Project Summary Flow cytometry is widely used in basic and translational research and in clinical diagnostic assays. Spectral flow cytometry represents a revolutionary change in the approach to flow cytometry by using full spectral information from each fluorophore—by collecting the emission over the full spectrum and using multiple excitation sources for each fluorophore to obtain a spectral “signature” or “fingerprint”. The resulting spectral signature is used in automated spectral unmixing that is simpler and more effective than current compensation methods. As a result, even fluorophores with highly overlapping spectra can be used simultaneously, simplifying panel construction and enlarging the maximum panel size; subtraction of cell autofluorescence is also improved. These factors combine to allow greater resolution of cell subpopulations. In addition, hardware can be simplified since a particular configuration of filter sets is not needed for a particular set of dyes. Spectral flow cytometry has progressed to the mainstream, with several commercial spectral flow systems now available (Cytek Aurora, Sony ID7000, BD Symphony A5 SE, Thermo Bigfoot). However, the development of dyes has not kept pace with the development of spectral flow systems. For example, Sony’s ID7000 system includes a 320 nm excitation source for which no dyes have been specifically developed. We and others have developed a new family of ultrabright fluorescent nanoparticles called Pdots, which are semiconducting polymers collapsed into 5–30 nm (tunable size) nanoparticles. Pdots are bright and photostable, and are unique in exhibiting a long, tunable Stokes shift, enabling the creation of a large color panel. Here we propose to expand the multi-color panel of Pdots from 37 to 75 by adding 38 new colors, including a new series of 18 Pdots excited at 320 nm (no previous Pdots have been excitable at 320 nm), as well as 20 new Pdots excited at 355, 405, 488, 561, and 640 nm, filling gaps between the emission peaks of existing Pdots with the same excitation while preserving a separation of at least 20 nm between emission peaks with most emission peaks separated by 30 nm or larger. We will demonstrate the utility of these Pdots in spectral flow cytometry by developing and validating a 75-parameter panel for deep immunophenotyping of major cell subsets in human peripheral blood. The 75-color panel of Pdots will also be useful in high multiplex tissue imaging (e.g., histopathology).

Project Narrative / Public Health Relevance Statement Personalized medicine requires technologies for measuring many disease biomarkers simultaneously for use in diagnostic tests; one widely used technology for clinical single-cell assays is flow cytometry, which is undergoing a transformation to greater multiplexing using a dramatically different technological approach called “spectral flow cytometry.” We have previously developed ultrabright high-multiplexing fluorescent probes called Pdots and have commercialized these Pdots for flow cytometry. Here we propose to expand our multi-color panel of Pdots to 75 for use in high multiplex spectral flow cytometry; this multi-color panel will also be useful in high multiplex tissue imaging (e.g., histopathology).