Ultra Bright Fluorescent Probes for Flow Cytometry

Project Summary For modern flow cytometry high throughput multiplexing is extremely important because of the great need in analyzing a large number of biomolecules on and in a single cell The multiplexing performance of flow cytometry is hindered by the probe performance including their fluorescence brightness and emission bandwidth However there is renewed interest and effort to develop highly multiplexed e g probes fluorescence based flow cytometry analysis of cells For this high degree of multiplexing with fluorescence both probe brightness and narrow emission are critical specifically because of how compensation for spectral spillover works a very bright probe greatly facilitates multiplexing by minimizing spillover spreading which is a key driver for extremely bright probes in flow cytometry The ability to detect low expression antigens is also another important capability provided by very bright probes Recently we and others have developed fluorescent nanoparticles based on semiconducting polymers called Pdots The motivation of adapting fluorescent semiconducting polymers into nanoparticle labels stems from a number of favorable characteristics such as large absorptivity high quantum yield fast emission rates and excellent photostability The resulting Pdots exhibit extraordinarily high fluorescence brightness under both one photon and two photon excitations a factor of higher than conventional fluorescent dyes and a factor of higher than Qdots depending on the particle size We have successfully designed and prepared a Pdot series with nm excitation Following on this successful development we propose to design three additional Pdot series for flow cytometry excitable at nm nm and nm thus matching the laser wavelengths of existing flow cytometers Project Narrative Flow cytometry is a high throughput technology that is extensively employed in biology and medicine for quantifying cellular features cell lineages and cell signaling pathways Some applications of flow cytometry include the cell cycle and surface analysis of cancers monitoring the progression of AIDS and the response to treatment by measuring CD lymphocyte levels in blood the study of surface markers of lymphomas and leukemias and characterization of immune cells for immuno therapy and precision medicine all of which are of significant value for diagnostics and prognostics This proposal aims to develop a new class of reagents for flow cytometry