Abstract: 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.

Abstract: 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.

Abstract: Non-parametric transforms such as t-distributed stochastic neighbor embedding (tSNE) are used to analyze multi-parametric data such as data derived from flow cytometry or other particle analysis systems and methods. These transforms may be included for dimensionality reduction and identification of subpopulations (e.g., gating). By nature, non-parametric transforms cannot transform new observations without training a new transformation based on the entire dataset including the new observations. The features described parameterize non-parametric transforms using a neural network thereby allowing a small training dataset to be transformed using non-parametric techniques. The training dataset may then be used to generate an accurate parametric model for assessing additional events in a manner consistent with the initial events.

Abstract: A cell sorting system that automatically generates a sorting strategy based on examples of target events provided by an operator. The target events can be selected using measurements ranging from traditional flow cytometry measurements to derived measurements that are computationally expensive to complex measurements such as images.

Abstract: Aspects of the present disclosure include methods for determining baseline noise of a photodetector (e.g., in a light detection system of a particle analyzer). Methods according to certain embodiments include irradiating a sample having particles in a flow stream, detecting light with the photodetector from the irradiated flow stream, generating data signals from the detected light and calculating a moving average mean squared error of the generated data signals to determine the baseline of the photodetector. Systems (e.g., particle analyzers) having a light source and a light detection system that includes a photodetector for practicing the subject methods are also described. Integrated circuits and non-transitory computer readable storage medium are also provided.

Abstract: 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.

Abstract: 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.

Abstract: A cell sorting system that automatically generates a sorting strategy based on examples of target events provided by an operator. The target events can be selected using measurements ranging from traditional flow cytometry measurements to derived measurements that are computationally expensive to complex measurements such as images.

Abstract: 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.

Abstract: In one aspect, a method of sorting cells in a flow cytometry system is disclosed, which includes illuminating a cell with radiation having at least two optical frequencies shifted from one another by a radiofrequency to elicit fluorescent radiation from the cell, detecting the fluorescent radiation to generate temporal fluorescence data, and processing the temporal fluorescence data to arrive at a sorting decision regarding the cell without generating an image (i.e., a pixel-by-pixel image) of the cell based on the fluorescence data.. In other words, while the fluorescence data can contain image data that would allow generating a pixel-by-pixel fluorescence intensity map, the method arrives at the sorting decision without generating such a map. In some cases, the sorting decision can be made with a latency less than about 100 microseconds, In some embodiments, the above method of sorting cells can have a sub-cellular resolution, e.g.

Abstract: Aspects of the present disclosure include methods for flow cytometrically sorting a sample with particles, such as cells, based on order of identification. Methods according to certain embodiments include introducing the sample into a flow cytometer; flowing the introduced sample in a flow stream; irradiating the sample in the flow stream with a light source; detecting light from cells in the sample flowing in the flow stream; identifying phenotypes of cells in the sample flowing in the flow stream based on one or more data signals generated from the detected light; and dynamically sorting into partitions cells of the sample that have a phenotype of a collection of predetermined phenotypes based on order of identification. Systems for practicing the subject methods are also provided. Non-transitory computer readable storage mediums are also described.

Abstract: In one aspect, a method of sorting cells in a flow cytometry system is disclosed, which includes illuminating a cell with radiation having at least two optical frequencies shifted from one another by a radiofrequency to elicit fluorescent radiation from the cell, detecting the fluorescent radiation to generate temporal fluorescence data, and processing the temporal fluorescence data to arrive at a sorting decision regarding the cell without generating an image (i.e., a pixel-by-pixel image) of the cell based on the fluorescence data. In some cases, the sorting decision can be made with a latency less than about 100 microseconds. In some embodiments, the above method of sorting cells can have a sub-cellular resolution. In some embodiments, a single radiofrequency shift is employed to separate the optical frequencies while in other such embodiments a plurality of different radiofrequency shifts are employed.

Abstract: Aspects of the present disclosure include methods for spectrally resolving light from fluorophores having overlapping fluorescence spectra in a sample. Methods according to certain embodiments include detecting light with a light detection system from a sample having a plurality of fluorophores having overlapping fluorescence spectra and spectrally resolving light from each fluorophore in the sample. In some embodiments, methods include estimating the abundance of one or more of the fluorophores in the sample, such as on a particle. In certain instances, methods include identifying the particle in the sample based on the abundance of each fluorophore and sorting the particle. Methods according to some embodiments includes spectrally resolving the light from each fluorophore by calculating a spectral unmixing matrix for the fluorescence spectra of each fluorophore. Systems and integrated circuit devices (e.g., a field programmable gate array) for practicing the subject methods are also provided.

Abstract: Aspects of the present disclosure include methods for spectrally resolving light from fluorophores having overlapping fluorescence spectra in a sample. Methods according to certain embodiments include detecting light with a light detection system from a sample having a plurality of fluorophores having overlapping fluorescence spectra and spectrally resolving light from each fluorophore in the sample. In some embodiments, methods include estimating the abundance of one or more of the fluorophores in the sample, such as on a particle. In certain instances, methods include identifying the particle in the sample based on the abundance of each fluorophore and sorting the particle. Methods according to some embodiments includes spectrally resolving the light from each fluorophore by calculating a spectral unmixing matrix for the fluorescence spectra of each fluorophore. Systems and integrated circuit devices (e.g., a field programmable gate array) for practicing the subject methods are also provided.

Abstract: In one aspect, the present teachings provide a system for performing cytometry that can be operated in three operational modes. In one operational mode, a fluorescence image of a sample is obtained by exciting one or more fluorophore(s) present in the sample by an excitation beam formed as a superposition of a top-hat-shaped beam with a plurality of beams that are radiofrequency shifted relative to one another. In another operational mode, a sample can be illuminated successively over a time interval by a laser beam at a plurality of excitation frequencies in a scanning fashion. The fluorescence emission from the sample can be detected and analyzed, e.g., to generate a fluorescence image of the sample. In yet another operational mode, the system can be operated to illuminate a plurality of locations of a sample concurrently by a single excitation frequency, which can be generated, e.g., by shifting the central frequency of a laser beam by a radiofrequency.

Abstract: In one aspect, a system for performing flow cytometry is disclosed, which comprises a laser for generating laser radiation for illuminating a sample, at least one detector for detecting at least a portion of a radiation emanating from the sample in response to said illumination so as to generate a temporal signal corresponding to said detected radiation, and an analysis module for receiving said temporal signal and performing a statistical analysis of said signal based on a forward model to reconstruct an image of said sample.

Abstract: In one aspect, a system for performing flow cytometry is disclosed, which comprises a laser for generating laser radiation for illuminating a sample, at least one detector for detecting at least a portion of a radiation emanating from the sample in response to said illumination so as to generate a temporal signal corresponding to said detected radiation, and an analysis module for receiving said temporal signal and performing a statistical analysis of said signal based on a forward model to reconstruct an image of said sample.

Abstract: In one aspect, the present teachings provide a system for performing cytometry that can be operated in three operational modes. In one operational mode, a fluorescence image of a sample is obtained by exciting one or more fluorophore(s) present in the sample by an excitation beam formed as a superposition of a top-hat-shaped beam with a plurality of beams that are radiofrequency shifted relative to one another. In another operational mode, a sample can be illuminated successively over a time interval by a laser beam at a plurality of excitation frequencies in a scanning fashion. The fluorescence emission from the sample can be detected and analyzed, e.g., to generate a fluorescence image of the sample. In yet another operational mode, the system can be operated to illuminate a plurality of locations of a sample concurrently by a single excitation frequency, which can be generated, e.g., by shifting the central frequency of a laser beam by a radiofrequency.

Abstract: 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.

Abstract: 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.