We request funds to upgrade a shared two-photon/confocal laser scanning microscope (Carl Zeiss LSM710 NLO) to the high-efficiency LSM780 NLO with the Coherent Chameleon Vision II-S Ti:Sapphire laser, which will provide dispersion compensation and narrow pulse widths to allow maximal depth penetration. The current system has 6 major users focused in three 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, and the proposed improvements in the system are crucial for the currently-funded work. The existing microscope was installed in 2008, and has served very well for many live cell and tissue experiments. However, we have discovered that spectral imaging is critical to take advantage of newly-available multi-fluorescent protein labeled cells and tissues, and that the scientific questions being asked also need with two-photon excitation for deep tissue imaging. To assure experimental continuity, we propose to upgrade both the LSM and the Ti:Sapphire laser to permit the deep-tissue spectral imaging required for the funded projects of the major users. In addition, we are requesting software upgrades needed to extract the maximal amount of information from the data via Correlation Spectroscopy as needed by several major and minor users. 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 contracts for two-photon excitation microscopes (LSM510 from 1999 to 2008, and LSM710 since 2008) over the last 12 years, and we foresee no difficulty in continuing that arrangement for the LSM780/Chameleon Vison II-S system. In addition, the CISR will also 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 to 10 fold deeper than with confocal microscopy. The proposed instrument will continue to be the only generally available two-photon excitation imaging system available at Vanderbilt University.
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. Understanding the temporal and spatial organization of these components requires us to monitor multiple signals simultaneously. Recent advances in microscopy, such as the high-efficiency spectral detectors combined with two-photon excitation microscopy as requested here, allow us to watch these arrangements and movements in living tissues and whole animals with minimal effects on cell viability.
|Benninger, Richard K P; Piston, David W (2014) Cellular communication and heterogeneity in pancreatic islet insulin secretion dynamics. Trends Endocrinol Metab 25:399-406|
|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|
|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|