Deep Immunophenotyping in tissue imaging

PROJECT SUMMARY There is a growing demand for deep phenotyping of single cells in tissue, giving rise to the field of spatial biology. High multiplex imaging technologies such as cyclic immunofluorescence and mass spectrometry imaging are poised to transform spatial biology and pathology workflows. However, these technologies are limited by their cost, complexity, and long sample-to-result time. Iterative fluorescence approaches are limited to 3–6-plex per round of staining, imaging, and de-staining, while mass spectrometry imaging can provide ~50 plex but with lower throughput and much higher costs. This proposal’s objective is to address these bottlenecks by significantly expanding fluorescence spectral multiplexing in tissue imaging—the maximum number of protein markers per fluorescence imaging cycle—from 3–6 plex to 56 plex, by developing a new type of ultrabright semiconducting polymer dot (Pdot) with simultaneously narrow absorption and emission bands. We will accomplish this objective via the following aims: (1) Develop a 56-color panel of Pdots with simultaneously narrow absorption and emission bands; (2) Demonstrate 56-plex imaging applied to immune profiling of cells in tissue samples and evaluate performance; (3) Demonstrate even higher multiplex imaging (168 plex) via cyclic imaging (with only 3 cycles) for extremely deep cell immuno-phenotyping, useful in clinical discovery and research applications. The narrow-absorption Pdots will be immediately applicable as they can be used with existing commercial instruments. These Pdots will also enable spectral imaging—a new technology inspired by spectral flow cytometry, in which the full spectrum of dye emission is collected to better distinguish dyes and facilitate higher multiplexing. The proposed project is significant because it addresses the need for detecting tens to hundreds of protein markers in tissue samples in a simple, timely, and affordable manner.