highlighted the intricacies of protocol development in achieving reliable and reproducible results.

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Fluorescence imaging is a powerful technique that allows scientists to visualize and analyze biological structures and processes with high specificity and sensitivity. The method relies on the principle of fluorescence, where certain molecules (called fluorophores) absorb light at a specific wavelength and then emit light at a longer wavelength. By tagging these fluorophores to specific proteins, nucleic acids, or other molecules of interest, researchers can track their location and activity within cells or tissues. Modern fluorescence imaging systems use filters to isolate the excitation and emission wavelengths, enabling precise detection of the fluorescent signal against a dark background. Advanced microscopy systems, such as confocal or two-photon microscopes, enhance this technique by providing high-resolution, three-dimensional imaging of biological samples.

Fluorescence imaging is used in a wide variety of experiments to study cellular and molecular processes. For instance, it plays a critical role in immunocytochemistry, where fluorescently labeled antibodies bind to specific proteins to reveal their distribution within a cell. It is also used for live-cell imaging to observe dynamic events such as cell signaling, membrane trafficking, and cytoskeletal changes. Calcium imaging is another application, where dyes like Fura-2 are used to monitor calcium ion fluctuations in real-time, providing insights into neuronal signaling, muscle contraction, and other physiological processes. In molecular biology, techniques like fluorescence in situ hybridization (FISH) use fluorescent probes to detect specific DNA or RNA sequences, aiding in gene mapping and diagnostics.

The versatility of fluorescence imaging makes it an indispensable tool in both basic and applied sciences. Researchers use it to study disease mechanisms, test drug candidates, and even develop novel therapeutic approaches. For example, in cancer research, fluorescence imaging can identify tumor markers or visualize the distribution of cancer cells in tissues. In neuroscience, it enables the tracking of neurotransmitter release and receptor activity. The ability to label multiple targets with different fluorophores also allows for multiplexing, where researchers can study the interplay of several molecules simultaneously. This wide-ranging applicability ensures that fluorescence imaging is a key tool to push scientific boundries.

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It IS important to be a Jack of all trades.

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It IS important to be a Jack of all trades.

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One of the most fascinating moments during this process of learning the skill was witnessing cells active in real time, a perspective that brought biology to life in a way I had never seen before. The process leading up to imaging was equally engaging—I learned how to culture and split cells, gaining an appreciation for the great effort and timing required to keep them alive and thriving. Every step of the preparation felt like an essential piece of the larger puzzle, emphasizing the importance of precision and care in experimental science.

What stood out to me most about fluorescence imaging was how it integrates multiple disciplines of science into one cohesive technique. My PI’s deep understanding of physics, engineering, and chemistry was particularly inspiring. Watching him troubleshoot and repair the imaging equipment when it failed highlighted the necessity of a multidisciplinary approach. His ability to apply knowledge from these fields to solve complex problems demonstrated how essential it is to develop expertise beyond one’s primary area of study.

This experience has reshaped how I think about learning in science. It’s not just about mastering my major but also about building a strong foundation in parallel disciplines that contribute to the bigger picture. Fluorescence imaging has taught me that groundbreaking research often lies at the intersection of fields, and the effort I put into understanding these connections will only enhance my ability to contribute to meaningful scientific projects.

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References

Ettinger, A., & Wittmann, T. (2014). Fluorescence live cell imaging. Methods in Cell Biology, 123, 77–94. https://doi.org/10.1016/b978-0-12-420138-5.00005-7

Wen, H., Gwathmey, J. K., & Xie, L.-H. (2020). Role of Transient Receptor Potential Canonical Channels in Heart Physiology and Pathophysiology. Frontiers in Cardiovascular Medicine, 7. https://doi.org/10.3389/fcvm.2020.00024

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Failure and redirection are not just inevitable in science—they are essential for growth and discovery. This lesson became especially clear to me during research journey. I was initially surprised, even unsettled, by Dr. Obukhov's (my PI) unwavering resolve when a multi-year experiment started looking like it yielded no scientifically significant results. While I expected disappointment, my PI instead saw an opportunity to refine our approach and deepen our understanding, demonstrating that persistence and adaptability are just as critical as technical expertise. In a field where there can be immense pressure to produce significant findings, my PI's commitment to integrity set a powerful example.

This semester further reinforced the importance of flexibility. I entered the term with the expectation that I would be working primarily on a new fluorescence imaging project, excited to explore a fresh area of research. However, as the semester progressed, we shifted focus to revisit and re-analyze data from an ongoing three-year study on atherosclerosis. At first, this redirection felt like a detour from the exciting new work I had anticipated. But as we delved deeper into the data, I came to appreciate the value of this effort—not only in strengthening our conclusions but also in teaching me how important it is to adapt to the needs of the research.

These experiences have shown me that science is rarely straightforward and often demands a willingness to embrace uncertainty. Failure doesn’t signify the end of a project but rather an opportunity to approach questions from new angles and uncover insights that might have been missed. It has taught me to view setbacks not as obstacles, but as valuable stepping stones in the process of discovery.

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I’ve always been drawn to science, especially the idea of exploring questions and uncovering answers through research. The process of forming hypotheses, designing experiments, and interpreting results deeply resonated with me, as it felt like a tangible way to contribute to solving real-world problems. In my freshman year of high school, this curiosity motivated me to reach out to researchers, and I was fortunate to secure a position at the IU School of Medicine. Over the past three years, this experience has been incredibly fulfilling, giving me the opportunity to engage in hands-on research and deepen my understanding of how science can be applied to tackle complex challenges.