A 3D super-resolved platform for rapid and highly multiplexed proteomics

Project Summary Addressing the complexities of brain function and disorders at the cellular and molecular level requires a spatial omics technology, capable of mapping the 3D distribution of specific cell markers within tissue at the subcellular scale. While spatial proteomics technologies have revolutionized in situ molecular profiling of tissues, limitations in resolution, epitope accessibility, sample degradation, and 3D imaging capability have hindered their widespread impact. This is the focus of our project, which proposes to combine two groundbreaking microscopy technologies, pan-Single Step Expansion Microscopy (pan-SSExM) and FLASH-PAINT, into a revolutionary new method named pan-OptiX S3D. Our optimized pan-SSExM protocol offers tunable physical sample expansion between 1.5-fold and 8-fold linearly, while retaining the proteome for subsequent labeling. FLASH-PAINT, on the other hand, allows rapid and unlimited 3D multiplexed protein labeling without any sample damage. By synergizing these technologies, pan-OptiX S3D promises subcellular and multicolor optical imaging, in 3D, across whole tissue sections, with no label limitations due to degradation or steric hindrance. Panluminate, Inc. is a leading company in the Spatial Biology and Tissue Expansion fields. Our proposed project aims to develop pan-OptiX S3D and validate it in rodent brain tissue to enable analysis of neural networks and pathologies in the brain. We specifically propose to (1) develop novel FLASH probes specifically designed for compatibility with 5-fold tissue expansion imaging, and (2) automate sample expansion and microfluidics multiplexing with 3D microscopy and software integration. Ultimately, the developments proposed here will enable panluminate to disseminate pan-OptiX S3D sample preparation and imaging to every research and biotech lab around the world.