Development of multiplexed nanoscopy for ultrastructural phenotyping of retinal tissue

About This Grant

Project Summary Addressing the complexities of vision at the cellular and molecular level necessitates an advanced spatial omics technology, capable of mapping the 3D distribution of specific molecular markers within tissue from the cellular level down to the ultrastructural 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-Expansion Microscopy (pan-ExM) and FLASH- PAINT, into a revolutionary new method named pan-OptiX. Pan-ExM enhances spatial resolution ~20x by expanding samples physically, while retaining the proteome for subsequent labeling. FLASH-PAINT, on the other hand, allows rapid and efficient multiplexed protein labeling with minimal sample damage. By engineering these technologies for compatibility, pan-OptiX promises multi-scale and multi-color imaging, in 3D, across whole tissue sections, with no label limitations due to degradation or steric hindrance. Panluminate, Inc. is a leading spatial proteomics company which holds an exclusive license to both technologies from Yale. Our proposed project aims to develop pan-OptiX and apply it to map the circuitry of the mammalian retina. We specifically propose to (1) validate a second-generation pan-ExM protocol compatible with post-expansion DNA-conjugated antibody labeling and multiplexing, (2) develop novel FLASH-PAINT probes compatible with expansion imaging, (3) build an automated light sheet microscope designed specifically to image pan-OptiX samples, and (4) characterize the molecular and ultrastructural heterogeneity of the mouse retina. Ultimately, the developments proposed here will enable Panluminate to disseminate pan-OptiX sample preparation and imaging to every research and biopharma lab around the world.