Systems and methods for color-scalable flow cytometry with Raman tags
Inventors
HIRAMATSU, Kotaro • NISHIYAMA, Ryo • Goda, Keisuke • DODO, Kosuke • KAWAMURA, Shintaro • Sodeoka, Mikiko • Suzuki, Hideyusi • Zhang, Charlie • Terziyan, William Yang
Assignees
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Publication Number
US-11982612-B2
Publication Date
2024-05-14
Expiration Date
Abstract
Systems and methods for flow cytometry. The methods comprise: labeling cells of a sample with Raman tags; causing the sample to flow through a microfluidic channel of a flow cytometer through which a laser beam passes; detecting Raman signals emitted from the Raman tags while being illuminated by the laser beam; and determining characteristics of the cells based on the detected Raman signals.
Core Innovation
The invention provides a method for flow cytometry that uses a plurality of different Raman-active dots. Each Raman-active dot includes a nanoparticle containing a plurality of Raman tags incorporated therein, where the Raman tags provide multiple peaks in a Raman shift region of 400–1600 cm−1. Cells of a sample are exposed to the plurality of different Raman-active dots and the sample and dots flow through a microfluidic channel of a flow cytometer as a laser beam passes.
The laser beam is generated by a single excitation laser, and Raman signals emitted from the Raman tags are detected while being illuminated by the single laser beam. Cell characteristics are measured based on the detected Raman signals, enabling distinguishing among multiple Raman-active dots by using Raman tags having multiple peaks in the 400–1600 cm−1 region. The disclosed approach supports multiplexing through Raman signals associated with different Raman-active dots.
The Raman tags are generated by substituting hydrogens in a tetraene unit of a cyanine dye by deuterium and by introducing at least one substituent to an IR740 skeleton at a respective one of a plurality of different positions. The substituent includes chlorine, bromine, or fluorine, and the introduction is performed without changing an absorption wavelength of the cyanine dye. The described system also includes a computing device that determines characteristics of the cells based on detected Raman signals.
Claims Coverage
Two independent claims are provided, one directed to a method for flow cytometry and one directed to a system. Both claims share the same core inventive structure: multiplex Raman-active dots in a microfluidic flow cytometer, illuminated by a single excitation laser, with Raman tags produced by tetraene deuteration in a cyanine dye and position-wise IR740-skeleton substitution using chlorine, bromine, or fluorine without changing absorption wavelength.
Single-excitation Raman flow cytometry with microfluidic channel
A method/system in which cells labeled with a plurality of different Raman-active dots flow through a microfluidic channel of a flow cytometer while illuminated by a laser beam generated by a single excitation laser, and Raman signals emitted from the Raman tags of the plurality of different Raman-active dots are detected to determine characteristics of the cells.
Raman-tag multiplexing using multiple peaks in 400–1600 cm−1 region
Raman-active dots include Raman tags having multiple peaks in a Raman shift region of 400–1600 cm−1, enabling measurement of cell characteristics based on the detected Raman signals emitted by the Raman tags of different Raman-active dots.
Deuterated tetraene cyanine Raman tags on IR740 skeleton positions
The plurality of different Raman tags are generated by substituting hydrogens in a tetraene unit of a cyanine dye by deuterium and introducing at least one substituent to an IR740 skeleton at a respective one of a plurality of different positions without changing an absorption wavelength of the cyanine dye.
Chlorine, bromine, or fluorine substituents on IR740 skeleton
The at least one substituent comprising chlorine, bromine, or fluorine is introduced to the IR740 skeleton at respective positions for producing different Raman tags.
Across the independent claims, the inventive concept combines a single-excitation microfluidic Raman flow cytometer with multiplex Raman-active dots whose Raman tags provide multiple peaks in the 400–1600 cm−1 region. The tags are produced by deuterating a tetraene unit in a cyanine dye and performing position-wise IR740-skeleton substitution using chlorine, bromine, or fluorine without changing the cyanine dye absorption wavelength, with a detector and computing device determining cell characteristics from the detected Raman signals.
Stated Advantages
Enables multiplexing of Raman signals using multiple narrow peaks in the 400–1600 cm−1 Raman shift region for distinguishing different Raman-active dots.
Increases color scalability for multiplex Raman flow cytometry via Raman tag design that supports different Raman tags without changing the absorption wavelength of the cyanine dye.
Documented Applications
High-throughput multiplex Raman flow cytometry for measuring characteristics of cells using labeled Raman-active dots and Raman signals.
Time-course endocytosis analysis encoded via sequential Raman-active dots exposed to cells, with inhibition used to match expected dynamics [procedural detail omitted for safety].
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