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The emergence of single molecule localization microscopy (SMLM) techniques made the imaging of cells at resolutions far beyond the diffraction barrier possible. However, the usual approach of tagging a protein of interest (PoI) with a primary antibody, and tagging this one with a fluorophore-carrying secondary antibody, introduces a significant displacement of the signal from the PoI. Here, the generation and application of an αGFP-nanobody is described which, through its reduced size and direct fluorophore labeling, leads to a much higher co-localization of signal and PoI and qualifies for dSTORM imaging of nuclear proteins., Student Undergraduate Research E-journal!, Vol 4 (2018): Student Research Conference 2018

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm 3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS , but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia., Competing Interests: Competing Interests Statement: A pending patent application “COMPUTATIONAL TECHNIQUES FOR THREE-DIMENSIONAL RECONSTRUCTION AND MULTI-LABELING OF SERIALLY SECTIONED TISSUE” was filed on 6/24/2022 by authors AK, RHH, PHW, DW, and LDW. The other authors report no competing interests.