CORE 1: Cellular diagnostics/imaging core

Massachusetts General Hospital, Boston MA

Investigators

Linked publications & trials

Abstract

ABSTRACT The mission of the cellular diagnostics/imaging core continues to be two-fold. 1. 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. 2. 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 (IVM), instrumentation design and fabrication, complemented by expertise in image processing and data analysis. Our laboratory has been at the forefront of the development in IVM and associated technologies, particularly for imaging the bone marrow (BM). We also have a record of productive collaborations with the project investigators in this PPG, as evidenced in our joint publications. To continue the mission of the Core, we propose two Specific Aims. In Aim 1, 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 continue to refine and optimize methods for i) structural and functional imaging of the BM vasculature, ii) analysis of hematopoietic cells (their spatial localization and clonal distribution), iii) spatial transcriptomic analysis of BM cells (both hematopoietic and stromal) using Image-seq, and iv) analysis of large 3D volumetric imaging data sets. These tools will be integrated in the workflow of the Projects in this PPG to a) image the physical relationships of hematopoietic and stromal elements (P1, P2, P4), b) examine the pairing of hematopoietic and stromal clonal outgrowth in mice with CVD (P1), c) characterize and profile newly assembled (angiogenic) BM vascular sites post MI (P2), d) visualize the location and behavior of different mutants (identified in P4) in the BM during CVD, and e) map the three-dimensional brain activity during cardiovascular disease (P3). In Aim 2, we will develop new tools for studying hematopoiesis in CVD. We will develop a new assay for BM stromal cell activation based on calcium signaling in the stromal network. Imaging the network calcium activity will provide a direct readout of the stromal response to stimulation or silencing of specific brain regions during CVD (in collaboration with P3). A similar assay will be developed to study BM vascular endothelial cell activation in collaboration with P2. We will implement three-photon (3P) microscopy for deep imaging into the BM and develop an optical platform for simultaneous optogenetic stimulation of the brain and imaging of the overlying calvarium to assess its BM response (P3). The core expertise in the design and fabrication of optical instruments will give project investigators the flexibility to pursue new research ideas without the constraint of existing technologies.