We request funds to purchase a shared two-photon/confocal laser scanning microscope (Carl Zeiss LSM710 NLO), as a replacement for an aging Zeiss LSM510 system. The existing microscope was originally installed in 1998 and has been upgraded several times (for instance, adding the META detector in 2002), but we have now been informed that Carl Zeiss will discontinue service support for this older LSM510/Axiovert100 system in 2009. To assure experimental continuity, we will utilize much of our existing optics and accessory hardware on the new system, including the recently purchased Coherent Chameleon Ti:Sapphire laser. The current system has 6 major users focused in three broad research areas: cancer, diabetes, and neuroscience, and these users will constitute the major user group for the proposed instrument. All of these major users have qualifying NIH-funded projects that specifically include the use of two-photon excitation, so the guaranteed reliability of a system under service contract is crucial. As with the current instrument, the proposed instrument will be part of the Cell Imaging Shared Resource (CISR), of which Dr. Piston is the Scientific Director and Dr. Wells is the Managing Director. All major users will have access to the instrument and training through the established CISR infrastructure. Usage charges have supported the service contract for the existing LSM510 NLO over the last eight years, and we foresee no difficulty in continuing that arrangement for the LSM710. In addition, the CISR will also help identify and train new users of the two-photon instrument as their projects require. The Resource has an extensive track record of education, training, and productivity with over 300 lab groups at Vanderbilt University. Over the last 15 years, we have introduced shared access to confocal microscopy, live cell imaging, two-photon excitation, total internal reflection (TIRF) microscopy, fluorescence correlation spectroscopy, and deconvolution microscopy. These techniques all began with use by the more biophysical laboratories, but have become widely used by the general biomedical research community. For imaging of thick intact tissues or live animal models (such as the mouse), two-photon excitation is far superior to other approaches and permits high-resolution imaging at a level ~6 fold deeper than with confocal microscopy. The proposed instrument will be the only generally available two-photon excitation imaging system available at Vanderbilt University. Using two-photon excitation, we have developed in vivo quantitative laser scanning imaging protocols for NAD(P)H, Ca2+ indicator dyes, quantum dots, and GFP mutants, as well as photochemical activation protocols. Over the last few years, we have used these new approaches to elucidate many biological mechanisms involved in tumor growth and metastasis, diabetic pathophysiology, and neurotransmitter dynamics. Because of the imminent lack of service support for the current microscope, the requested instrumentation is critical to continue this research momentum.
Over the last decade, it has become apparent that the biological cell cannot be regarded as a """"""""bag of chemicals"""""""" that carries out reactions. The exact three-dimensional arrangement of the cellular components is tremendously important, as are the time-dependent changes in this arrangement during the life of the cell and upon interaction with external stimuli. Recent advances in microscopy, such as the two-photon excitation microscope requested here, allow us to watch these arrangements and movements in living cells with minimal effects on cell viability.
| Hughes, Jing W; Ustione, Alessandro; Lavagnino, Zeno et al. (2018) Regulation of islet glucagon secretion: Beyond calcium. Diabetes Obes Metab 20 Suppl 2:127-136 |
| Reissaus, Christopher A; Piston, David W (2017) Reestablishment of Glucose Inhibition of Glucagon Secretion in Small Pseudoislets. Diabetes 66:960-969 |
| Cranfill, Paula J; Sell, Brittney R; Baird, Michelle A et al. (2016) Quantitative assessment of fluorescent proteins. Nat Methods 13:557-62 |
| Liv, Nalan; van Oosten Slingeland, Daan S B; Baudoin, Jean-Pierre et al. (2016) Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy. ACS Nano 10:265-73 |
| Elliott, Amicia D; Ustione, Alessandro; Piston, David W (2015) Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreatic ?-cell by lowering cAMP. Am J Physiol Endocrinol Metab 308:E130-43 |
| Benninger, Richard K P; Piston, David W (2014) Cellular communication and heterogeneity in pancreatic islet insulin secretion dynamics. Trends Endocrinol Metab 25:399-406 |
| Benninger, Richard K P; Hutchens, Troy; Head, W Steven et al. (2014) Intrinsic islet heterogeneity and gap junction coupling determine spatiotemporal Ca²? wave dynamics. Biophys J 107:2723-33 |
| Schwetz, Tara A; Reissaus, Christopher A; Piston, David W (2014) Differential stimulation of insulin secretion by GLP-1 and Kisspeptin-10. PLoS One 9:e113020 |
| Schietinger, Andrea; Arina, Ainhoa; Liu, Rebecca B et al. (2013) Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer. Oncoimmunology 2:e26677 |
| Ustione, Alessandro; Piston, David W (2012) Dopamine synthesis and D3 receptor activation in pancreatic ?-cells regulates insulin secretion and intracellular [Ca(2+)] oscillations. Mol Endocrinol 26:1928-40 |
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