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As a pioneer in diabetes research, David Piston, Ph.D. of Vanderbilt University has spent almost two decades trying to answer fundamental questions that could lead to the cure of this devastating disease. More than 25 million children and adults in the United States – 8.3 percent of the population – are living with diabetes, making it one of the most prevalent diseases in the country. Because of – and in hopes of advancing – this important work, Nikon Instruments, Inc. is proud to announce Dr. Piston as the recipient of the prestigious 2011 Nikon Fellowship. In his lab at Vanderbilt in Nashville, Tennessee, Dr. Piston serves as a professor in the Department of Molecular Physiology and Biophysics and director of the W.M. Keck Free-Electron Laser Center. As a Fellow, Dr. Piston used Nikon’s most cutting-edge technology – in the form of a microscope system he helped customize - to explore the micro-biological events that hold the key to correcting the body’s production and use of insulin. This is critical to understanding and treating both type 1 and type 2 diabetes.

Small Events with Big Consequences

Diabetes affects the body in one of two ways. In type 1 diabetes, the body does not produce any of the energy-regulating hormone insulin. In type 2 diabetes, the body does not produce enough insulin or ignores what is produced. In order to understand why this happens, Dr. Piston leads research on beta cells, the insulin-producing cells in the islets of the pancreas that are damaged by diabetes. Dr. Piston, his research staff and students had attempted experiments designed to study the lifetime of insulin granules and examine the relationship between insulin and calcium fluxes in pancreatic beta cells. However, they found that their equipment was not advanced enough to produce the imaging the experiment required.

In normal beta cells, insulin secretion correlates with intra-cellular free calcium activity. However, there are instances in which insulin secretion is blunted - even when calcium activity is normal. With his Fellowship, Dr. Piston wanted to replicate the experiments he attempted in his lab to determine at what point the insulin bottlenecking occurs in the beta cells. He believes this bottlenecking may be key to understanding why the insulin granules fail to be secreted.

Dr. Piston’s experiment required imaging the flattened pancreatic islets to count the insulin secretory events using TIRF and widefield fluorescence microscopy. This advanced technology allowed him to identify a sensor - which he also created - that shows when an insulin granule is secreted. The sensor was designed to change the insulin granule’s color based on the pH of the environment from inside to outside the cell, allowing Dr. Piston to count these events and compare secretion rates.

Seeing at the Cellular Level

To track secretion rates, Nikon supplied Dr. Piston with a customized version of its standard high-end TiE Perfect Focus TIRF/Photactivation system equipped with an ultra-sensitive Andor EMCCD camera. This system, which Dr. Piston worked hand-in-glove with an internal Nikon team to develop, allowed him to see these microscopic events, which previously eluded him and his team. Additionally, the TIRF system gave him an extremely high signal to background ratio so he could understand the context of events as they were happening. The Perfect Focus, the leading dynamic focus system in the market, helped maintain the validity of the experiment. When monitoring calcium signals, focus stability is essential to preventing false data because loss of focus or focus fluctuations can appear strikingly similar to a calcium signal.

The photoactivation system also allowed Dr. Piston to test a tangent theory on excess insulin in the body. Past research has shown that the body secretes a very small percentage (less than 10 percent) of insulin granules. The reason for this is unclear, but Dr. Piston hypothesized that long-lived granules actually form an organelle that acts as a signaling platform in the cells. Photoactivation of a subset of granules allowed him to watch where the insulin granules go as the cells are stimulated with glucose and correlate those granules that are secreted against those that are maintained in the cell.

Paving the Way for Future Research

For Dr. Piston, the project and his time as a Fellow marked an ability to get back to basics, conducting experiments in a hands-on environment, free from the meeting-filled days that come with running a high-profile laboratory. He also has the ability to course correct as he works. For example when discovering the need for even faster imaging to mark and capture secretion events, Nikon’s photometric team was able to install dual-view capabilities to his equipment on the fly.

The unique equipment Nikon provided gave Dr. Piston and his students the opportunity to start work on new projects and test ideas and strategies. “The biology didn’t always cooperate, as the label we started out using (called phogrin) turns out not to be on the very early insulin containing vesicles, but rather it seems to join onto the vesicles as they age,” said Dr. Piston. “We learned a lot about phogrin trafficking and formulated some ways around that issue.”

I had an opportunity to play with a lot of the other new systems that were brought into the MBL this summer, such as the Nikon SIM system,” said Dr. Piston. “That was informative and would not have been possible at home without a tremendous effort. There are also lots of folks doing very interesting experiments around and I had a good time exchanging ideas with all of them. I learned a lot, and I hope they did too.”

Perhaps most important, the work Dr. Piston completed as a Fellow provides him with preliminary data to get funding for further research and, possibly, a dissertation grant to purchase the TIRF equipment his experiment requires. With the end game of this vital work being more understanding on how we may cure a disease devastating to so many, Nikon is proud to support Dr. Piston and his work on insulin secretion and regulation.