The mission of the cellular diagnostics/imaging core is two fold. First, we will provide state-of-the art imaging technology and cell analysis tools to meet the scientific needs of the four projects in this PPG. We will work closely with project investigators to perform quantitative measurements and data analysis, customizing instrumentation and optimizing experimental protocols as necessary. Second, we will pursue new technical innovations that will lead to future scientific advances beyond what can be accomplished with existing technologies. The Core is a unique resource with deep expertise in optical technology, intravital microscopy, instrumentation design and fabrication, as well as image processing and data analysis. We also have a record of productive collaborations with multiple investigators in this PPG, including Dr. Scadden (Project 1), Dr. Nahrendorf (Project 2), and Dr. Swirski (Project 3). Our laboratories are all physically co-located in the same building, making the Core a common meeting place where team members from various laboratories converge, not just to use the facility but also to interact and exchange ideas. To carry out the mission of the Core, we propose the following Specific Aims.
In Aim 1, we will work with Projects 1 and 4 to perform clonal analysis of hematopoietic cells based on their expression of multi-color fluorescent proteins. We will expand the multi-color capability of our in vivo flow cytometer, a technology developed in our laboratory for real-time detection and quantification of fluorescent cells in the circulation of live animals without the need to draw blood samples, to enable noninvasive monitoring of the clonal dynamics in the peripheral circulating leukocyte population.
In Aim 2, we will work with Project 2 to assess how the bone marrow (BM) vasculature is altered by cardiovascular diseases (CVD). We will measure functional parameters such as blood flow, vascular permeability, vascular reactivity, and trans-endothelial migration, by performing multiphoton imaging of the BM vasculature together with second-harmonic imaging of the extracellular matrix component. We will also work with Project 3 to characterize hematopoietic stem cell (HSC) localization and dynamics in the BM in the settings of MI and atherosclerosis using gene-edited HSCs provided by the Genome Engineering Core.
In Aim 3 we propose to develop a new method to enable image-guided laser microdissection and extraction of live cells from the BM for single cell molecular profiling. We will initially focus on capturing BM vascular endothelial cells, as they form a critical component of the HSC niche and also regulate leukocyte trafficking and macromolecular transport. The technique will bridge the existing divide between single-cell analysis on the one hand, which provides molecular but no spatial information, and live imaging on the other, which supplies the 3D spatial context lacking in the molecular analysis. The technique can be extended in future studies to the analysis of other cell types and in other tissues.
The cellular diagnostics/imaging core we will provide state-of-the art imaging technology and cell analysis tools to meet the scientific needs of the projects in this PPG. We will also pursue new technical innovations that will lead to future scientific advances beyond what can be accomplished with existing technologies.
