Systems and methods for classifying T cells by activation state are disclosed. The system includes a cell analysis pathway, a time-resolved autofluorescence decay spectrometer, a processor, and a non-transitory computer-readable memory. The memory is accessible to the processor and has stored thereon instructions. The instructions, when executed by the processor, cause the processor to: a) receive the time-resolved autofluorescence decay signal; b) compute at least a first phasor coordinate at a first frequency and a second phasor coordinate at a second frequency from the time-resolved autofluorescence decay signal, wherein the first and second frequency are different; and c) compute an activation prediction for the T cell using at least the first phasor coordinate and the second phasor coordinate.

An apparatus and method for analyzing particulates in a sample is disclosed. The method includes placing the sample on a moveable stage in an apparatus having a tunable MIR light scanner and a visible imaging system, the stage moving between the MIR light scanner and the visible imaging system, providing a visible image of the sample, and receiving user input as to a region of the sample that is to be analyzed. The sample is then moved to the MIR light scanner, the MIR light scanner generating an MIR light beam that is focused to a point on the specimen and measuring light reflected from the specimen. The specimen is then scanned at a first MIR wavelength by moving the specimen relative to the MIR light beam, and particles are identified that meet a selection criterion. The MIR absorption spectrum of each of the identified particle is then automatically measured.

Methods for characterizing spillover spreading originating from a first fluorochrome in fluorescent flow cytometer data collected for a second fluorochrome are provided. In some embodiments, methods include partitioning the fluorescent flow cytometer data according to the intensity of the data relative to the first fluorochrome. In embodiments, methods also include estimating with a first linear regression a zero-adjusted standard deviation for the intensity of light collected from the second fluorochrome for each of the partitioned quantiles based on the assumption that the intensity of light collected from the first fluorochrome is zero, and obtaining with a second linear regression a spillover spreading coefficient from the zero-adjusted standard deviations. Systems and computer-readable media for characterizing spillover spreading originating from a first fluorochrome in fluorescent flow cytometer data collected for a second fluorochrome are also provided.

Aspects of the present disclosure include methods for adjusting sensitivity of a photodiode in a light detection system. Methods according to certain embodiments include determining a background data signal from a photodetector at a plurality of detector gains, irradiating the photodetector with a light source at a plurality of different light intensities, generating data signals from the photodetector for the plurality of light intensities at each detector gain and adjusting the photodetector to the detector gain that corresponds to the lowest light irradiation intensity that generates a data signal resolvable from the background data signal. 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. Non-transitory computer readable storage medium are also provided.

The present invention discloses a three-dimensional diffraction tomography microscopy imaging method based on LED array coded illumination. Firstly, acquiring the raw intensity images, three sets of intensity image stacks are acquired at different out-of-focus positions by moving the stage or using electrically tunable lens. And then, after acquiring the intensity image stacks of the object to be measured at different out-of-focus positions, the three-dimensional phase transfer function of the microscopy imaging system with arbitrary shape illumination is derived. Further, the three-dimensional phase transfer function of the microscopic system under circular and annular illumination with different coherence coefficients is obtained as well, and the three-dimensional quantitative refractive index is reconstructed by inverse Fourier transform of the three-dimensional scattering potential function. The scattering potential function is converted into the refractive index distribution. Thus, the quantitative three-dimensional refractive index distribution of the test object is obtained. The invention realizes high-resolution and high signal-to-noise ratio 3D diffraction tomography microscopic imaging of cells, tiny biological tissues and other samples.

Aspects of the present disclosure include methods for adjusting sensitivity of a photodiode in a light detection system. Methods according to certain embodiments include determining a background data signal from a photodetector at a plurality of detector gains, irradiating the photodetector with a light source at a plurality of different light intensities, generating data signals from the photodetector for the plurality of light intensities at each detector gain and adjusting the photodetector to the detector gain that corresponds to the lowest light irradiation intensity that generates a data signal resolvable from the background data signal. 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. Non-transitory computer readable storage medium are also provided.

Aspects of the present disclosure include flow cytometers configured to compensate for optical noise caused by operational change of a laser. Flow cytometers according to certain embodiments include a laser assessor configured to assess operational change of a laser. In embodiments, the laser assessor includes a reference detector and a mirror positioned between the reference detector and the flow cell that is configured to reflect forward scattered light to a forward scatter detector and allow non-scattered laser light to pass through to the reference detector. Methods for assessing laser functionality and, where desired, dynamically adjusting flow cytometer data, based on laser reference data from the laser assessor are also provided.

Methods for determining a parameter of a particle in a flow stream (e.g., in a particle analyzer of a flow cytometer) from scattered light are described. Methods according to certain embodiments include irradiating a particle in a flow stream with a frequency-modulated beam of laser light modulated at a reference frequency, detecting scattered light from the particle with a photodetector, generating a frequency-encoded data signal from the detected scattered light, synchronizing the frequency-encoded data signal with the reference frequency and determining one or more parameters of the particle based on the synchronized frequency-encoded data signal. Systems and non-transitory computer readable storage medium with instructions for practicing the subject methods are also provided.

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. For example, a horizontal extent of the sample can be illuminated by a laser beam at a single excitation frequency. The detected fluorescence radiation can be used to analyze the fluorescence content of the sample, e.g., a cell/particle.

A sample analyzer includes a sample preparation device, an impedance testing device, an optical testing device, and a controller. The controller is configured to control the impedance testing device to test a first test sample to obtain a first platelet counting result for a test blood sample based on electronic information of the first test sample; control the optical testing device to test a second test sample to obtain a second platelet counting result for the test blood sample based only on optical information of the second test sample or based on both the electronic information of the first test sample and the optical information of the second test sample; determine whether the first platelet counting result is unreliable due to abnormality of the test blood sample; and output the first and/or the second platelet counting result according to the result of the determination.