The Evans-Molina Lab utilizes a variety of imaging techniques to monitor β cell health and calcium signaling. These techniques include cytosolic imaging of glucose-induced calcium responses and organelle-specific calcium measurements using FRET and FLIM to study how loss of ER and Golgi calcium impact β cell function. The lab is working with the Slak Rupnik lab to analyze fresh pancreas tissue slices used in functional multicellular calcium imaging. This workflow allows unprecedented insight into time resolved calcium dynamics and regulation at subcellular, cellular, and cell collective spatial scales in situ.
[Pictured: Micrograph of the rat insulinoma-derived pancreatic β cell expressing gene-encoded ER calcium indicator (Red) and Mitochondrial calcium indicator (Green). Cells were treated with 15 mM glucose. Magnification = 400X. Photograph courtesy of Tatsuyoshi Kono, PhD, and Carmella Evans-Molina, MD, PhD.]
More recently, through collaboration with Dr. Fang Huang at Purdue University, the lab has begun to address how changes in the structure and composition of key β cell organelles contribute to the loss of insulin secretory capacity in the β cell during diabetes development. Dr. Huang's group has developed a novel super resolution imaging technique known as whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN) which allows for 10- to 20-nm 3D resolution across the thickness of the entire mammalian cell due to its ability to overcome the diffraction limit of light. The lab is using this technique to image the ultrastructural architecture of the ER in the β cell. By leveraging the nanometer resolution and localization capabilities of W-4PiSMSN, the precise meshwork of the ER can be visualized and the tubule diameter can be quantified.
[Pictured: Representative super-resolution image of the rat insulinoma-derived pancreatic β cell line with the endoplasmic reticulum labeled using a Sec61β-GFP plasmid and GFP-Booster Alexa Fluor 647 nanobody (shown in orange). Scale bar = 5 micrometers. Photograph courtesy of Madeline McLaughlin, Fang Huang, PhD, and Carmella Evans-Molina, MD, PhD.]
