Biosensor Development and Delivery Core
Genetically encoded biosensor proteins enable the detection of the spatial and temporal characteristics of specific cell signaling or metabolic events in single living cells. When used in combination with intravital microscopy (IVM), the changing signals from the biosensor probes can be monitored from the tissues of living animals, supporting unique studies of cell biology in the relevant physiological context of the living animal.During the previous grantcycle the IU O’Brien Center Probe Development Core developed novel fluorescent biosensor reagents and protocols for imaging these probes in vivo using multiphoton microscopy. In parallel, the IU O’Brien Center Probe Delivery Core developed methods of in vivo gene transfer to provide investigators with a flexible approach for utilizing fluorescent protein biosensors, obviating the time and costs associated with generation of transgenic animals.
During the upcoming period we propose to apply, refine and extend the approaches developed during the previous grant period to provide renal investigators with effective new reagents and protocols to support unique studies of renal cell biology in vivo. The new Biosensor Development and Delivery Core will develop several important resources for the renal research community. We will provide optimized biosensor probes that have been validated for IVM, provide these probes in viral vectors optimized for in vivo gene transfer and provide protocols for delivery and useof probes. We will develop novel rat models providing titratable expression of introduced genes and transgenic rats to support novel in vivo studies of the rat glomerulus.
Three-dimensional tissue imaging core
Recent developments in optical sectioning microscope systems have provided biomedical researchers with the capability to conduct high-resolution, high-content 3D microscopy at the scale of millimeters, if not centimeters in biological tissues. Image volumes of this size and complexity are enormously rich in potential information. However, microscopy at this scale introduces new sets of challenges with respect to data handling, image visualization and quantitative analysis that limit the ability of most researchers to realize this potential. The overarching goal of the 3-dimensional Tissue Imaging (3DTI) core is to make large-scale, quantitative 3D microscopy accessible and productive for renal researchers, regardless of their institutional infrastructure or their expertise in imaging.
Service of the 3DTI core will consist of two components. First, the core will provide imaging services, which will consist of preparing a sample for fluorescence microscopy, collecting high-resolution image volumes by confocal microscopy, and combining these image volumes into a single hyper-volume. Second, the core will provide access, training and support to investigators in the use of a high-performance image analysis system that can be used by remote investigators in a client-server format. Taking advantage of the advanced computer and networking infrastructure of Indiana University, this system will be designed to provide remote investigators with a powerful, yet easy-to-use tool for interactive exploration and analysis of their image data.