With support from the NCRR COBRE Program Project and the Cystic Fibrosis Foundation Research Development Program, the Lung Biology Center offers the following core services.
The goal of the Live Cell Imaging Core is to facilitate research on biological mechanisms that contribute to lung disease and to accelerate efforts to identify candidate therapeutic targets. The infrastructure and expertise of the Core supports live cell imaging, as well as protein localization and host-microbe interaction studies.
Advanced imaging technique development, including the recent application of bimolecular fluorescent complementation to study protein-protein interaction and development of a fusion assay to study bacterial toxin delivery in live airway epithelia cells.
Confocal microscopy to monitor intracellular protein trafficking and localization.
Hands-on training by Dr. Yu and trainee participation in extramural workshops and short courses (e.g. Cold Spring Harbor, MDIBL and MBL). In addition, the Core partners with affiliated Departments to invite outside speakers with expertise in advanced imaging techniques.
Live cell imaging using a wide field Olympus fluorescent microscope or a Nikon swept-field confocal LSCM microscope. The Nikon LSC has 488, 563, and 633 nm laser lines for confocal microscopy. The system also can perform TIRF imaging using the 488 nm laser line, using an Apo TIRF 100X or 63X oil immersion 1.49 NA objectives. The Nikon LSC system is equipped with a Piezo Z stage, allowing rapid multi-position and 3D image capture. The Sweptfield Confocal system is also suited with Perfect Focus, an auto focus mechanism, critical to TIRF imaging, that permits highly accurate 3D imaging over time by memorizing the distance of the sample from the coverslip, thus preventing drift in the z axis. A full incubation chamber controlled for temperature, CO2 levels, and humidity maintains optimal conditions for cells during the experiment. The system is equipped with QuantEM:512sc camera and CoolSnapHQ Monochrome cameras (Photometrics, Tuscon, AZ). These cameras allow extremely rapid image acquisition of low intensity signals, while linearly amplifying signal to allow quantitation of detected fluorescence. This system employs Nikon Elements 2.2 Research Duo software with particle tracking module (Nikon, Inc.) to analyze images.Both live cell-imaging platforms are customized for the analysis of bacterial biofilms grown on abiotic or biotic surfaces (i.e., confluent, polarized human airway epithelial monolayers), for analyzing bacterial adhesion to biotic surfaces, and for conducting cell migration assays.
Technical support is provided by Dr. Qianru Yu. She assists investigators to obtain preliminary data for pilot projects and grant applications, with follow-up training to enable the investigator to perform his/her imaging.