Becton Dickinson and Company Limited

Bd Biosciences

Matthew Bahr

Keegan Owsley

Eric D Diebold

Jonathan Lin

Jizuo Zou

Majid Mehrpouyan

Disclosed herein include embodiments of a system, a device, and a method for sorting a plurality cells of a sample. A plurality of raw images comprising pixels of complex values in a frequency space can be generated from a plurality of channels of fluorescence intensity data of fluorescence emissions of fluorophores, the fluorescence emissions being elicited by fluorescence imaging using radiofrequency-multiplexed excitation in a temporal space. Spectral unmixing can be performed on the raw images prior to a sorting decision being made.

Spectral unmixing of fluorescence imaging using radiofrequency-multiplexed excitation data

Aspects of the present disclosure include methods for characterizing particles of a sample in a flow stream. Methods according to certain embodiments include generating frequency-encoded fluorescence data from a particle of a sample in a flow stream; and calculating phase-corrected spatial data of the particle by performing a transform of the frequency-encoded fluorescence data with a phase correction component. In certain embodiments, methods include generating an image of the particle in the flow stream based on the phase-corrected spatial data. Systems having a processor with memory operably coupled to the processor having instructions stored thereon, which when executed by the processor, cause the processor to calculate phase-corrected spatial data from frequency-encoded fluorescence data of a particle a flow stream are also described. Integrated circuit devices (e.g., field programmable gate arrays) having programming for practicing the subject methods are also provided.

Phase-correction of radiofrequency-multiplexed signals

Aspects of the present disclosure include methods for modulating an intensity profile of a laser beam. Methods according to certain embodiments include irradiating an acousto-optic device with a laser to generate an output laser beam having a plurality of angularly deflected laser beams, capturing an image of the output laser beam, determining an intensity profile of the output laser beam along a horizontal axis from the captured image and adjusting one or more parameters of a waveform inputted into the acousto-optic device in response to the determined intensity profile to generate an output laser beam having a modulated intensity profile. Systems having a laser, an acousto-optic device, an imaging sensor and a waveform generator as well as non-transitory computer readable storage medium with instructions for practicing the subject methods are also described.

Methods for modulating an intensity profile of a laser beam and systems for same

Aspects of the present disclosure include methods for characterizing particles of a sample in a flow stream. Methods according to certain embodiments include detecting light from a sample having cells in a flow stream, generating an image of an object in the flow stream in an interrogation region and determining whether the object in the flow stream is an aggregate based on the generated image. Systems having a processor with memory operably coupled to the processor having instructions stored thereon, which when executed by the processor, cause the processor to generate an image of an object in a flow stream and to determine whether the object is an aggregate are also described. Integrated circuit devices (e.g., field programmable gate arrays) having programming for practicing the subject methods are also provided.

Parameters for use in particle discrimination

Light detection systems for measuring light (e.g., in a flow stream) are described. Light detection systems according to embodiments include a light scatter detector, a brightfield photodetector and an optical adjustment component configured to convey light to the light scatter detector and to the brightfield photodetector. Systems and methods for measuring light emitted by a sample (e.g., in a flow stream) and kits having a light scatter detector, a brightfield photodetector and a beam splitter component are also provided.

Light detection systems and methods of use thereof

Aspects of the present disclosure include a method for sorting cells of a sample based on an image of a cell in a flow stream. Methods according to certain embodiments include detecting light from a sample having cells in a flow stream, generating an image mask of a cell from the sample and sorting the cell based on the generated image mask. Systems having a processor with memory operably coupled to the processor having instructions stored thereon, which when executed by the processor, cause the processor to generate an image mask of a cell in a sample in a flow stream and to sort the cell based on the generated image mask are also described. Integrated circuit devices (e.g., field programmable gate arrays) having programming for generating an image mask and for determining one or more features of the cell are also provided.

Systems for cell sorting based on frequency-encoded images and methods of use thereof

Aspects of the present disclosure include methods for producing an output laser beam having two or more angularly deflected laser beams (e.g., for irradiating a sample in a flow stream) with a predetermined intensity profile. Systems for practicing the subject methods having a laser, an acousto-optic device, a radiofrequency generator and a controller for adjusting the amplitude of the radiofrequency drive signals to produce an output laser beam of angularly deflected laser beams with a predetermined intensity profile are also described.

Flow cytometer with optical equalization

Aspects of the present disclosure include methods for phase correcting signals from a light detection system (e.g., in a flow cytometer). Methods according to certain embodiments include detecting light from a sample having particles in a flow stream with a light detection system that includes a brightfield photodetector configured to generate a brightfield data signal and a fluorescence detector configured to generate a fluorescence data signal and calculating a phase correction for the fluorescence detector based on the relative phase between the brightfield data signal and the fluorescence data signal. Systems having a processor with memory operably coupled to the processor having instructions stored thereon, which when executed by the processor, cause the processor to calculate a phase correction for a fluorescence detector based on a brightfield data signal and a fluorescence data signal from the fluorescence detector are also described. Integrated circuit devices (e.g., field programmable gate arrays) having programming for practicing the subject methods are also provided.

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Fremont, CA, United States of America

Matthew Bahr