NSF Award
2430743
Super-resolution microscopy has revolutionized the study of biology, but crucial technical barriers remain to image cells in 3D at the extremely small length scales needed to understand the fundamental structure and function of the genome, such as double-strand DNA. Current imaging techniques require the use of fluorescent labels which can disrupt natural cellular processes, presenting a challenge for understanding how the molecular machinery underpinning cellular behavior and many disease processes operates. This project will develop a new label-free nanoimaging platform to image the genome of intact fixed cells in 3D, with 2 nanometer resolution while providing chemical and molecular information, along with comprehensive structural and functional information. This may lead to a deeper understanding of the 3D structure of cells in their natural state and eventually to better understanding of multiple significant disease processes at the fundamental molecular level. In addition, the project will provide training and educational opportunities to a diverse audience through public symposia and workshops, mentorship, outreach to K-12 students, and by recruiting undergraduate students from minority-serving institutions in the Chicago region who may not have the opportunity to gain exposure or access to cutting-edge scientific research projects.<br/><br/>This project will develop a new label-free nanoimaging platform, 3D DNA spectroscopic photon-localization intrinsic-contrast nanoscopy (3D DNA-SPIN), to image chromatin in 3D in intact fixed cells with 2 nm resolution while providing chemical and molecular information. Bringing the spatial resolution below 10 nm would enable imaging of chromatin at the nucleosomal scale. Enhancing resolution to ~2 nm would allow thus far unattainable imaging of chromatin at its most fundamental level, the double-strand DNA. If developed, this technique may answer the long-standing question of the 3D conformation of the chromatin polymer in its native state. The ability to record stochastic excitation-emission spectra will increase the spatial resolution of DNA localization and provide chemico-functional information about emitting DNA such as nucleotide sequences. Finally, 3D DNA-SPIN will be co-registered with existing single molecule localization microscopy (SMLM) modalities for superresolution molecular imaging of histone states, polymerases, locations of specific genes, and other molecular events, together providing comprehensive structural, functional, and molecular information, which in the longer term may help elucidate the interplay between chromatin, epigenetics, and phenotype. This project aims to 1) develop 3D DNA-SPIN for 3D imaging of cells with spatial resolution approaching 2 nm, 2) characterize photophysical properties of label-free, endogenous DNA photoswitching, and 3) develop and validate algorithms for molecular recognition using 3D DNA-SPIN. Based on the photochemical characterization in the second aim, machine/deep learning algorithms will be developed to use DNA-SPIN emission-excitation data to distinguish nucleotide sequences, including AT- versus CG-rich parts of the genome, which are mostly associated with repressed vs gene-rich parts of the genome. The project will also explore the feasibility of generating other functional data including DNA and nucleosomal conformations, volume concentration, and surrounding ionic environment. Cross-validation experiments on fixed cells will be performed to verify the accuracy, reliability, and robustness of the algorithm. In addition, the project will provide training and educational opportunities to a diverse audience through public symposia and workshops, mentorship, outreach to K-12 students, and by recruiting undergraduate students from<br/>minority-serving institutions in the Chicago region who may not have the opportunity to have exposure or access to cutting-edge scientific research projects.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
