This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): We propose to acquire a newly developed high-speed laser scanning_slit-confocal microscope (LSM 5 Live, Zeiss), to support research of five NIH-funded investigators at Vanderbilt University that require imaging of rapid dynamic events in live samples. No other microscope on campus has equivalent capabilities, therefore, this system offers significant potential for a large number of investigators. The new instrument will be incorporated into the Cell Imaging Shared Resource (CISR). The CISR provides expert optical microscopy services for about 250 VUMC laboratories, most are NIH-funded, including seven research centers; trained users can access the facility 24 hours/day, 7 days a week. The CISR has a 12-year history of outstanding service, reliability and self-sufficiency; microscope usage is currently about 9000 hours/year. During a recent evaluation we confirmed that the LSM 5 Live can acquire viable confocal images up to 120 (512x512) frames/sec. We imaged flowing blood cells in microvasculature of mouse muscle at rates of 100- 250 frames/sec. We also imaged motions of all individual cardiac cells throughout the beating heart of an embryonic zebrafish by combining piezo-objective focusing with high frame rates to obtain 3-D images (20slices/40microns) at a rate of three z-series/sec. The signal collection efficiency of the instrument was excellent and photobleaching was minimal under the conditions required for our feasibility experiments. Importantly, we visualized numerous biological processes without obvious, significant photo-damage, e.g., calcium oscillations in pancreatic islets were persistent following >12,000 frames of data. The signal-to- bleaching ratio appeared more favorable compared to a point-confocal laser scanning system. Five Major Users with immediate, essential and appropriate need for the requested instrument will utilize 70% of total instrument activity. They will use it to study: 1) zebrafish models of cellular and molecular mechanisms of cardiovascular development and congenital heart disease; 2) C. elegans models of meiosis and molecular processes of reproduction; 3) stimulus-secretion coupling in intact functioning pancreatic islets; 4) mouse models of fuel metabolism and it's relationship to diabetes and heart disease; and 5) mouse models of neo-vascularization and functionality of transplanted islets. Placing an LSM 5 Live in the established and widely used CISR will not only advance the research of the Major Users in this proposal, but also provide significant opportunity to advance the NIH-sponsored research of many more investigators studying features of living models of biology and disease processes previously inaccessible to scientific observation. Justification of need: The proposed LSM510Live scanning slit confocal will be located in a large pre-existing optical core facility with a solid track record. This facility has four state-of-the-art Zeiss 510 confocals, including a 510Meta purchased through a recent NIH shared equipment grant prepared by the PI. Data documenting the current usage on each of these existing confocals was not supplied and would have strengthened the application. Five major users propose to utilize the requested system for rapid live tissue/cell imaging. The proposed experiments make use of the special capabilities of the 510Live though not all users presented preliminary data directly related to their proposed experiments. Technical Expertise: The imaging center is run by the PI (not one of the major users) who trained with Watt Webb before working at Molecular Probes and BioRad. David Piston (a major user) is the scientific director of the center. The imaging center also employs several other staff to run day-to-day operations. The technical support available for the proposed instrument is excellent. Research Projects: Five major users proposed projects that all involve fast, dynamic imaging of living preparations and thus make good use of the 510Live. The ability of the 510Live to stabilize images of moving blood cells (Wasserman preliminary data) is especially impressive. Two major users (Greenstein and Powers) did not provide 510Live preliminary data directly related to their proposed uses. This was a negative point for the application since many of the users proposed using the 510Live to image intracellular calcium dynamics-a non-trivial task at the proposed high temporal speeds. Only one major user (Piston) provided Ca imaging data from the requested system. Administration: The proposed instrument will be housed in a modern imaging core with electronic access devices, on-line sign-up, etc. The center has an advisory board that represents a broad array of departments and procedures are in place to prevent the instrument from being monopolized by a small group of users. The long-term financial plan is workable as long as the institution continues to pay most of the salary costs. The proposed charge-back rate ($25/hr) is probably too low to recover the costs associated with this specialized instrument without those subsidies. Institutional Commitment: The applicant's institution provides a strong commitment to the proposed instrument and to its imaging core facility by providing a majority of the salary funds for the imaging center director and staff members. Overall Benefit: This is a strong proposal for a rather specialized system to be used predominately by five users. For these users, this machine will certainly open up new experimental approaches that were not possible with their existing confocals. This opportunity is balanced against the relatively large number of existing modern confocals in their imaging center and the absence of data demonstrating how extensively these systems are presently used (log sheets, etc) and the limited set of preliminary data provided with the application. Budget: The standard Zeiss environmental chamber appears to be used by only one of the major users with two other users requiring specialized chambers. The nature of these chambers was not presented in the application (modification of the Zeiss chamber or a different design?)
| Kumar, Ankur N; Short, Kurt W; Piston, David W (2013) A motion correction framework for time series sequences in microscopy images. Microsc Microanal 19:433-50 |
| Nyman, Lara R; Ford, Eric; Powers, Alvin C et al. (2010) Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo. Am J Physiol Endocrinol Metab 298:E807-14 |
| Nyman, Lara R; Wells, K Sam; Head, W Steve et al. (2008) Real-time, multidimensional in vivo imaging used to investigate blood flow in mouse pancreatic islets. J Clin Invest 118:3790-7 |
