Technology Development Unit: PROJECT SUMMARY The overall goal of the U54 Northwestern University Center for Chromatin Nanoimaging in Cancer (NU-CCNIC) is to develop and deploy a multi-scale chromatin nanoimaging platform together with molecular analyses and computational modeling to characterize chromatin structure and transcriptional patterns associated with cancer stem cells (CSCs) and the chemoresistance phenotype. We seek to fill three technology gaps in developing such a nanoimaging platform. (1) The multi-scale challenge stems from the need to investigate how chromatin regulates gene expression across a wide range of scales, from the diameter of DNA (~2 nm) to the size of nucleosomes (~10 nm) to chromatin domains (~100 nm) to cell nuclei (~10 µm) and to a population of cells to account for intercellular heterogeneity. (2) The multiplexed molecular imaging challenge arises due to a large number of critical genes and molecular regulators that need to be co-registered with the 3D chromatin structural data. And (3) The temporal dynamics challenge requires the ability to work across a wide range of time scales to study short-term processes that may occur within minutes, such as chromatin remodeling, and long-term processes that may progress over weeks, such as the emergence of chemoresistance in cancer cells. No single technique satisfies all three requirements. Our solution to address these three challenges is to organically correlate three families of technologies: chromatin scanning transmission electron microscopy (ChromSTEM), spectroscopic single-molecule localization microscopy (sSMLM), and partial wave spectroscopic (PWS) nanosensing microscopy. After registering these technologies with 1 nm precision, they will collectively satisfy the multi-scale, multiplexing, and dynamics requirements. We will achieve three objectives in TECH. (1) Develop next-generation sSMLM that enables, in principle, unlimited multiplexing with the highest photon utilization and at 1 nm spatial precision. (2) Develop correlative ChromSTEM-sSMLM-PWS imaging that enables cross-modality registration at 1 nm precision assisted by novel quantum dot-based nano assembly fiduciary markers. And (3) Develop a multi-scale molecular modeling platform to elucidate the etiological relationship between chromatin structure and transcriptional reprogramming in CSCs imaged with the nanoimaging platform. The proposed technology development is driven by the needs of the Research Test-Bed unit (RTB) to elucidate epigenetic and chromatin drivers of transcriptional plasticity in CSC processes, in particular the development of adaptive resistance to chemotherapy, and to explore the feasibility of reprogramming CSCs out of the stem-states as a therapeutic strategy. Continuous feedback from RTB investigators will be integrated with the TECH platform's optimization, enabling, in the longer-term, our U54 technologies to impact the broader cancer research community within and beyond the Cell...