Nearly two meters of DNA are packed into every cell nucleus. Within this volume, gene expression is regulated by the binding of transcription factors, cofactors, and RNA polymerase machinery to the promoters of target genes. However, how a transcription factor navigates through roughly 3.2 billion base pairs to find a 100-1000 base pair long promoter is an open question. Nor is it clear how the underlying organization, composition, and dynamics of chromatin within the nucleus regulates transcription factor search dynamics. Answering these questions requires technologies that are capable of directly visualizing transcription factors, which diffuse in milliseconds, over the hours to days required for cellular fate specification.
With support from the NIH New Innovator Program we are developing a new multifunctional lightsheet microscope. This Multimodal Optical microScope with Adaptive Imaging Correction (MOSAIC) instrument permits rapid cellular imaging under several different types of diffraction limited and super-resolution modalities. In parallel, we are developing microfluidic systems that are compatible with the angled orientation of lightsheet microscopy objectives. We fully characterize the optical performance (aberration, transmission, and polarization effects) of these chips and demonstrate that they are compatible with high resolution lightsheet, single-molecule imaging, and structured illumination microscopy. Additionally, we demonstrate that they support imaging of both adherent and non-adherent cells, allow for rapid exchange of reagents while imaging, and maintain cell growth and sample sterility over multiple days of imaging.
We are applying these advances to visualize the single-molecule dynamics of transcription factor search and directly visualize how they navigate the genome at different stages of cell differentiation. These studies will provide a novel window into how genes are regulated in both normal and disease settings.
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