Methods and systems for co-linear particle imaging

Abstract

Described herein are methods and systems for imaging one or more particles flowing through a fluidic channel. The system includes a first light source optically coupled to a first beam resizing optical element, a second light source optically coupled to a second beam resizing optical element, and a data processing device for processing optical data from light scanners.

Description

METHODS AND SYSTEMS FOR CO-LINEAR PARTICLE IMAGINGCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of US provisional application no. 63 / 712,269, filed October 25, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Particles traveling through a fluidic system, although constrained by a channel or flow cell of the fluidic system travel through such a system with non-uniform velocities and continuously rotating orientation. Such issues complicate accurately imaging and analyzing particles for purposes of identification of the particle, image registration across channels and sorting of the particles as they flow through the fluidic system. Thus, there exists an unmet need for an imaging system and / or device capable of rapidly imaging flowing particles illuminated by multiple wavelengths of light to accurately identify particles and sort them as necessary for further downstream biological assays or analysis.SUMMARY

[0003] An aspect of the present disclosure provides a particle imaging system, comprising: an imaging device comprising: a first light source optically coupled to a first beam resizing optical element, wherein an output of the first beam resizing optical element is optically coupled to a first light source scanner configured to scan a first beam of the first light source along an axis of a particle to obtain a first data set comprising fluorescent information or label-free image information of the particle, a second light source optically coupled to a second beam resizing optical element, wherein an output of the second beam resizing optical element is optically coupled to a second light source scanner configured to scan a second beam of the second light source along the axis of the particle to obtain a second data set comprising fluorescent information or label-free image information of the particle, wherein the particle is flowing in a channel at an angle to an optical axis of the first beam of the first light source and an optical axis of the second beam of the second light source, and wherein the first beam resizing optical element and the second beam resizing optical element differ; and a data processing device in communication with the imaging device, wherein the data processing device comprises a processor configured to process the first data set, the second data set, or a combination thereof, obtained by the imaging device to determine one or more properties associated with the particle. In some embodiments, the first beam resizing optical element comprises a first anamorphic prism pair or a first cylindrical lens, and wherein the second beam resizing optical element comprises asecond anamorphic prism pair or a second cylindrical lens. In some embodiments, the first data set, the second data set, or a combination thereof, is generated by a detector configured to detect one or more optical signals emitted, attenuated, or scattered by the particle in response to the illumination of particle by the first scanned beam, the second scanned beam, or a combination thereof, and wherein the one or more optical signals emitted or scattered by the particle are emitted, attenuated, or scattered along an optical axis towards an emission of the first light source, the second light source, or a combination thereof. In some embodiments, the one or more optical signals emitted or scattered by the particle along the optical axis towards the emission of the first light source, the second light source, or a combination thereof, comprise, darkfield optical signals, back scattered optical signals, epifluorescent optical signals, or a combination thereof. In some embodiments, the detector comprises a camera, one or more photomultiplier tubes, one or more photodiodes, avalanche photodetector, array of photomultiplier tubes, a fiber coupled detector, or a combination thereof. In some embodiments, the one or more photomultiplier tubes are optically coupled to the one or more optical signals emitted, or scattered by the particle through a beam splitter. In some embodiments, the first scanned beam of light is coupled to a third anamorphic prism pair, and wherein the second scanned beam of light is coupled to a fourth anamorphic prism pair. In some embodiments, the first anamorphic prism pair and the third anamorphic prism pair magnify a scanned field of view of the first scanned beam provided by the first light source scanner, and wherein the second anamorphic prism pair and the fourth anamorphic prism pair magnify a scanned field of view of the second scanned beam provided by the second light source scanner. In some embodiments, the first anamorphic prism pair, the second anamorphic prism pair, or a combination thereof, comprise a reversed optical orientation compared with the third anamorphic prism pair, the fourth anamorphic prism pair, or a combination thereof. In some embodiments, the first light source scanner, the second light source scanner, or a combination thereof, comprise an Acousto-Optic Deflector (AOD). In some embodiments, the first scanned beam of light is coupled to a first cylindrical lens, wherein the second scanned beam of light is coupled to a second cylindrical lens, and wherein the first cylindrical lens and the second cylindrical lens differ. In some embodiments, the first cylindrical lens compensates for a first astigmatism of the first scanned beam of light, and wherein the second cylindrical lens compensates for a second astigmatism of the second scanned beam of light.

[0004] In some embodiments, the system comprises a third light source optically coupled to a fifth anamorphic prism pair and a third cylindrical lens, wherein a light stripe of the third light source outputted by the third cylindrical lens illuminates the particle thereby producing a third data set comprising fluorescent information or label-free image information of the particle. Insome embodiments, the light stripe of the third light source does not spatially overlap with the first scanned beam of the first light source, the second scanned beam of the second light source, or a combination thereof. In some embodiments, the data processing device processes the third data set in comparison to the first data set, the second data set, or a combination thereof, to determine a speed of the particle flowing in the channel. In some embodiments, the speed of the particle flowing in the channel is determined by conducting autocorrelation, thresholding, or a combination thereof, with the first data set, the second data set, the third data set, or a combination thereof. In some embodiments, the third light source comprises a light emitting diode, a laser, super luminescent diode, white light source, or any combination thereof.

[0005] In some embodiments, the system comprises a mirror, wherein the mirror comprises an annular reflective region and a transmissive region, wherein the annular reflective region and the transmission region are spatially separated, wherein the first scanned beam, the second scanned beam, the light stripe, or a combination thereof, are reflected from the annular reflective region toward the particle, and wherein the one or more optical signals emitted or scattered by the particle transmit through the transmission region. In some embodiments, the transmission region comprises an anti -reflective coating. In some embodiments, the light stripe of the third light source is coupled to an optical axis of the first scanned beam, the second scanned beam, or a combination thereof, with a polarization beam splitter. In some embodiments, the imaging device comprises a bright field detector, a dark field detector, a speed detector, or any combination thereof, optically coupled to the particle, wherein the bright field detector, the dark field detector, the speed detector, or any combination thereof, is configured to detect one or more scattered or attenuated optical signals of the particle in response to the illumination of the particle by the first scanned beam, the second scanned beam, or a combination thereof, and wherein the one or more optical signals emitted or scattered by the particle are emitted or scattered along an optical axis away from an emission of the first light source, the second light source, or a combination thereof. In some embodiments, the imaging device comprises a bright field detector, a dark field detector, or a combination thereof, optically coupled to the particle, wherein the bright field detector, the dark field detector, or a combination thereof, is configured to detect one or more scattered or attenuated optical signals of the particle in response to the illumination of the particle by the first scanned beam, the second scanned beam, the light stripe, or any combination thereof, and wherein the one or more optical signals emitted or scattered by the particle are emitted or scattered along an optical axis away from an emission of the first light source, the second light source, the third light source, or any combination thereof. In some embodiments, the one or more optical signals emitted or scattered by the particle along the optical axis away from the emission of the first light source, the second light source, third light source, or any combination thereof,comprise brightfield microscopy optical signals, forward scattered optical signals, or a combination thereof. In some embodiments, the processor is in electrical communication with memory storing one or more predictive models configured to process the first data set, the second data set, the third data set, or a combination thereof, to provide a classification of the particle. In some embodiments, the one or more predictive models comprise clustering, classifying, or a combination thereof predictive models. In some embodiments, the one or more predictive models cluster the first data set, the second data set, the third data set, or any combination thereof, based on one or more morphologic features of the first data set, the second data set, the third data set, or a combination thereof. In some embodiments, the classification of the particle comprises a classification of one or more regions or one or more segments of the particle. In some embodiments, the particle comprises a cell, and wherein the one or more regions or the one or more segments of the particle comprise one or more subcellular components of the cell. In some embodiments, the one or more predictive models comprise a convolutional neural network, a logistic regression model, sequential perceptron model, k-nearest neighboring model, support vector machine model, adaboost model, random forest model, or any combination thereof. In some embodiments, the convolutional neural network comprises a structure of a UNet predictive model, a convolutional autoencoder, a fused UNet predictive model, or any combination thereof. In some embodiments, the first scanned beam of the first light source and the second scanned beam of the second light source spatially overlap. In some embodiments, the first scanned beam of the first light source and the second scanned beam of the second light source do not spatially overlap. In some embodiments, an intensity of the first light source comprises a first intensity modulation frequency 180 degrees out of phase with a second intensity modulation frequency of the second light source. In some embodiments, the first light source or the second light source comprises a center wavelength of about 355nm to about 785 nm. In some embodiments, the first light source scanner, the second light source scanner, or a combination thereof, is provided an amplitude modulated chirp driving signal to scan the first beam, the second beam, or a combination thereof. In some embodiments, the amplitude modulated chirp driving signal provides an output power of at least 5 mW of the first scanned beam, the second scanned beam, or a combination thereof, across a field of view of the first light source scanner, the second light source scanner, or a combination thereof. In some embodiments, the data processing device comprises a field programmable gate array (FPGA), graphical processing unit (GPU), neural processing unit (NPU), or any combination thereof. In some embodiments, the first light source, the second light source, or a combination thereof, comprise fiber coupled lasers. In some embodiments, the particle comprises one or more cells. In some embodiments, the one or more properties associated with the particle comprise a gradient root mean square, contrast, particlecentroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof. In some embodiments, the one or more properties are used to align the fluidic channel and the imaging device. In some embodiments, the one or more properties are determined by cross-correlation of the first data set and the second data set, the first data set and the third data set, the second data set and the third data set, or a combination thereof. In some embodiments, the first data set, the second data set, the third data set, or a combination thereof, comprise an image data set of the particle. In some embodiments, the first scanned beam and the second scanned beam are sequentially scanned along the axis of the particle. In some embodiments, when the first scanned beam is scanned along the axis of the particle, the second scanned beam is directed to a first beam capturing optical component, and wherein when the second scanned beam is scanned along the axis of the particle, the first scanned beam is directed to a second beam capturing optical component. In some embodiments, the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, comprise a beam dump. In some embodiments, a background signal is removed or subtracted from the first data set, the second data set, or a combination thereof, wherein the background signal comprises one or more optical signals detected, obtained, and / or collected when the channel is illuminated with the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, the first data set, the second data set, or a combination thereof, is phase shifted by one or more obtained data samples to synchronize the scanning of the first light source scanner and obtaining the first data set and to synchronize the scanning of the second light source scanner and obtaining the second data set. In some embodiments, the first light source scanner and the second light scanner differ. In some embodiments, the data processing device sorts the particle based at least on the one or more properties associated with the particle. In some embodiments, the data processing device sorts the particle with a piezo electric sorting apparatus. In some embodiments, the data processing device sorts the particle based at least on the one or more properties associated with the particle determined by gating the first data set or the second data set. In some embodiments, the label-free image information comprises non-fluorescent image information of the particle.

[0006] Another aspect of the present disclosure provides a method for scanning a particle, comprising: moving a particle along a first direction; generating a first scanned beam of a first light source with a first light source scanner and a second scanned beam of a second light source with a second light source scanner, wherein an output of the first light source is optically coupledto a first beam resizing optical element and an output of the second light source is optically coupled to a second beam resizing optical element, wherein an output of the first beam resizing optical element is optically coupled to the first light source scanner and an output of the second beam resizing optical element is optically coupled to the second light source scanner, and wherein the first beam resizing optical element and the second beam resizing optical element differ; and scanning the particle with the first scanned beam of the first light source and the second scanned beam of the second light source in a second direction at an angle to the first direction. In some embodiments, the first beam resizing optical element comprises a first anamorphic prism pair or a first cylindrical lens, and wherein the second beam resizing optical element comprises a second anamorphic prism pair or a second cylindrical lens. In some embodiments, the method comprises detecting a first data set of the particle from one or more optical signals emitted, attenuated, or scattered by the particle in response to illumination of the particle with the first scanned beam, and detecting a second data set of the particle from one or more optical signals emitted or scattered by the particle in response to illumination of the particle with the second scanned beam. In some embodiments, the first data set, the second data set, or a combination thereof comprise an image data set of the particle. In some embodiments, the method comprises processing the first data set and the second data set to determine one or more properties associated with the particle. In some embodiments, the one or more properties of the particle comprise speed, fluorescent signal, morphologic feature, or a combination thereof. In some embodiments, the morphologic feature data is adjusted based on the particle speed. In some embodiments, the one or more optical signals emitted or scattered by the particle in response to illumination of the particle by the first scanned beam, the second scanned beam, or a combination thereof, are emitted or scattered along an optical axis towards an emission of the first light source, the second light source, or a combination thereof. In some embodiments, the one or more optical signals emitted, attenuated, or scattered by the particle in response to the illumination along the optical axis towards the emission of the first light source, the second light source, or a combination thereof, comprise darkfield optical signals, back scattered optical signals, epifluorescent optical signals, or a combination thereof. In some embodiments, the one or more optical signals emitted or scattered by the particle in response to the illumination of the particle with the first scanned beam, the second scanned beam, or a combination thereof, is detected by a camera, one or more photomultiplier tubes, one or more photodiodes, a fiber coupled detector, or any combination thereof. In some embodiments, the first scanned beam is coupled to a third anamorphic prism pair, and wherein the second scanned beam is coupled to a fourth anamorphic prism pair. In some embodiments, the first anamorphic prism pair and the third anamorphic prism pair magnify a scanned field of view of the first scanned beam provided by the first light sourcescanner, and wherein the second anamorphic prism pair and the fourth anamorphic prism pair magnify a scanned field of view of the second beam provided by the second light source scanner. In some embodiments, the first anamorphic prism pair, the second anamorphic prism pair, or a combination thereof, comprise a reversed optical orientation compared with the third anamorphic prism pair, the fourth anamorphic prism pair, or a combination thereof. In some embodiments, the first light source scanner, the second light source scanner, or a combination thereof, comprise an Acousto-Optical Deflector (AOD). In some embodiments, the first scanned beam of light is coupled to a first cylindrical lens, wherein the second scanned beam of light is coupled to a second cylindrical lens, and wherein the first cylindrical lens and the second cylindrical lens differ. In some embodiments, the first cylindrical lens compensates for a first astigmatism of the first scanned beam of light, and wherein the second cylindrical lens compensates for a second astigmatism of the second scanned beam of light. In some embodiments, a third light source is optically coupled to a fifth anamorphic prism pair and a third cylindrical lens, wherein a light stripe of the third light source outputted by the third cylindrical lens illuminates the particle thereby producing a third data set of the particle. In some embodiments, the light stripe of the third light source does not spatially overlap with the first scanned beam of the first light source, the second scanned beam of the second light source, or a combination thereof. In some embodiments, the method comprises processing the third data set of the particle in comparison to the first data set, the second data set, or a combination thereof, to determine a speed of the particle flowing in the first direction. In some embodiments, the speed of the particle flowing in the first direction is determined with a processing latency of less than about 100 milliseconds. In some embodiments, the speed of the particle flowing in the first direction is determined by conducting autocorrelation, cross-correlation, thresholding, or a combination thereof, with the first data set, second data set, third data set, or any combination thereof. In some embodiments, the method comprises detecting one or more optical signals scattered, attenuated, or emitted from the particle with a bright field detector, dark field detector, a speed detector, or a combination thereof, in response to the illumination of the particle by the first scanned beam, the second scanned beam, the light stripe, or a combination thereof, wherein the bright field detector, the dark field detector, the speed detector, or any combination thereof, detects the one or more optical signals emitted, attenuated, or scattered by the particle along an optical axis away from an emission of the first light source, the second light source, the third light source, or any combination thereof. In some embodiments, the one or more optical signals emitted, attenuated, or scattered by the particle along the optical axis away from the emission of the first light source, the second light source, the third light source, or any combination thereof, comprise brightfield microscopy optical signals, forward scatter optical signals, or a combination thereof. In someembodiments, the method comprises processing the first data set, the second data set, the third data set, or a combination thereof, with one or more predictive models to provide a classification of the particle. In some embodiments, the one or more predictive models comprise clustering, classifying, or a combination thereof predictive models. In some embodiments, the one or more predictive models cluster the first data set, the second data set, the third data set, or a combination thereof, based on one or more morphologic features of the first data set, the second data set, the third data set, or a combination thereof. In some embodiments, the classification of the particle comprises a classification of one or more regions or one or more segments of the particle. In some embodiments, the particle comprises a cell, and wherein the one or more regions or the one or more segments of the particle comprise one or more subcellular components of the cell. In some embodiments, the one or more predictive models comprise a convolutional neural network. In some embodiments, the convolutional neural network comprises a structure of a UNet predictive model, a convolutional autoencoder, a fused UNet predictive model, or any combination thereof. In some embodiments, the method comprises directing the first scanned beam, the second scanned beam, the light stripe, or a combination thereof, to the particle with a mirror, wherein the mirror comprises an annular reflective region and a transmissive region, wherein the annular reflective region and the transmission region are spatially separated, and wherein the one or more optical signals emitted or scattered by the particle in response to the illumination of the particle with the first scanned beam, the second scanned beam, or a combination thereof, transmit through the transmission region. In some embodiments, the transmission region comprises an anti -reflective coating. In some embodiments, the light stripe of the third light source is coupled to an optical axis of the first scanned beam, the second scanned beam, or a combination thereof, with a polarization beam splitter In some embodiments, the first scanned beam of the first light source and the second scanned beam of the second light source spatially overlap. In some embodiments, the first scanned beam of the first light source and the second scanned beam of the second light source do not spatially overlap. In some embodiments, the first data set, the second data set, the third data set, or any combination thereof, is processed by one or more processors. In some embodiments, the one or more processors comprises a field programmable gate array (FPGA), graphic processing unit (GPU), neural processing unit (NPU), or any combination thereof. In some embodiments, the first light source or the second light source comprises a center wavelength of about 355nm to about 785nm. In some embodiments, the first light source scanner, the second light source scanner, or a combination thereof, is provided an amplitude modulated chirp driving signal to generate the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, the amplitude modulated chirp driving signal provides an output power of at least about 5mW of the first scanned beam,the second scanned beam, or a combination thereof, across a field of view of the first light source scanner, the second light source scanner, or a combination thereof. In some embodiments, the first light source, the second light source, or a combination thereof, comprise fiber coupled lasers. In some embodiments, the particle comprises one or more cells. In some embodiments, the one or more properties associated with the particle comprise gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof. In some embodiments, the one or more properties associated with the particle are used to align a channel the particle is moving through along the first direction with the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, a background signal is removed or subtracted from the first data set, the second data set, or a combination thereof, wherein the background signal comprises one or more optical signals detected, obtained, and / or collected when the channel is illuminated with the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, the first data set, the second data set, or a combination thereof, is phase shifted by one or more detected data samples to synchronize the scanning of the first light source scanner and detecting the first data set and to synchronize the scanning of the second light source scanner and detecting the second data set. In some embodiments, the one or more properties associated with the particle are determined by cross-correlation of the first data set and the second data set, the first data set and the third data set, the second data set and the third data set, or any combination thereof. In some embodiments, the first data set, the second data set, the third data set, or a combination thereof, comprise an image data set of the particle. In some embodiments, the first scanned beam and second scanned beam are sequentially scanned in the second direction. In some embodiments, when the first scanned beam is scanned in the second direction, the second scanned beam is directed to a first beam capturing optical component, and wherein when the second scanned beam is scanned in the second direction, the first scanned beam is directed to a second beam capturing optical component. In some embodiments, the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, comprises a beam dump. In some embodiments, the first light source scanner and the second light source scanner differ. In some embodiments, the method comprises sorting the particle based at least on the one or more properties associated with the particle.

[0007] Another aspect of the present disclosure provides a system for adjusting a display of a particle, or the particle properties comprising: one or more processors, and memory storing oneor more programs for execution by the one or more processors, the one or more programs comprising instructions to: receive or obtain a first data set of a particle generated by scanning the particle with a first light source optically coupled to a first beam resizing optical element and a first light source scanner, or a second data set of the particle generated by scanning the particle with a second light source optically coupled to a second beam resizing optical element and a second light source scanner; process the first data set and the second data set with one or more processing parameters; and adjust the display of the particle or the particle’s features, when the one or more processing parameters are changed. In some embodiments, the first beam resizing optical element comprises a first anamorphic prism pair, and wherein the second beam resizing optical element comprises a second anamorphic prism pair. In some embodiments, the one or more processing parameters comprise compensation, ellipse fit based aspect ratio, or a combination thereof. In some embodiments, a user of the system changes the one or more processing parameters with a user interface of the system. In some embodiments, the one or more processing parameters are changed in real-time when the first data set, the second data set, or a combination thereof, is received or obtained. In some embodiments, real-time comprises a rate of at least about 30 events per second, wherein each event comprises receiving or obtaining and processing the first data set, the second data set, or a combination thereof. In some embodiments, the particle comprises a plurality of particles. In some embodiments, the plurality of particles comprise cell clusters, spheroids, organelles, or a combination thereof. In some embodiments, the display of the particle comprises a scatter plot of one or more properties of the particle, wherein the one or more properties of the particle are determined from the first data set, the second data set, or a combination thereof. In some embodiments, the display of the particle comprises an image of the particle. In some embodiments, the particle comprises a cell.

[0008] Another aspect of the present disclosure provides a method of training a predictive model to sort a particle, comprising: receiving or obtaining a first particle image data set of a first particle and a second particle image data set of a second particle; processing the first particle image data set or the second particle image data set to obtain a first one or more properties of the first particle image data set or a second one or more properties of the second particle image data set; gating the first particle image data set or the second particle image data set to separate the first one or more properties of the first particle image data set from the second one or more properties of the second particle image data set, thereby indicating a gated first one or more properties of the first particle image data, and a gated second one or more properties of the second particle image data set; and training the predictive model to sort the first particle and the second particle, wherein the predictive model is trained with the gated first one or more properties of the first particle image data set and the gated second one or more properties of thesecond particle image data set. In some embodiments, gating the first particle image data set or the second particle image data set comprises displaying the first one or more properties of the first particle image data set and the second one or more properties of the second particle image data set on a plot and selecting or providing a gate line by the user on the plot to separate the first one or more properties of the first particle image data and the second one or more properties of the second particle image data set. In some embodiments, the first one or more properties comprise morphological features of the first particle, wherein the second one or more properties comprise morphological features of the second particle. In some embodiments, the morphologic feature of the particles is adjusted by the speed of the particles. In some embodiments, the first one or more properties, the second one or more properties, or a combination thereof, comprise gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof. In some embodiments, the method comprises displaying a feature importance rank, feature heatmap, or a combination thereof, of the gated first one or more properties or the gated second one or more properties that separate or distinguish the first particle from the second particle. In some embodiments, the predictive model comprises a convolutional neural network, a logistic regression model, sequential perceptron model, k-nearest neighboring model, support vector machine model, adaboost model, random forest model, or any combination thereof.

[0009] Another aspect of the present disclosure provides a system comprising one or more processors. In some embodiments, the one or more computer processors are coupled to a computer memory coupled thereto. In some embodiments, the one or more processors comprise one or more computer processors, a field programmable gate array (FPGA), a graphics processing unit (GPU), neural processing unit (NPU), or any combination thereof. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0010] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG ”, “FIGS ” herein), of which:

[0012] FIGS. 1A-1C show a schematic overview of an imaging system and / or device for imaging a flowing particle and determining a speed correction for the flowing particle. FIG. 1A shows an optical schematic diagram of the imaging system and / or device scanning components, light sources, etc., with coupling to backward detection optics, as described in some embodiments herein. FIG. IB shows an optical schematic diagram of the image system and / or device forward detection optics, as described in some embodiments herein. FIG. 1C shows an optical schematic diagram of the image system and / or device backward detection optical components including, e.g., one or more detectors and spectrum separation components, as described in some embodiments herein.

[0013] FIGS.2A-2B show an amplitude modulated chirp scanner driving waveform (FIG.2A), and resulting transmission intensity profile outputted by the optical scanner as a function of deflection angle of the scanner (FIG.2B), as described in some embodiments herein.

[0014] FIGS.3A-3E show an imaging configuration schematic of two spatially separate regions along a path of a particle flowing through a channel (FIG.3A), temporal light transmission intensity signal of the particle traversing through the two spatially separate regions using one detector (FIGS.3B and 3D), and autocorrelation of the temporal light transmission intensity signal of the particle to determine particle speed (FIGS.3C and 3E), as described in some embodiments herein.

[0015] FIGS. 4A-4B show a temporal light transmission intensity of stable windowed and unstable portions of emitted light from the optical scanner (FIG. 4A), and light intensity thresholding of an integral of the temporal light transmission signal to distinguish stable and unstable portions of the scanned beams (FIG. 4B), as described in some embodiments herein.

[0016] FIG. 5 shows an optical schematic of a beam resizing optical element and interaction with an optical scanner, as described in some embodiments herein.

[0017] FIGS. 6A-6C show a temporal interleaved scanning voltage waveform of a combined first light source and second light source (FIG. 6A), scanning voltage waveform for a first light source scanner (FIG. 6B), and scanning voltage waveform for a second light source scanner (FIG. 6C), as described in some embodiments herein.

[0018] FIG. 7 shows a fluidic cartridge and fluid path connecting a particle sorting junction coupled thereto, as described in some embodiments herein.

[0019] FIGS. 8A-8J shows 15 pm and 7 pm bead raw transmission images acquired by the imaging system and / or device (FIGS. 8A and 8F, respectively); raw detector acquired signal for fluorescence, transmission, and particle temporal light transmission signal(s) (FIGS. 8B-C and 8G-H, respectively); processed particle temporal light transmission signal(s) (FIGS. 8C and 8H, respectively); autocorrelation of the processed temporal light transmission signal(s) (FIGS. 8D and 81, respectively); and corrected trans illumination image with calculated speed (FIGS. 8E and 8J, respectively), as described in some embodiments herein.

[0020] FIGS. 9A-9G show 16 pm bead diameter transmission images acquired by a 488 nm and 561 nm spatially separate laser illumination (FIG. 9A), corresponding background subtracted images (FIG. 9B), raw temporal light transmission signal (FIG. 9C), inverted and background and / or baseline removed temporal light transmission signal (FIG. 9D), cross correlation of the inverted temporal light transmission signal (FIG. 9E), and speed resized images of the 16 pm bead generated by the 488 nm and 561 nm light sources (FIGS. 9F-9G), as described in some embodiments herein.

[0021] FIGS. 10A-10G show 10 pm bead diameter transmission images acquired by a 488 nm and 561 nm spatially separate laser illumination (FIG. 10A), corresponding background subtracted images (FIG. 10B), raw temporal light transmission signal (FIG. 10C), inverted and background and / or baseline removed temporal light transmission signal (FIG. 10D), cross correlation of the inverted temporal light transmission signal (FIG. 10E), and speed resized images of the 10 pm bead generated by the 488 nm and 561 nm light sources (FIGS. 10F-10G), as described in some embodiments herein.

[0022] FIGS. 11A-11G show 3 pm bead diameter transmission images acquired by a 488 nm and 561 nm spatially separate laser illumination (FIG. 11 A), corresponding background subtracted images (FIG. 11B), raw temporal light transmission signal (FIG. 11C), inverted and background and / or baseline removed temporal light transmission signal (FIG. 11D), cross correlation of the inverted temporal light transmission signal (FIG. HE), and speed resized images of the 5 pm beads generated by the 488 nm and 561nm light sources (FIGS. 11F-11G), as described in some embodiments herein.

[0023] FIGS. 12A-12F show 16 pm bead diameter background subtracted transmission images acquired by a 488 nm and 561 nm light source (FIG. 12A and 12B, respectively), particle temporal light transmission signal (FIG. 12C), autocorrelation of the temporal light transmission signal (FIG. 12D), and speed resized images of the 16 pm bead generated by the 488 nm and 561 nm light sources (FIGS. 12E and 12F, respectively), as described in some embodiments herein.

[0024] FIGS. 13A-13F show 7 pm bead diameter background subtracted transmission images acquired by a 488 nm and 561 nm light source (FIG. 13A and 13B, respectively), particle temporal light transmission signal (FIG. 13C), autocorrelation of the temporal light transmission signal (FIG. 13D), and speed resized images of the 7 pm beads generated by the 488 nm and 561 nm light sources (FIGS. 13E and 13F, respectively), as described in some embodiments herein.

[0025] FIGS. 14A-14F show 5 pm bead diameter background subtracted transmission images acquired by a 488 nm and 561 nm light source (FIG. 14A and 14B, respectively), particle temporal light transmission signal (FIG. 14C), autocorrelation of the temporal light transmission signal (FIG. 14D), and speed resized images of the 5 pm bead generated by the 488 nm and 561 nm light sources (FIGS. 14E and 14F, respectively), as described in some embodiments herein.

[0026] FIGS. 15A-15F show 3 pm bead diameter background subtracted transmission images acquired by a 488 nm and 561 nm light sources (FIG. 15A and 15B, respectively), particle temporal light transmission signal (FIG. 15C), autocorrelation of the temporal light transmission signal (FIG. 15D), and speed resized images of the 3 pm bead generated by the 488 nm and 561nm light sources (FIGS. 15E and 15F, respectively), as described in some embodiments herein.

[0027] FIGS. 16A-16F show particle speed distributions for various sample and sheath flow rates. FIG. 16A shows a particle speed distribution for 12 pL / min sample flow rate and a sheath flow rate of 120 pL / min, as described in some embodiments herein. FIG. 16B shows a particle speed distribution for 16 pL / min sample flow rate and a sheath flow rate of 120 pL / min, as described in some embodiments herein. FIG. 16C shows a particle speed distribution for 24 pL / min sample flow rate and a sheath flow rate of 120 pL / min, as described in some embodiments herein. FIG. 16D shows a particle speed distribution for 12 pL / min sample flow rate and a sheath flow rate of 100 pL / min, as described in some embodiments herein. FIG. 16E shows a particle speed distribution for 16 pL / min sample flow rate and a sheath flow rate of 100 pL / min, as described in some embodiments herein. FIG. 16F shows a particle speed distribution for 24 pL / min sample flow rate and a sheath flow rate of 160 pL / min, as described in some embodiments herein.

[0028] FIGS. 17A-17D show an empirical measurement of correction speed of a particle flowing in a channel. FIG. 17A shows the measured relationship of the height and width ratio of 15 pm diameter spherical beads and the cross-correlation delay determined from the speed autocorrelation calculation, as described in some embodiments herein. FIG. 17B shows the raw fluorescence and bright field images of the particle without speed corrections, as described in some embodiments herein. FIG. 17C shows a best fit of the bright field measured bead waveform intensity super imposed on the measured bead temporal light transmission signal, asdescribed in some embodiments herein. FIG. 17D shows the speed corrected images for the bead fluorescence image and bead brightfield image, as described in some embodiments herein.

[0029] FIGS. 18A-18F show beads fluorescent and transmission images generated with and without a cylindrical lens at a scan rate of 200kHz for a 15 pm diameter bead (FIGS. 18B and 18A, respectively), for a 7 pm diameter bead (FIGS. 18D and 18C, respectively), and for a 1 pm diameter bead (FIGS. 18F and 18E, respectively), as described in some embodiments herein.

[0030] FIGS. 19A-19F show beads fluorescent and transmission images generated with and without a cylindrical lens at a scan rate of 400kHz for a 15 pm diameter bead (FIGS. 19B and 19A, respectively), for a 7 pm diameter bead (FIGS. 19D and 19C, respectively), and for a 1 pm diameter bead (FIGS. 19F and 19E, respectively), as described in some embodiments herein.

[0031] FIGS. 20A-20C show images acquired with the particle image systems and / or devices, described elsewhere herein. FIG. 20A shows images of 10 pm beads, as described in some embodiments herein. FIG. 20B shows images of Vericells, as described in some embodiments herein. FIG. 20C shows images of HEK cells with 1 pm beads, as described in some embodiments herein.

[0032] FIGS. 21A-21H show brightfield, fluorescent stained, and overlaid images of 15 pm green fluorescent beads (FIG. 21A), 7 pm green fluorescent beads (FIG. 21B), CHO-ES cells with Vybrant Dy eCycle e Green DNA staining (FIG. 21C), MCF7 cells with Mito View Green mitochondrial staining (FIG. 21D), Human iPSC with Calcein AM staining (FIG.21E), Human granulocytes with BB515 immunostaining (FIG. 21F), Human lymphocytes with PE immunostaining (FIG. 21G), and Human monocytes with BB515 immunostaining (FIG. 21H), acquired with the image system and / or device, as described in some embodiments herein.

[0033] FIGS. 22A-22D show brightfield, forward scattering, back scattering, and fluorescence images of HEK293 cells with 1 pm red fluorescent beads (FIG. 22A); unstained HeLa, SKNO-1, and MCF7 human cancer cell lines (FIG. 22B); Mitochondrial and lysosome intracellular localization during MCF7 cell mitosis (FIG. 22C); and 4-color human leukocytes immune-phenotyping (FIG. 22D), acquired with the imaging system and / or device, as described in some embodiments herein.

[0034] FIGS. 23A-23C show a schematic structure of a predictive model and / or machine learning for processing one or more properties of a particle imaged by the imaging system and / or device described elsewhere herein, as described in some embodiments herein.

[0035] FIG. 24 shows an image processing workflow that analyzes one or more images of particles and / or cells obtained by the imaging system and / or device, described elsewhere herein, where the analysis then directs sorting of the one or more particles and / or cells, as described in some embodiments herein.

[0036] FIG.25 shows a workflow for training a predictive model and / or a machine learning model with one or more properties extracted from reconstructed images of particles and / or cells, where the trained predictive model and / or the trained machine learning model sorts a plurality of particles and / or cells based on the one or more properties of the particles and / or cells, as described in some embodiments herein.

[0037] FIGS.26A-26F show a distribution of two populations of cells plotted against properties determined from transillumination images of the cells acquired with the imaging system and / or device, namely area of the cell (FIG.26A), integrated intensity of the cell (FIG.26B), maximum intensity of the cell (FIG.26C), aspect ratio of the cell (FIG.26D), gradient root mean square of the cell (FIG.26E), and contrast of the cell (FIG.26F), as described in some embodiments herein.

[0038] FIGS.27A-27E show combined training and validation accuracy and loss plots across one or more epochs for the machine learning and / or predictive models described elsewhere herein (FIG.27A), the training accuracy and loss plots across one or more epoch and one or more folds (FIGS.27B-27C), and the validation accuracy and loss plots across one or more epochs and one or more folds (FIGS.27D-27E), as described in some embodiments herein.

[0039] FIGS.28A-28D show visualization of a high dimensionality parameters space for two cell populations using t-distributed stochastic neighbor embedding algorithm (t-SNE) (FIG. 28A), feature importance of the one or more image parameters of cells that distinguish the two populations of cells using mean decrease in impurity (FIG.28B), feature importance of the one or more image parameters that distinguish the two population of particles with a fully trained model (FIG.28C), and a corresponding correlation heatmap of properties between the two cells (FIG. 28D), as described in some embodiments herein.

[0040] FIG.29 shows a workflow for a method of scanning a particle and / or a cell, as described in some embodiments herein.

[0041] FIG.30 shows a workflow for a method of training a predictive model to sort a particle and / or cell, as described in some embodiments herein.

[0042] FIG.31 shows a computer system that is programmed and / or otherwise configured to implement methods provided herein, as described in some embodiments herein.

[0043] FIG.32 shows a flow diagram schematic of acoustic and electrical delays between initiating scanning of one or more light sources of an imaging systems, and acquiring and / or detecting and processing optical signals transmitted, scattered, and / or emitted by a particle illuminated by the one or more light sources, as described in some embodiments herein.

[0044] FIGS.33A-33F shows a graphical representation of signal scanning, data acquisition, and data processing of the imaging systems and methods described herein, that have an acousticand / or electrical delay (FIGS. 33A-33C) and where the first data set and / or the second data set are shifted by one or more sampled data points to compensate for the acoustic and / or electrical delay (FIGS. 33D-33F), as described in some embodiments herein.

[0045] FIG. 34, shows an example beam alignment and offset error correction, as described in some embodiments herein.DETAILED DESCRIPTION

[0046] Provided herein are imaging systems, devices, and / or methods of imaging one or more particles and / or cells (as shown in FIGS. 20A-22D) flowing through a fluidic system and / or fluidic channel of e.g., a fluidic cartridge. In some cases, the particles and / or cells may comprise extracellular vesicles, microorganisms, viruses, sub-cellular components, pollen, spores, or any combination thereof. The optical structure of the imaging systems, devices, and / or methods of imaging one or more particles and / or one or more cells addresses the aforementioned unmet needs of imaging one or more particles and / or one or more cells flowing through a fluidic system that would otherwise rotate and / or move with varying speed through both imaging system and / or device optical components. The imaging systems, devices, and / or methods described elsewhere herein may optimize a fluidic network flow path and use one or more predictive models trained on one or more properties of one or more populations of particles to identify, analyze and / or sort one or more particles, one or more cells, one or more population of particles, and / or one or more population of cells. In some embodiments, the one or more properties may comprise one or more image features. In some cases, the imaging systems and / or device described elsewhere herein, may image one or more particles and / or one or more cells despite the particle(s) and / or cell(s) non-uniform traveling speed and / or rotation as the particle(s) and / or cell(s) traverses through the fluidic system by interleave scanning multiple imaging beams of light across a fluidic channel of a fluidic system at a high scanning rate e.g., at least about 200 kHz or at least about 400 kHz to minimize the rotation and / or non-linearities (e.g., changes in speed) in the movement of the particle and / or the cell that may occur when the particle is traversing through e.g., a channel of a fluidic system.

[0047] In some embodiments, the imaging system and / or device may comprise one or more beam resizing optical elements coupled to one or more scanners that scan one or more beams across a fluidic channel that maintain a beam quality (e.g., size and shape) of the one or more beams while maintaining a full extent of the scan angles achieved by the one or more scanners. Increasing the scan angle of the one or more scanner while maintain the beam size of the imaging beam may enable e.g., the imaging of particles and / or cells of a plurality of diameters despite movement and / or rotation of such particles as they traverse through the fluidic system. In some embodiments, the imaging system and / or device may utilize a method to determine a speed ofone or more particles flowing through a fluidic channel of the fluidic system based on one or more optical signals from one or more particle imaging beams and / or one or more particle speed beams as the particle traverses and / or flows through an optical path of the one or more particle imaging beams and / or one or more particle speed beams, described elsewhere herein. For example, a particle flowing and / or traversing through a fluidic channel of a fluidic system may travel through the channel with a varying speed in time that distorts an image of the particle collected over time. The methods utilized and / or employed by the imaging system and / or device, described elsewhere herein, may determine the speed of the particle, and compensate and / or correct for a distortion and / or elongation and / or compression of an image of particle that is collected and / or obtained with the imaging systems and / or devices, described elsewhere herein. In some embodiments, the inner diameter and length of fluidic tubing 710 coupled to a fluidic cartridge 188 may be utilized in the fluidic systems imaged by the imaging systems and / or devices, described elsewhere herein, to prevent local fluidic turbulence and to reduce a variation of travel time from a first point in the fluidic system to a second point (e.g., a particle sorting junction) to increase an accuracy of sorting a plurality of particles into a plurality of wells and / or vessels for further processing.Imaging System

[0048] In some embodiments, the imaging system and / or device (100, 190, 162), as shown in FIGS. 1A-1C, may comprise: an imaging system and / or device comprising: a first light source 106 optically coupled to a first beam resizing optical element (112, 114, 118, 120), where an output of the first beam resizing optical element is optically coupled to a first light source scanner 116 configured to scan a first beam of the first light source 106 along an axis of a particle 304 to obtain a first data set comprising fluorescent information and / or label-free information (e.g., structure, fluorophore content, texture, granularity, morphology, shape, etc., or any combination thereof, described elsewhere herein) of the particle 304; a second light source 130 optically coupled to a second beam resizing optical element (136, 138, 142, 144), where an output of the second beam resizing optical element is optically coupled to a second light source scanner 140 configured to scan a second beam of the second light source 130 along the axis of the particle 304 to obtain a second data set comprising fluorescent information and / or label-free information of the particle 304, where the particle may be flowed 302 through a fluidic channel 300 (e.g., of a fluidic cartridge 188) at an angle with respect to an optical axis of the first beam of the first light source 106 and / or an optical axis of the second beam of the second light source 130, and where the first beam resizing optical element (112, 114, 118, 120) and the second beam resizing optical element (136, 138, 142, 144) differ; and a data processing device (e.g., a computer system 1100,as shown in FIG. 31, described elsewhere herein) in communication with the imaging device, where the data processing device 1100 comprises one or more processor 1102 to process the first data set, the second data set, or a combination thereof, obtained by the imaging system and / or device to determine one or more properties associate with the particle 304. In some embodiments, the angle of the particle flowing through the fluidic channel with respect to the optical axis may comprise an orthogonal angle. In some embodiments, the angle of the particle flowing through the fluidic channel 300 may comprise an angle of about 45 degrees to about 135 degrees. In some embodiments, the first beam resizing optical element (112, 114, 118, 120) and the second beam resizing optical element (136, 138, 142, 144) may comprise one or more beam expanding optical elements, one or more beam reducing optical elements, or a combination thereof.

[0049] In some embodiments, the fluidic channel 300 may be disposed and / or provided in an imaging region 700 of the fluidic cartridge 188. In some embodiments, the first beam resizing optical element (112, 114, 118, 120) may operate with a first wavelength or in a first wavelength band, where the second beam resizing optical element (136, 138, 142, 144) may operate with a second wavelength or a second wavelength band, and where the first wavelength or the first wavelength band is different from the second wavelength or the second wavelength band. In some embodiments, the data processing device 1100 may comprise a FPGA, GPU, NPU, or any combination thereof. In some embodiments, the particle may comprise one or more particles. In some embodiments, the particle may comprise a plurality of particles. In some embodiments, the particle may comprise one or more cell. In some embodiments, the particle may comprise a plurality of cells. In some embodiments, the label-free image information may comprise non-fluorescent image information of the particle.

[0050] In some embodiments, the data processing device may sort and / or dispense the particle based at least on the one or more properties associated with the particle. In some embodiments, the data processing device may sort the particle based at least on the one or more properties associated with the particle determined by gating the first data set and / or gating the second data set. In some instances, the data processing device may actuate and / or control a sorting actuator 706 to sort one or more particles, as shown in FIG. 7. In some cases, the sorting actuator 706 may be embedded within the fluidic cartridge 188. In some embodiments, the sorting actuator 706, may be fluidically coupled to an imaging region 700, one more fluidic channels 702, one or more output channels (707, 708) , or any combination of one or more thereof. In some embodiments, the sorting actuator 706 may comprise a piezo electric sorting apparatus. In some embodiments, the one or more output channels (707, 708) may dispense one or more sorted particles flowing in a fluid into one or more wells (e.g., of a well plate), one or more vessels,and / or one or more reservoirs. In some embodiments, a first output channel 707 of the one or more output channels may be fluidically coupled to a first well, and where a second output channel 708 of the one or more output channels may be fluidically coupled to a second well where the first well and the second well are different wells. In some embodiments, the sorting actuator 706 may be disposed and / or provided adjacent to the imaging region 700 of the fluidic cartridge 188. In some embodiments, the one or more output channels (707, 708) may be fluidically coupled to tubing 710. In some embodiments, a first output channel In some embodiments, the tubing coupled to the one or more output channels (707, 708) may comprise one or more segments of tubing. In some embodiments, the one or more segments of tubing may comprise a length of up to about 1 inch. In some embodiments, the tubing may comprise an inner diameter of about 50 pm to about 400 pm. In some embodiments, the tubing may comprise an inner diameter of about 50 pm to about 60 pm, about 50 pm to about 75 pm, about 50 pm to about 80 pm, about 50 pm to about 90 pm, about 50 pm to about 100 pm, about 50 pm to about 150 pm, about 50 pm to about 200 pm, about 50 pm to about 250 pm, about 50 pm to about 300 pm, about 50 pm to about 350 pm, about 50 pm to about 400 pm, about 60 pm to about 75 pm, about 60 pm to about 80 pm, about 60 pm to about 90 pm, about 60 pm to about 100 pm, about 60 pm to about 150 pm, about 60 pm to about 200 pm, about 60 pm to about 250 pm, about 60 pm to about 300 pm, about 60 pm to about 350 pm, about 60 pm to about 400 pm, about 75 pm to about 80 pm, about 75 pm to about 90 pm, about 75 pm to about 100 pm, about 75 pm to about 150 pm, about 75 pm to about 200 pm, about 75 pm to about 250 pm, about 75 pm to about 300 pm, about 75 pm to about 350 pm, about 75 pm to about 400 pm, about 80 pm to about 90 pm, about 80 pm to about 100 pm, about 80 pm to about 150 pm, about 80 pm to about 200 pm, about 80 pm to about 250 pm, about 80 pm to about 300 pm, about 80 pm to about 350 pm, about 80 pm to about 400 pm, about 90 pm to about 100 pm, about 90 pm to about 150 pm, about 90 pm to about 200 pm, about 90 pm to about 250 pm, about 90 pm to about 300 pm, about 90 pm to about 350 pm, about 90 pm to about 400 pm, about 100 pm to about 150 pm, about 100 pm to about 200 pm, about 100 pm to about 250 pm, about 100 pm to about 300 pm, about 100 pm to about 350 pm, about 100 pm to about 400 pm, about 150 pm to about 200 pm, about 150 pm to about 250 pm, about 150 pm to about 300 pm, about 150 pm to about 350 pm, about 150 pm to about 400 pm, about 200 pm to about 250 pm, about 200 pm to about 300 pm, about 200 pm to about 350 pm, about 200 pm to about 400 pm, about 250 pm to about 300 pm, about 250 pm to about 350 pm, about 250 pm to about 400 pm, about 300 pm to about 350 pm, about 300 pm to about 400 pm, or about 350 pm to about 400 pm. In some embodiments, the tubing may comprise an inner diameter of about 50 pm, about 60 pm, about 75 pm, about 80 pm, about 90 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about350 pm, or about 400 pm. In some embodiments, the tubing may comprise an inner diameter of at least about 50 pm, about 60 pm, about 75 pm, about 80 pm, about 90 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, or about 350 pm. In some embodiments, the tubing may comprise an inner diameter of at most about 60 pm, about 75 pm, about 80 pm, about 90 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, or about 400 pm.

[0051] In some embodiments, the fluid flow rate may comprise a flow rate of about 20 pL / min to about 550 pL / min. In some embodiments, the fluid flow rate may comprise a flow rate of about 20 pL / min to about 40 pL / min, about 20 pL / min to about 50 pL / min, about 20 pL / min to about 70 pL / min, about 20 pL / min to about 80 pL / min, about 20 pL / min to about 100 pL / min, about 20 pL / min to about 150 pL / min, about 20 pL / min to about 200 pL / min, about 20 pL / min to about 250 pL / min, about 20 pL / min to about 300 pL / min, about 20 pL / min to about 500 pL / min, about 20 pL / min to about 550 pL / min, about 40 pL / min to about 50 pL / min, about 40 pL / min to about 70 pL / min, about 40 pL / min to about 80 pL / min, about 40 pL / min to about 100 pL / min, about 40 pL / min to about 150 pL / min, about 40 pL / min to about 200 pL / min, about 40 pL / min to about 250 pL / min, about 40 pL / min to about 300 pL / min, about 40 pL / min to about 500 pL / min, about 40 pL / min to about 550 pL / min, about 50 pL / min to about 70 pL / min, about 50 pL / min to about 80 pL / min, about 50 pL / min to about 100 pL / min, about 50 pL / min to about 150 pL / min, about 50 pL / min to about 200 pL / min, about 50 pL / min to about 250 pL / min, about 50 pL / min to about 300 pL / min, about 50 pL / min to about 500 pL / min, about 50 pL / min to about 550 pL / min, about 70 pL / min to about 80 pL / min, about 70 pL / min to about 100 pL / min, about 70 pL / min to about 150 pL / min, about 70 pL / min to about 200 pL / min, about 70 pL / min to about 250 pL / min, about 70 pL / min to about 300 pL / min, about 70 pL / min to about 500 pL / min, about 70 pL / min to about 550 pL / min, about 80 pL / min to about 100 pL / min, about 80 pL / min to about 150 pL / min, about 80 pL / min to about 200 pL / min, about 80 pL / min to about 250 pL / min, about 80 pL / min to about 300 pL / min, about 80 pL / min to about 500 pL / min, about 80 pL / min to about 550 pL / min, about 100 pL / min to about 150 pL / min, about 100 pL / min to about 200 pL / min, about 100 pL / min to about 250 pL / min, about 100 pL / min to about 300 pL / min, about 100 pL / min to about 500 pL / min, about 100 pL / min to about 550 pL / min, about 150 pL / min to about 200 pL / min, about 150 pL / min to about 250 pL / min, about 150 pL / min to about 300 pL / min, about 150 pL / min to about 500 pL / min, about 150 pL / min to about 550 pL / min, about 200 pL / min to about 250 pL / min, about 200 pL / min to about 300 pL / min, about 200 pL / min to about 500 pL / min, about 200 pL / min to about 550 pL / min, about 250 pL / min to about 300 pL / min, about 250 pL / min to about 500 pL / min, about 250 pL / min to about 550 pL / min, about 300 pL / min to about 500 pL / min, about 300 pL / min to about 550 pL / min, or about 500 pL / min to about 550 pL / min. Insome embodiments, the fluid flow rate may comprise a flow rate of about 20 pL / min, about 40 pL / min, about 50 pL / min, about 70 pL / min, about 80 pL / min, about 100 pL / min, about 150 pL / min, about 200 pL / min, about 250 pL / min, about 300 pL / min, about 500 pL / min, or about 550 pL / min. In some embodiments, the fluid flow rate may comprise a flow rate of at least about 20 pL / min, about 40 pL / min, about 50 pL / min, about 70 pL / min, about 80 pL / min, about 100 pL / min, about 150 pL / min, about 200 pL / min, about 250 pL / min, about 300 pL / min, or about 500 pL / min. In some embodiments, the fluid flow rate may comprise a flow rate of at most about 40 pL / min, about 50 pL / min, about 70 pL / min, about 80 pL / min, about 100 pL / min, about 150 pL / min, about 200 pL / min, about 250 pL / min, about 300 pL / min, about 500 pL / min, or about 550 pL / min.

[0052] In some embodiments, the one or more segments of tubing may reduce a standard deviation of a transit time of one or more particles traveling and / or flowing from a first location of the fluidic cartridge 188 (e.g., the imaging region 700) to a second location (e.g., a dispensing region 710) of the sorting actuator 706. The reduced standard deviation of transit time of one or more particles traveling and / or flowing from the first location to the second location may improve dispensing efficiency to at least about 70% for dispensing single 7 pm, 10 pm, and / or 15 pm particles and / or cells in one or more wells of a well plate.

[0053] In some embodiments, the first light source 106, the second light source 130, or a combination thereof, may comprise a fiber coupled laser. In some cases, the first light source 106 may couple a first emission of the first light source into a first fiber 108. In some embodiments, the second light source 130 may couple a second emission of the second light source 130 into a second fiber 132. In some cases, the first emission of the first light source 106 emitted from the first fiber 108 may be coupled into a first fiber collimator 110. In some embodiments, the second emission of the second light source 130 emitted from the second fiber 132 may be coupled into a second fiber collimator 134. The first emission emitted from the first fiber collimator 110 may be optically coupled to a first corrective lens 112. In some embodiments, the second emission emitted from the second fiber collimator 134 may be optically coupled to a second corrective lens 136. In some embodiments, the first emission emitted by the first corrective lens 112 may be optically coupled to the first beam resizing optical element (114, 118, 120). In some embodiments, the second emission emitted by the second corrective lens 136 may be coupled to the second beam resizing optical element (136, 138, 142, 144). In some instances, the first light source 106, the first fiber 108, the first fiber collimator 110, the first corrective lens 112, the first beam resizing optical element (114, 118, 120), the first optical scanner 116, or any combination thereof, may be housed within a first light source housing 102. In some embodiments, the second light source 130, the second fiber 132, the second fiber collimator 134, the second corrective lens136, the second beam resizing optical element (138, 142, 144), the second optical scanner 140, or any combination thereof may be housed within a second light source housing 104. In some embodiments, the first beam resizing optical element (112, 114, 118, 120) may comprise a first anamorphic prism pair 114 and / or a first cylindrical lens 118. In some embodiments, the second beam resizing optical element (138, 142, 144) may comprise a second anamorphic prism pair 138 and / or a second cylindrical lens 142. In some embodiments, the first emission emitted from the first beam resizing optical element 118 may be optically coupled to a third anamorphic prism pair 120. In some embodiments, the second emission emitted from the second beam resizing optical element 142 may be optically coupled to a fourth anamorphic prism pair 144. In some embodiments, the first emission emitted from the third anamorphic prism pair 120 may be optically coupled to a first lens 122 that forms a set of relay lenses with a second lens 148. In some embodiments, the second emission emitted from the fourth anamorphic prism pair 144 may be directed to a first mirror 146 to steer, reflect, direct, and / or fold the second emission emitted from the fourth anamorphic prism pair to a third lens 128 that forms a set of relay lenses with the second lens 148. In some embodiments, the first emission emitted from the first lens 122 and the second emission emitted from the third lens 128 may be combined into a combined emission with a first dichroic mirror 124. The combined emission of the first and / or second emission may be optically coupled to a second mirror 126 to steer, reflect, direct, and / or fold an optical path of the combined emission to the second lens 148 of the relay lens set. The combined emission emitted by the second lens 148 may be optically coupled to a first beam splitter 150. In some embodiments, the first beam splitter may comprise an optical power splitting ratio of about 90 to about 10, about 80 to about 20, about 70 to about 30, about 60 to about 40, or about 50 to about 50. The combined emission emitted from the first beam splitter 150 may be optically coupled to a polarization beam splitter 152. In some embodiments, the combined emission emitted from the polarization beam splitter 152 may be optically coupled to a quarter wave plate 154. In some embodiments, the quarter wave plate 154 may convert a linearly polarized light of the combined emission of the first emission and / or the second emission to circularly polarized light, which provides and / or produces consistent and / or stable scattered and / or transmitted photons (e.g., back scattered, forward scattered, and / or transmitted photons) of the particle 304 when the combined emission of the first scanned beam and / or the second scanned beam are incident on the particle. In some embodiments, the combined emission emitted from the quarter wave plate 154 may be optically coupled to a mirror 164 comprising an annular reflective region and a transmissive region. In some embodiments, the combined emission emitted from the quarter wave plate 154 may be incident on the annular reflective region of the mirror 164 to steer, reflect, direct, and / or fold an optical path of the combined emission towards the particle 304 flowing in a fluidicchannel 300 of the cartridge 188. In some embodiments, the combined emission incident on the annular reflective region of the mirror 164 may be optically coupled to a fourth lens 168 and a fifth lens 184. In some embodiments, the fourth lens 168 and fifth lens 184 may form an optical relay. In some embodiments, the combined emission emitted from the fifth lens 184 may be optically coupled to an objective lens 186. In some embodiments, the combined emission emitted from the objective lens 186 may scan across an axis of the particle 304 flowing through a fluidic channel 300 of the fluidic cartridge 188.

[0054] In some embodiments, the objective lens 186 may comprise a numerical aperture of about 0.2 to about 0.9. In some embodiments, the objective lens 186 may comprise a numerical aperture of about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 to about 0.6, about 0.2 to about 0.75, about 0.2 to about 0.8, about 0.2 to about 0.9, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 0.6, about 0.3 to about 0.75, about 0.3 to about 0.8, about 0.3 to about 0.9, about 0.4 to about 0.5, about 0.4 to about 0.6, about 0.4 to about 0.75, about 0.4 to about 0.8, about 0.4 to about 0.9, about 0.5 to about 0.6, about 0.5 to about 0.75, about 0.5 to about 0.8, about 0.5 to about 0.9, about 0.6 to about 0.75, about 0.6 to about 0.8, about 0.6 to about 0.9, about 0.75 to about 0.8, about 0.75 to about 0.9, or about 0.8 to about 0.9. In some embodiments, the objective lens 186 may comprise a numerical aperture of about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.75, about 0.8, or about 0.9. In some embodiments, the objective lens 186 may comprise a numerical aperture of at least about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.75, or about 0.8. In some embodiments, the objective lens 186 may comprise a numerical aperture of at most about 0.3, about 0.4, about 0.5, about 0.6, about 0.75, about 0.8, or about 0.9.

[0055] In some embodiments, the objective lens 186 may comprise a magnification of up to about 50X. In some embodiments, the first lens 122, the second lens 148 and / or the third lens 128 may comprise a focal length of up to about 60 millimeters (mm). In some embodiments, the fourth lens 168 may comprise a focal length of up to about 30 mm. In some embodiments, the fifth lens 184 may comprise a focal length of up to about 40mm.

[0056] In some embodiments, the first data set, the second data set, or a combination thereof, may be generated by one or more detectors (192, 226, 206, 236, 242, 248, 254, 260, 264). In some embodiments, the first light source scanner 116, the second light source scanner 140, or a combination thereof, may comprise one or more Acousto-Optic Deflectors (AOD), one or more galvanometers, one or more piezo electric elements, one more rotating prisms, or any combination thereof. In some cases, the first light source scanner 116 and the second light source scanner 140 may differ. In some embodiments, the first light source scanner 116, the second light source scanner 140, or a combination thereof, may be provided an amplitude modulated chirpdriving signal, as shown in FIG. 2A, to scan the first beam, the second beam, or a combination thereof. In some cases, the amplitude modulated chirp driving signal may provide an output power of about 1 milliwatt (mW) to about 500 mW of the first scanned beam, the second scanned beam, or a combination thereof, across a field of the view of the first light source scanner, the second light source scanner, or a combination thereof, as shown in FIGS. 2B and 6A-6C. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of about 1 mW to about 5 mW, about 1 mW to about 10 mW, about 1 mW to about 20 mW, about 1 mW to about 40 mW, about 1 mW to about 75 mW, about 1 mW to about 100 mW, about 1 mW to about 150 mW, about 1 mW to about 200 mW, about 1 mW to about 350 mW, about 1 mW to about 400 mW, about 1 mW to about 500 mW, about 5 mW to about 10 mW, about 5 mW to about 20 mW, about 5 mW to about 40 mW, about 5 mW to about 75 mW, about 5 mW to about 100 mW, about 5 mW to about 150 mW, about 5 mW to about 200 mW, about 5 mW to about 350 mW, about 5 mW to about 400 mW, about 5 mW to about 500 mW, about 10 mW to about 20 mW, about 10 mW to about 40 mW, about 10 mW to about 75 mW, about 10 mW to about 100 mW, about 10 mW to about 150 mW, about 10 mW to about 200 mW, about 10 mW to about 350 mW, about 10 mW to about 400 mW, about 10 mW to about 500 mW, about 20 mW to about 40 mW, about 20 mW to about 75 mW, about 20 mW to about 100 mW, about 20 mW to about 150 mW, about 20 mW to about 200 mW, about 20 mW to about 350 mW, about 20 mW to about 400 mW, about 20 mW to about 500 mW, about 40 mW to about 75 mW, about 40 mW to about 100 mW, about 40 mW to about 150 mW, about 40 mW to about 200 mW, about 40 mW to about 350 mW, about 40 mW to about 400 mW, about 40 mW to about 500 mW, about 75 mW to about 100 mW, about 75 mW to about 150 mW, about 75 mW to about 200 mW, about 75 mW to about 350 mW, about 75 mW to about 400 mW, about 75 mW to about 500 mW, about 100 mW to about 150 mW, about 100 mW to about 200 mW, about 100 mW to about 350 mW, about 100 mW to about 400 mW, about 100 mW to about 500 mW, about 150 mW to about 200 mW, about 150 mW to about 350 mW, about 150 mW to about 400 mW, about 150 mW to about 500 mW, about 200 mW to about 350 mW, about 200 mW to about 400 mW, about 200 mW to about 500 mW, about 350 mW to about 400 mW, about 350 mW to about 500 mW, or about 400 mW to about 500 mW. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of about 1 mW, about 5 mW, about 10 mW, about 20 mW, about 40 mW, about 75 mW, about 100 mW, about 150 mW, about 200 mW, about 350 mW, about 400 mW, or about 500 mW. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of at least about 1 mW, about 5 mW, about 10 mW, about 20 mW, about 40 mW, about 75 mW, about 100 mW, about 150 mW, about 200 mW, about 350 mW, or about 400 mW. In some cases, the amplitude modulated chirp driving signalmay provide an optical output power of at most about 5 mW, about 10 mW, about 20 mW, about 40 mW, about 75 mW, about 100 mW, about 150 mW, about 200 mW, about 350 mW, about 400 mW, or about 500 mW.

[0057] In some embodiments, the first light source scanner 116, the second light source scanner 140, or a combination thereof, may comprise one or more portions of a scanning waveform, signal, and / or trajectory 408 that may be erratic and / or unusable to scan the first beam of the first light source and / or the second beam of the second light source, as shown in FIG. 4A. In some embodiments, the one or more erratic and / or unusable portions of the scanning waveform, signal, and / or trajectory 408 may be interleaved with one or more scans of the first scanned beam 400 and / or the second scanned beam 406. In some embodiments, a threshold level 420 of a detected and windowed integrated and / or summed intensity 416 of the first scanned beam 400 and / or a detected integrated and / or summed intensity 416 of the second scanned beam 406 may be used to start image acquisition when the integrated and / or summed intensity 416 passes 414 the threshold 420, as shown in FIG. 4B. In some cases, the width and / or duration, and offset of the window 410 may comprise a width and / or duration and offset with respect to the start and / or beginning of scanning the first scanned beam 400 and / or the start and / or beginning of scanning the second scanned beam 406. In some cases, the width and / or duration and offset of the window 410 may be fixed for the first scanned beam 400, the second scanned beam 406, or a combination thereof.

[0058] In some embodiments, the first cylindrical lens 118 and the second cylindrical lens 142 may differ in size, focal length, working distance, or any combination thereof. In some embodiments, the first cylindrical lens 118 may compensate for a first astigmatism of the first scanned beam of light. In some embodiments, the second cylindrical lens 142 may compensate for a second astigmatism of the second scanned beam of light.

[0059] In some embodiments, the first scanned beam of light may be coupled to a third anamorphic prism pair 120. In some embodiments, the second scanned beam of light may be coupled to a fourth anamorphic prism pair 144. In some embodiments, the first anamorphic prism pair 114 and the third anamorphic prism pair 120 may magnify a scanned field of view of the first scanned beam provided by the first light source scanner 116. In some embodiments, the second anamorphic prism pair 138 and the fourth anamorphic prism pair 144 may magnify a scanned field of view of the second scanned beam provided by the second light source scanner 140. In some embodiments, the first anamorphic prism pair 114, the second anamorphic prism pair 138, or a combination thereof, may comprise a reversed optical orientation compared with the third anamorphic prism pair 120, the fourth anamorphic prism pair 144, or a combination thereof, as shown in FIG. 5. In some cases, the reversed optical orientation of the first anamorphic prism pair 114, the second anamorphic prism pair 138, or a combination thereof, inview of optical orientation of the third anamorphic prism pair 120, the fourth anamorphic prism pair 144, or a combination thereof, may expand 504 a diameter of an input beam of light 500 from the first light source 106 and / or the second light source 130 prior to light incident on the first optical scanner 116 and / or the second optical scanner 140 to magnify and / or increase a scanning range of an emitted beam of light 508 of the first light source 106 and / or the second light source 130 outputted by the first optical scanner 116 and / or the second optical scanner 140.In some cases, the reversed optical orientation of the third anamorphic prism pair 120, the fourth anamorphic prism pair 144, or a combination thereof, with respect to the optical orientation of the first anamorphic prism pair 114, the second anamorphic prism pair 138, or a combination thereof, may reduce a diameter of the outputted the magnified scanned beam 512 to the diameter of the original beam diameter of the emitted beam of light from the first light source 106 and / or the second light source 140. The reduction of the diameter of the outputted magnified scanned beam 512 increases the scan range while maintaining beam spot size to increase an imaging resolution of the imaging system and / or device, described elsewhere herein.

[0060] In some embodiments, the one or more detectors, described elsewhere herein, may comprise a camera 176, one or more photomultiplier tubes (206, 236, 242, 248, 254, 260, 264), one or more photomultiplier tube arrays, one or more avalanche photodetectors, one or more arrays of photomultiplier tubes, a fiber coupled detector, one or more photodiodes (226, 192), or any combination of one or more thereof. In some cases, the one or more photomultiplier tubes (206, 236, 242, 248, 254, 260, 264), may be optically coupled to the one or more optical signals emitted and / or scattered by the particle through a second beam splitter 223, one or more dichroic mirrors (200, 202, 232, 238, 244, 250, 256, 262), one or more dispersive elements (e.g., gratings or prisms), or any combination thereof. In some cases, the one or more photodiodes (192, 226), may be optically coupled to the one or more optical signals emitted and / or scattered by the particle through a second beam splitter 223, one or more dichroic mirrors (200, 202), or any combination thereof. In some embodiments, the camera 176 may comprise a two-dimensional sensor comprising a plurality of photodiode sensors. In some embodiments, the camera 176 may be optically coupled to the first beam splitter 150, described elsewhere herein, by a sixth 172 and a seventh 170 lens. In some embodiments, the sixth lens 172 and the seventh lens 170 may form an optical relay. In some embodiments, the camera 176 may detect a light emission of an alignment light source 220, after the alignment light source traverses and / or passes through the cartridge 188. In some embodiments, the alignment light source may comprise an output center wavelength of about 635nm. In some embodiments, the alignment light source may comprise a light emitting diode (LED). In some embodiments, the detected light emission of the alignmentlight source 220 by camera 176 may be used to align the image system and / or device (100, 190, 162) with respect to the cartridge 188.

[0061] The one or more detectors (192, 226, 206, 236, 242, 248, 254, 260, 264) may detect one or more optical signals emitted and / or scattered by the particle in response to the illumination of the particle by the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, the one or more optical signals may be emitted and / or scattered by the particle along an optical axis towards an emission of the first light source 106, the second light source 130, or a combination thereof. In some embodiments, the one or more optical signals emitted and / or scattered by the particle along an optical axis towards an emission of the first light source 106, the second light source 130, or a combination thereof, may comprise one or more back scattered photons, one or more epi -fluorescent optical signals, one or more fluorescent optical signals, or any combination thereof. In some cases, the scattered and / or emitted photons from the particle traveling through and / or flowing through the fluidic cartridge 188, described elsewhere herein, may be collected by a collection optical component 158 (e.g., a lens and / or a collimator), optically coupled (e.g., via an optical path 160) to a back scatter detection optical system 162, shown in FIG. 1C. In some cases, the scattered and / or emitted photons from the particle may be optically coupled to collection optical component 158 through an eighth lens 156 optically coupled to the transmissive region of the mirror 164, the sixth lens 168, the seventh lens 184, the objective lens 186, or any combination thereof. In some cases, the eighth lens 156 may comprise a focal lens of 12 mm. In some instances, the optical path 160 may comprise one or more optical fibers. In some embodiments, the one or more optical fibers may comprise one or more single and / or one or more multi-mode optical fibers. In some embodiments, the optical path 160 may be optically coupled to a fiber ferrule 228. In some embodiments, the back scattered photons emitted from the fiber ferrule 228 may be optically coupled to a thirteenth lens 230. In some embodiments, the thirteenth lens 230 may be optically coupled to a fourth dichroic mirror 232, a fifth dichroic mirror 238, a sixth dichroic mirror 244, a seventh dichroic mirror 250, an eighth dichroic mirror 256, a ninth filter 262, or any combination of one or more thereof. In some embodiments, the fourth dichroic mirror 232 may be optically coupled to a fourth filter 234. In some embodiments, the fourth dichroic mirror 232 may reflect and / or the fourth filter 234 may transmit and / or pass one or more back scatter photons emitted and / or scattered from the particle with a center wavelength of about 488nm that may be detected by the detector 236. In some embodiments, the fifth dichroic mirror 238 may be optically coupled to a fifth filter 240. In some embodiments, the fifth dichroic mirror 238 may reflect and / or the fifth filter 240 may transmit and / or pass one or more back scatter photons emitted and / or scattered from the particle with a center wavelength of about 535nm that may be detected by the detector 242. In someembodiments, the sixth dichroic mirror 244 may be optically coupled to a sixth filter 246. In some embodiments, the sixth dichroic mirror 244 may reflect and / or the sixth filter 246 may transmit and / or pass one or more back scatter photons emitted and / or scattered from the particle with a center wavelength of about 585nm that may be detected by the detector 248. In some embodiments, the seventh dichroic mirror 250 may be optically coupled to a seventh filter 252. In some embodiments, the seventh dichroic mirror 250 may reflect and / or the seventh filter 252 may transmit and / or pass one or more back scatter photons emitted and / or scattered from the particle with a center wavelength of about 620nm that may be detected by the detector 254. In some embodiments, the eighth dichroic mirror 256 may be optically coupled to an eighth filter 258. In some embodiments, the eighth dichroic mirror 256 may reflect and / or the eighth filter 258 may transmit and / or pass one or more back scatter photons emitted and / or scattered from the particle with a center wavelength of about 710 nm that may be detected by the detector 260. In some embodiments, the eighth dichroic mirror 256 may be optically coupled to a ninth filter 262. In some embodiments, the nineth filter 262 may transmit and / or pass one or more back scattered photons emitted and / or scattered from the particle with a center wavelength of about 810 that may be detected by the detector 264.

[0062] In some embodiments, the one or more optical signals transmitted, emitted, and / or scattered by the particle 304 may be attenuated, emitted and / or scattered from the particle along an optical axis away from an emission of the first light source 106, the second light source 130, the third light source 180, or any combination thereof. In some embodiments, the one or more optical signals transmitted, emitted, and / or scattered by the particle 304 along the optical axis away from the emission of the first light source 106, the second light source 130, the third light source 180, or any combination thereof, comprise brightfield microscopy optical signals, forward scattered optical signals, or a combination thereof. In some cases, the one or more optical signals emitted, transmitted, and / or scattered by the particle may comprise one or more forward scattered, transmitted, and / or emitted photons collected and / or detected by a forward and / or transmission detection optical system 190, as shown in FIG. IB. In some embodiments, the forward and / or transmission detection optical system 190 may comprise a bright field detector 226, a dark field detector 206, a particle speed detector 192, or any combination thereof, optically coupled to the particle 304, as shown in FIG. IB. In some embodiments, the bright field detector 226, the dark field detector 206, or a combination thereof, may be configured to detect one or more attenuated transmitted, scattered and / or emitted optical signals of the particle 304 in response to the illumination of the particle 304 by the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, the light stripe306 of the third light source 180, or any combination thereof. In some embodiments, the one or moreattenuated transmitted, forward scattered, and / or emitted optical signals of the particle 304 flowing through a fluidic channel 300 of the fluidic cartridge 188 may be optically coupled to a ninth lens 190. In some embodiments, the ninth lens 190 may be optically coupled to a third mirror 204 to steer, reflect, direct, and / or fold one or more forward scattered and / or transmitted photons scattered, transmitted, and / or emitted from the particle 304. In some embodiments, the third mirror 204 may steer, reflect, direct, and / or fold one or more forward scattered and / or transmitted photons scattered, transmitted, and / or emitted from the particle 304 to a second dichroic mirror 202 optically coupled to the third mirror 204. In some embodiments, the second dichroic mirror 202 may split and / or divide the one or more forward scattered and / or transmitted photons scattered, transmitted, and / or emitted from the particle to a first filter 212 optically coupled to the second dichroic mirror 202. In some embodiments, the first filter 212 may be optically coupled to the second beam splitter 223, where the second beam splitter 223 may split and / or divide the filtered one or more attenuated, forward scattered, transmitted, and / or emitted photons from the particle to a dark field blocker 210 and / or a bright field blocker 222. In some embodiments, the dark field blocker 210 may be optically coupled to a nineth lens 208, where the nineth lens 208 may focus the one or more forward scattered and / or transmitted photons scattered, transmitted, and / or emitted from the particle transmitted through the dark field blocker 210 to a detector 206 (e.g., a photomultiplier tube). In some embodiments, the bright field blocker 222 may be optically coupled to a tenth lens 224, where the tenth lens 224 may focus the photons attenuated, transmitted, scattered and / or emitted from the particle transmitted through the bright field blocker 222 to a detector 226 (e.g., a photodiode). In some embodiments, the detectors (206, 226) may detect one or more attenuated transmitted photons and / or forward scattered photons of the particle generated as a result of illumination of the particle with the first scanned beam of the first light source and / or as a result of illumination of the particle with the second scanned beam of the second light source.

[0063] In some embodiments, the second dichroic mirror 202 may be optically coupled to a third dichroic mirror 200, where the third dichroic mirror 200 may reflect a light emission of an alignment light source 220 and / or transmit and / or pass one or more attenuated, transmitted and / or forward scattered photons scattered and / or emitted from the particle. In some embodiments, an emission of the alignment light source 220 may be optically coupled to an aperture 218. The emission of the alignment light source 220 emitted by the aperture 218, may be optically coupled to a twelfth lens 216. In some embodiments, the twelfth lens 216 may expand and / or collimate the emission of the alignment light source 220. In some embodiments, the emission of the alignment light source emitted by the twelfth lens 216 may be optically coupled to a third filter 214. In some embodiments, the third filter 214 may be optically coupled to the third dichroicmirror 200. In some embodiments, the emission of the alignment light source 220 emitted from the twelfth lens 216 may be optically coupled to the third dichroic mirror 200.

[0064] In some embodiments, the third dichroic mirror 200 may be optically coupled to a second filter 198. In some embodiments, the forward scattered and / or transmitted photons emitted by the filter 198 may be optically coupled to a blocker bar 196. In some embodiments, the forward scattered and / or transmitted photons emitted by the blocker bar 196 may be optically coupled to an eleventh lens 194, where the eleventh lens 194 may be optically coupled to a detector 192 (e.g., a photodiode). In some embodiments, the detector 192 may be configured to detect one or more forward scattered, transmitted, and / or emitted photons from the particle when the particle is illuminated by the light stripe of the third light source 180. In some embodiments, the detected one or more forward scattered and / or transmitted photons from the particle detected by the detector 192 may be utilized and / or processed to determine a speed of the particle flowing in a fluidic channel of the fluidic cartridge, described elsewhere herein.

[0065] In some embodiments, a third emission of the third light source 180 may be optically coupled to a third fiber 178. In some embodiments, the third emission emitted from the third fiber 178 may be optically coupled to a third fiber coupler 174. In some embodiments, the third emission emitted from the third fiber coupler 174 may be optically coupled to a fifth anamorphic prism pair 182. The third emission emitted from the fifth anamorphic prism pair 182 may be optically coupled to a third cylindrical lens 166. The third emission emitted from the third cylindrical lens may be optically coupled to the polarization beam splitter 152 and combined with the combined emission of the first emission of the first light source 106 and / or the second emission of the second light source 130, described elsewhere herein.

[0066] In some embodiments, the imaging system and / or device 100 may comprise a third light source 180 optically coupled to a fifth anamorphic prism pair 182 and / or a third cylindrical lens 166. In some embodiments, the third light source 180 may comprise a light emitting diode, a laser, a super luminescent diode, a white light source, or any combination thereof. In some embodiments, the third light source 180 may comprise an output center wavelength of about 660nm. In some cases, the third light source 180 may output a light stripe when an emitted beam of the third light source 180 traverses through the third cylindrical lens 166. In some embodiments, the light stripe of the third light source may couple to an optical axis of the first scanned beam, the second scanned beam, or a combination thereof, with a beam splitter 152. In some embodiments, the first beam splitter may comprise a polarization beam splitter. In some cases, the light stripe of the third light source 180 may illuminate the particle and produce a third data set comprising fluorescent information and / or label-free image information of the particle, as described elsewhere herein. In some cases, the light stripe 306 generated from an emittedbeam of light of the third light source 180 may not spatially overlap with the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, as shown in FIGS. 3A and 34. In some embodiments, the light stripe 306 may be separated by a distance 310 from the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof.

[0067] In some embodiments, the distance 310 between the light stripe 306 and the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, may comprise a distance of about 1 pm to about 1,000 pm. In some embodiments, the distance 310 between the light stripe 306 and the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, may comprise a distance of about 1 pm to about 5 pm, about 1 pm to about 10 pm, about 1 pm to about 25 pm, about 1 pm to about 50 pm, about 1 pm to about 100 pm, about 1 pm to about 200 pm, about 1 pm to about 300 pm, about 1 pm to about 700 pm, about 1 pm to about 800 pm, about 1 pm to about 900 pm, about 1 pm to about 1,000 pm, about 5 pm to about 10 pm, about 5 pm to about 25 pm, about 5 pm to about 50 pm, about 5 pm to about 100 pm, about 5 pm to about 200 pm, about 5 pm to about 300 pm, about 5 pm to about 700 pm, about 5 pm to about 800 pm, about 5 pm to about 900 pm, about 5 pm to about 1,000 pm, about 10 pm to about 25 pm, about 10 pm to about 50 pm, about 10 pm to about 100 pm, about 10 pm to about 200 pm, about 10 pm to about 300 pm, about 10 pm to about 700 pm, about 10 pm to about 800 pm, about 10 pm to about 900 pm, about 10 pm to about 1,000 pm, about 25 pm to about 50 pm, about 25 pm to about 100 pm, about 25 pm to about 200 pm, about 25 pm to about 300 pm, about 25 pm to about 700 pm, about 25 pm to about 800 pm, about 25 pm to about 900 pm, about 25 pm to about 1,000 pm, about 50 pm to about 100 pm, about 50 pm to about 200 pm, about 50 pm to about 300 pm, about 50 pm to about 700 pm, about 50 pm to about 800 pm, about 50 pm to about 900 pm, about 50 pm to about 1,000 pm, about 100 pm to about 200 pm, about 100 pm to about 300 pm, about 100 pm to about 700 pm, about 100 pm to about 800 pm, about 100 pm to about 900 pm, about 100 pm to about 1,000 pm, about 200 pm to about 300 pm, about 200 pm to about 700 pm, about 200 pm to about 800 pm, about 200 pm to about 900 pm, about 200 pm to about 1,000 pm, about 300 pm to about 700 pm, about 300 pm to about 800 pm, about 300 pm to about 900 pm, about 300 pm to about 1,000 pm, about 700 pm to about 800 pm, about 700 pm to about 900 pm, about 700 pm to about 1,000 pm, about 800 pm to about 900 pm, about 800 pm to about 1,000 pm, or about 900 pm to about 1,000 pm. In some embodiments, the distance 310 between the light stripe 306 and the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, may comprise a distance of about 1 pm, about 5 pm, about 10 pm, about 25 pm, about 50 pm, about100 pm, about 200 pm, about 300 pm, about 700 pm, about 800 pm, about 900 pm, or about 1,000 pm. In some embodiments, the distance 310 between the light stripe 306 and the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, may comprise a distance of at least about 1 pm, about 5 pm, about 10 pm, about 25 pm, about 50 pm, about 100 pm, about 200 pm, about 300 pm, about 700 pm, about 800 pm, or about 900 pm. In some embodiments, the distance 310 between the light stripe 306 and the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, may comprise a distance of at most about 5 pm, about 10 pm, about 25 pm, about 50 pm, about 100 pm, about 200 pm, about 300 pm, about 700 pm, about 800 pm, about 900 pm, or about 1,000 pm.. In some embodiments, the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, may scan across an axis of the particle 304 within an imaging plane and / or region 320. In some cases, the position of the first scanned beam 308 of the first light source 106, the position of the second scanned beam 308 of the second light source 130, or a combination thereof, may be adjusted along a y-axis 314 of the imaging plane and / or region 320 and / or along an x-axis 316 of the imaging plane and / or region 320 to adjust the distance 310 between the light stripe 306 and the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, as shown in FIG. 34. In some embodiments, the light stripe 306 may comprise a detection margin distance 318 between the edge of the imaging plane and / or region 320 and the position of the light stripe 306, where the detection margin distance prevents the photons of the light stripe 306 from being detected in the imaging plane and / or region 320 where photons transmitted, scattered, and / or emitted by the particle as the particle is illuminated by the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof, are detected. Spacing the light stripe 306 by the detection margin distance may increase a signal to noise ratio of a detected one or more photons transmitted, emitted, and / or scattered by the particle when the particle is illuminated by the first scanned beam 308 of the first light source 106, the second scanned beam 308 of the second light source 130, or a combination thereof.

[0068] In some cases, the first scanned beam 308 of the first light source 106 and the second scanned beam 308 of the second light source 130 may be co-linear and / or spatially overlap. In some instances, the first scanned beam of the first light source 106 and the second scanned beam of the second light source 130 may not spatially overlap. In some embodiments, an intensity of an emitted beam of the first light source 106 may comprise a first intensity modulation frequencyabout 90 degrees out of phase with a second intensity modulation frequency of the second light source 130

[0069] In some cases, the first scanned beam 600 of the first light source 106 and the second scanned beam 602 of the second light source 130 may be sequentially scanned along the axis of the particle 304, as shown in FIGS. 6A-6C. As shown in FIGS. 6A-6B, the first scanned beam 600 may be scanned across the scan range of the first light source scanner 116 from about time ti,o to about time ti, after which the second scanned beam 602 may be scanned across the scan range of the second light source scanner 140 beginning at about time t2,oup to about time t2, as shown in FIGS. 6A-6C. In some embodiments, when the first scanned beam 600 is scanned along the axis of the particle 304, the second scanned beam 602 may be directed to a first beam capturing optical component. In some embodiments, when the second scanned beam 602 is scanned along the axis of the particle, the first scanned beam 600 may be directed to a second beam capturing optical component. In some embodiments, the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, may comprise an optical beam dump, a black coated absorbing material, or a combination thereof.

[0070] In some cases, the data processing device 1100 may process the third data set in comparison to the first data set, the second data set, or a combination thereof, to determine a speed of the particle 304 flowing 302 in a channel 300, as described elsewhere herein. In some embodiments, the speed of the particle 304 flowing 302 in the channel may be determined by conducting autocorrelation, thresholding, or a combination thereof, described elsewhere herein, with the first data set, the second data set, the third data set, or a combination thereof. The speed of the particle may be utilized to correct and / or compensate for any distortion in an image of the particle generated by one or more detected optical signals scattered and / or emitted from the particle when the particle is illuminated by the first scanned beam 308 of the first light source 106 and the second scanned beam 308 of the second light source 130.

[0071] In some embodiments, the imaging system and / or device 100 may comprise a mirror 164 where the mirror may comprise an annular reflective region and a transmissive region. In some embodiments, the annular reflective region and the transmission region may be spatially separated, where the first scanned beam, the second scanned beam, the light stripe, or a combination thereof may be reflected from the annular reflective region towards the particle 304.In some embodiments the one or more optical signals emitted and / or scattered by the particle may transmit through the transmission region of the mirror 164. In some embodiments, the mirror 164 may comprise an annulus mirror. In some embodiments, the transmission region of the mirror may comprise an anti -reflective coating.

[0072] In some embodiments, the one or more properties associated with the particle may comprise a gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof. In some embodiments, the one or more properties may be used to align the fluidic channel 300 of e.g., the fluidic cartridge 188 and the imaging system and / or device. In some embodiments, the one or more properties may be determined by cross-correlation of the first data set and the second data set, first data set and the third data set, the second data set and third data set, or a combination thereof. In some embodiments, the first data set, the second data set, the third data set, or any combination thereof, may comprise an image data set of the particle.

[0073] In some embodiments, a background signal may be removed and / or subtracted from the first data set, the second data set, or a combination thereof. In some embodiments, the background signal may comprise one or more optical signals detected, obtained, and / or collected when the fluidic channel 300 is illuminated with the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments the first data set, the second data set, or a combination thereof, may be phase shifted by one or more obtained data samples to synchronize the scanning of the first light source scanner and obtaining the first data set and / or to synchronize the scanning of the second light source scanner and obtaining the second data set.Methods

[0074] In some embodiments, aspects of the disclosure provided herein describe a method for scanning a particle 1000, as seen in FIG. 29. In some embodiments, the method 1000 may comprise: moving a particle along a first direction 1002; generating a first scanned beam of a first light source with a first light source scanner and a second scanned beam of a second light source with a second light source scanner, where an output of the first light source is optically coupled to a first beam resizing optical element and an output of the second light source is optically coupled to a second beam resizing optical element, where an output of the first beam resizing optical element is optically coupled to the first light source scanner and an output of the second beam resizing optical element is optically coupled to the second light source scanner, and where the first beam resizing optical element and the second beam resizing optical element differ 1004; and scanning the particle with the first scanned beam of the first light source and the second scanned beam of the second light source in a second direction at an angle to the first direction 1006. In some embodiments, the angle may comprise a perpendicular angle. In some embodiments, the angle may comprise an angle between about 45 degrees and 135 degrees. Insome embodiments, the particle may comprise a plurality of particles. In some embodiments, the particle may comprise a cell. In some embodiments, the plurality of particles may comprise a plurality of cells. In some embodiments, the first beam resizing optical element may comprise a first anamorphic prism pair or a first cylindrical lens, and where the second beam resizing optical element may comprise a second anamorphic prism pair or a second cylindrical lens. In some embodiments, the method 1000 may further comprise detecting a first data set of the particle from one or more optical signals emitted and / or scattered by the particle in response to illumination of the particle with the first scanned beam, and / or detecting a second data set of the particle from one or more optical signals emitted and / or scattered by the particle in response to illumination of the particle with the second scanned beam. In some embodiments, the first data set, the second data set, or a combination thereof, may comprise an image data set of the particle. In some embodiments, the first light source and / or the second light source may comprise a center wavelength of about 355nm to about 785nm. In some embodiments, the first light source, the second light source, or a combination thereof, may comprise a fiber coupled laser. In some embodiments, method 1000 may comprise sorting the particle based at least on the one or more properties associated with the particle.

[0075] In some embodiments, the method 1000 may comprise processing the first data set and / or the second data set to determine one or more properties associated with the particle. In some embodiments, the one or more properties of the particle may comprise speed, fluorescent signal, morphologic feature(s), or any combination thereof. In some embodiments, the morphologic feature data may be adjusted based on the particle speed. In some embodiments, the one or more optical signals emitted and / or scattered by the particle in response to illumination of the particle by the first scanned beam, the second scanned beam, or a combination thereof, may be emitted and / or scattered along an optical axis towards an emission of the first light source, the second light source, or a combination thereof. In some embodiments, the one or more optical signals emitted and / or scattered by the particle in response to the illumination along the optical axis towards the emission of the first light source, the second light source, or a combination thereof, may comprise scattered optical signals, back scattered optical signals, transmitted optical signals, epifluorescence optical signals, or a combination thereof. In some embodiments, the one or more optical signals emitted and / or scattered by the particle in response to the illumination of the particle with the first scanned beam, the second scanned beam, or a combination thereof, may be detected by a camera, one or more photomultiplier tubes, one or more photodiodes, a fiber coupled detector, or any combination thereof.

[0076] In some embodiments, the first scanned beam may be coupled to a third anamorphic prism pair, and wherein the second scanned beam may be coupled to a fourth anamorphic prismpair. In some embodiments, the first anamorphic prism pair and / or the third anamorphic prism pair may magnify a scanned field of view of the first scanned beam provided by the first light source scanner. In some embodiments, the second anamorphic prism pair and the fourth anamorphic prism pair may magnify a scanned field of view of the second beam provided by the second light source scanner. In some embodiments, the first anamorphic prism pair, the second anamorphic prism pair, or a combination thereof, may comprise a reversed optical orientation compared with the third anamorphic prism pair, the fourth anamorphic prism pair, or a combination thereof.

[0077] In some embodiments, the one or more properties associated with the particle may be used to align a channel the particle is moving, flowing, and / or transporting through along the first direction with the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, the one or more properties associated with the particle may comprise gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof.

[0078] In some embodiments, a background signal may be removed and / or subtracted from the first data set, the second data set, or a combination thereof, wherein the background signal comprises one or more optical signals detected, obtained, and / or collected when the channel is illuminated with the first scanned beam, the second scanned beam, or a combination thereof. In some embodiments, the one or more detected data samples may be shifted by e.g., a delay in the form of time and / or number of samples, to synchronize the scanning of the first light source scanner and detecting the first data set and / or to synchronize the scanning of the second light source scanner and detecting the second data set. The synchronization may compensate and / or reduce an acoustic and / or electrical delay generated and / or introduced within the electrical and / or acoustic signal transmission of the imaging system shown in FIG. 32. In some cases, the electrical delay may be generated by transmitting a scanner driving waveform (e.g., amplitude modulated chirp electrical driving signal, described herein) by a processor 1200 (e.g., a FPGA, GPU, NPU, or any combination thereof) electrically coupled to a digital analog converter 1202 that then transmits the scanning driving waveform via one or more electrical components 1204 to an acoustic scanner 1210 (e.g., an AOD). In some embodiments, the scanning driving waveform transmitted to the acoustic scanner 1210 may generate an acoustic delay when the scanning driving waveform induces an acoustic wave that travels a distance 1206 along an axis 1212 of the acoustic scanner 1210 to a position where a light source 1208 (e.g., a first light source and / or asecond light source, described herein) is incident on the acoustic scanner 1210. The varying position of the light source 1208 incident on the acoustic scanner 1210 due to an alignment of the light source 1208 may provide varying amounts of acoustic delay, described herein. In some cases, the acoustic wave that travels the distance 1206 along the axis 1212 may scan the light source 1208 incident on the scanner across a particle where photons of the light source may be emitted, transmitted, absorbed, and / or scattered 1214 and detected by a detector 1216 (e.g., a photodetector), described elsewhere herein. In some cases, an electrical delay may be generated from the detected photons of the light source emitted, transmitted, absorbed, and / or scattered 1214 from and / or by the particle when the photons are converted into one or more electrical signals and transmitted by one or more electrical components 1218 electrically coupled to an analog to digital convert 1220 and processed by a processor 1222. In some cases, the electrical and / or acoustic delay may result in a first scan position 1301 and the last scan position 1302 of the scanner spanning over a first and second row of a processed and / or displayed image, as shown in FIGS. 33A-33B. In some embodiments, the first data set, the second data set, or a combination thereof, may be shifted by one or more detected data samples 1303 e.g., one or more data samples collected over a period of time, to synchronize the scanning of the first light source scanner and detecting the first data set and / or to synchronize the scanning of the second light source scanner and detecting the second data set, as shown in FIGS. 33E-33F. In some cases, the one or more data samples may comprise up to about 125 data samples. In some instances, the one or more data samples may comprise one or more pixel data samples. In some cases, about 125 pixel data samples may be collected, acquired, and / or detected in about 1 microseconds (ps). In some instances, the first data set, the second data set, or a combination thereof, may be shifted by one or more rows of the first data set or the second data set. In some cases, a row of the first data set and / or the second data set may be collected, acquired, and / or detected in about 3 ps. In some cases, the one or more rows may comprise at least about 1.5 rows of the first data set or the second data set. In some cases, the one or more detected and / or processed data samples may be delayed by a temporal delay 1303 when being displayed or processed, as shown in FIG. 33D.

[0079] In some cases, the temporal delay 1303 may comprise about 1 ps to about 10 ps. In some cases, the temporal delay 1303 may comprise about 1 ps to about 2 ps, about 1 ps to about 3 ps, about 1 ps to about 4 ps, about 1 ps to about 5 ps, about 1 ps to about 6 ps, about 1 ps to about 7 ps, about 1 ps to about 8 ps, about 1 ps to about 9 ps, about 1 ps to about 10 ps, about 2 ps to about 3 ps, about 2 ps to about 4 ps, about 2 ps to about 5 ps, about 2 ps to about 6 ps, about 2 ps to about 7 ps, about 2 ps to about 8 ps, about 2 ps to about 9 ps, about 2 ps to about 10 ps, about 3 ps to about 4 ps, about 3 ps to about 5 ps, about 3 ps to about 6 ps, about 3 ps to about 7 ps, about 3 ps to about 8 ps, about 3 ps to about 9 ps, about 3 ps to about 10 ps, about 4 ps toabout 5 ps, about 4 jus to about 6 jus, about 4 jus to about 7 jus, about 4 jus to about 8 jus, about 4 ps to about 9 ps, about 4 jus to about 10 jus, about 5 jus to about 6 jus, about 5 jus to about 7 jus, about 5 ps to about 8 jus, about 5 jus to about 9 jus, about 5 jus to about 10 jus, about 6 jus to about 7 ps, about 6 jus to about 8 jus, about 6 jus to about 9 jus, about 6 jus to about 10 jus, about 7 jus to about 8 ps, about 7 jus to about 9 jus, about 7 jus to about 10 jus, about 8 jus to about 9 jus, about 8 ps to about 10 ps, or about 9 jus to about 10 ps. In some cases, the temporal delay 1303 may comprise about 1 ps, about 2 ps, about 3 ps, about 4 ps, about 5 ps, about 6 ps, about 7 ps, about 8 ps, about 9 ps, or about 10 ps. In some cases, the delay 1303 may comprise at least about 1 ps, about 2 ps, about 3 ps, about 4 ps, about 5 ps, about 6 ps, about 7 ps, about 8 ps, or about 9 ps. In some cases, the temporal delay 1303 may comprise at most about 2 ps, about 3 ps, about 4 ps, about 5 ps, about 6 ps, about 7 ps, about 8 ps, about 9 ps, or about 10 ps.

[0080] In some embodiments, the one or more structural features that vary in texture and / or position across an image of a particle and / or cell may be resolved by synchronizing the scanning of the first light source scanner and / or the second light source scanner with detecting the first data set and / or the second data set. For example, a resulting example image of a particle with shifted and / or compensated delay as shown in FIG. 33E maintains the original structure of the particle compared to distorted and / or wrapped image FIG. 33B of the same particle. The structural morphology of a particle and / or cell may be resolved with a higher accuracy of e.g., at least about 75%, at least about 80%, at least about 85%, or at least about 90%, compared to not synchronizing the scanning of the first light source scanner and / or the second light source scanner with the detection of the first data set and / or the second data set. In some cases, a fit 1304 of a regression of a particle and / or cell may be resolved with an accuracy of at least about 75%, at least about 80%, at least about 85%, or at least about 90% (as shown in FIGS. 33E-33F), compared to not synchronizing the scanning (as shown in FIGS. 33B-33C) of the first light source scanner and / or the second light source scanner with the detection of the first data set and / or the second data set. In some cases, the fit 1304 of the regression of the particle and / or cell of an image that is shifted and / or compensated with delay may determine and / or provide an aspect ratio of the particle and / or cells with an accuracy of at least about 75%, at least about 80%, at least about 85%, or at least about 90%, compared to not synchronizing the scanning of the first light source scanner and / or the second light source scanner with the detection of the first data set and / or the second data set.

[0081] In some embodiments, the first data set, the second data set, the third data set, or a combination thereof, may comprise an image data set of the particle. In some embodiments, the one or more properties associated with the particle may be determined by correlation (e.g., crosscorrelation, auto-correlation, described elsewhere herein) of the first data set and the second dataset, the first data set and the third data set, the second data set and the third data set, or any combination thereof.

[0082] In some embodiments, the first light source scanner and the second light source scanner may differ. In some embodiments, the first light source scanner, the second light source scanner, or a combination thereof, may comprise an AOD. In some embodiments, the first light source scanner, the second light source scanner, or a combination thereof, may be provided an amplitude modulated chirp driving signal to generate the first scanned beam, the second scanned beam, or a combination thereof.

[0083] In some cases, the amplitude modulated chirp driving signal may provide an optical output power of about 1 milliwatt (mW) to about 500 mW of the first scanned beam, the second scanned beam, or a combination thereof, across a field of the view of the first light source scanner, the second light source scanner, or a combination thereof. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of about 1 mW to about 5 mW, about 1 mW to about 10 mW, about 1 mW to about 20 mW, about 1 mW to about 40 mW, about 1 mW to about 75 mW, about 1 mW to about 100 mW, about 1 mW to about 150 mW, about 1 mW to about 200 mW, about 1 mW to about 350 mW, about 1 mW to about 400 mW, about 1 mW to about 500 mW, about 5 mW to about 10 mW, about 5 mW to about 20 mW, about 5 mW to about 40 mW, about 5 mW to about 75 mW, about 5 mW to about 100 mW, about 5 mW to about 150 mW, about 5 mW to about 200 mW, about 5 mW to about 350 mW, about 5 mW to about 400 mW, about 5 mW to about 500 mW, about 10 mW to about 20 mW, about 10 mW to about 40 mW, about 10 mW to about 75 mW, about 10 mW to about 100 mW, about 10 mW to about 150 mW, about 10 mW to about 200 mW, about 10 mW to about 350 mW, about 10 mW to about 400 mW, about 10 mW to about 500 mW, about 20 mW to about 40 mW, about 20 mW to about 75 mW, about 20 mW to about 100 mW, about 20 mW to about 150 mW, about 20 mW to about 200 mW, about 20 mW to about 350 mW, about 20 mW to about 400 mW, about 20 mW to about 500 mW, about 40 mW to about 75 mW, about 40 mW to about 100 mW, about 40 mW to about 150 mW, about 40 mW to about 200 mW, about 40 mW to about 350 mW, about 40 mW to about 400 mW, about 40 mW to about 500 mW, about 75 mW to about 100 mW, about 75 mW to about 150 mW, about 75 mW to about 200 mW, about 75 mW to about 350 mW, about 75 mW to about 400 mW, about 75 mW to about 500 mW, about 100 mW to about 150 mW, about 100 mW to about 200 mW, about 100 mW to about 350 mW, about 100 mW to about 400 mW, about 100 mW to about 500 mW, about 150 mW to about 200 mW, about 150 mW to about 350 mW, about 150 mW to about 400 mW, about 150 mW to about 500 mW, about 200 mW to about 350 mW, about 200 mW to about 400 mW, about 200 mW to about 500 mW, about 350 mW to about 400 mW, about 350 mW to about 500 mW, or about 400mW to about 500 mW. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of about 1 mW, about 5 mW, about 10 mW, about 20 mW, about 40 mW, about 75 mW, about 100 mW, about 150 mW, about 200 mW, about 350 mW, about 400 mW, or about 500 mW. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of at least about 1 mW, about 5 mW, about 10 mW, about 20 mW, about 40 mW, about 75 mW, about 100 mW, about 150 mW, about 200 mW, about 350 mW, or about 400 mW. In some cases, the amplitude modulated chirp driving signal may provide an optical output power of at most about 5 mW, about 10 mW, about 20 mW, about 40 mW, about 75 mW, about 100 mW, about 150 mW, about 200 mW, about 350 mW, about 400 mW, or about 500 mW.

[0084] In some embodiments, the first scanned beam may be coupled to a first cylindrical lens. In some embodiments, the second scanned beam may be coupled to a second cylindrical lens. In some embodiments, the first cylindrical lens and the second cylindrical lens may differ. In some embodiments, the first cylindrical lens may compensate for a first astigmatism of the first scanned beam. In some embodiments, the second cylindrical lens may compensate for a second astigmatism of the second scanned beam. In some embodiments, the first scanned beam 600 and second scanned beam 602 may be sequentially scanned in the second direction, as shown, for example, in FIGS. 6A-6C, described elsewhere herein.

[0085] In some embodiments, when the first scanned beam is scanned in the second direction, the second scanned beam may be directed to a first beam capturing optical component. In some embodiments, when the second scanned beam is scanned in the second direction, the first scanned beam may be directed to a second beam capturing optical component. In some embodiments, the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, may comprise a beam dump that absorbs an emission of the first scanned beam and / or an emission of the second scanned beam. In some embodiments, the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, may comprise a dark material and / or dark paint on a surface that absorbs an emission of the first scanned beam and / or an emission of the second scanned beam.

[0086] In some embodiments, a third light source may be optically coupled to a fifth anamorphic prism pair and / or a third cylindrical lens. In some embodiments, an emission of the third light source may be converted into a light stripe by providing the emission of the third light source to the third cylindrical lens. In some embodiments, the light stripe of the third light source output by the third cylindrical lens may illuminate the particle, thereby producing a third data set of the particle. In some embodiments, the light stripe of the third light source may not spatially overlap with the first scanned beam of the first light source, the second scanned beam of the second lightsource, or a combination thereof. In some embodiments, the method 1000 may comprise processing the third data set of the particle in comparison to the first data set, the second data set, or a combination thereof, to determine a speed of the particle flowing in the first direction. In some embodiments, the speed of the particle flowing in the first direction may be determined with a processing latency of less than about 100 milliseconds. In some embodiments, the speed of the particle flowing in the first direction may be determined by conducting autocorrelation, cross-correlation, thresholding, or any combination thereof, with the first data set, second data set, third data set, or any combination thereof, as shown in FIGS. 3B-3E. In some embodiments, the first data set, the second data set, the third data set, or any combination thereof may comprise a temporal light transmission intensity signal indicating the reduction in detected signal of the first scanned beam of the first light source, the second scanned beam of the second light source, and / or the light stripe of the third light source, as shown in FIGS. 3B and 3D, for example, for beads and HEK cells, respectively. In some embodiments, temporal light transmission intensity signal may be processed, e.g., by autocorrelation to determine a transit and / or transportation time of the particle 304 between a position of the light stripe 306 and a position of the first scanned beam 308, the second scanned beam 308, or a combination thereof, as shown in FIG. 3A, described elsewhere herein. In some embodiments, the speed may be determined and / or processed with the distance 310 between the light stripe 306 and the first scanned beam 308, the second scanned beam 308, or a combination thereof, divided by the transit and / or transportation time of the particle 304. In some embodiments, the first scanned beam 308 of the first light source and the second scanned beam 308 of the second light source may spatially overlap. In some embodiments, the first scanned beam of the first light source and the second scanned beam of the second light source may not spatially overlap. In some embodiments, the transmission intensity waveform may be inverted ahead of correlation and / or auto-correlation, as shown in FIGS. 9D, 10D, and 11D

[0087] In some embodiments, the method 1000 may further comprise detecting one or more optical signals attenuated, scattered, transmitted, and / or emitted from the particle with a bright field detector, dark field detector, a speed detector, or any combination thereof, in response to the illumination of the particle by the first scanned beam, the second scanned beam, the light stripe, or a combination thereof. In some embodiments, the bright field detector, the dark field detector, the speed detector, or any combination thereof, may detect the one or more optical signals emitted and / or scattered by the particle along an optical axis away from an emission of the first light source, the second light source, the third light source, or any combination thereof. In some embodiments, the one or more optical signals attenuated, emitted, transmitted, and / or scattered by the particle along the optical axis away from the emission of the first light source, the secondlight source, the third light source, or any combination thereof, may comprise brightfield microscopy optical signals, forward scattered optical signals, or a combination thereof.

[0088] In some embodiments, the method 1000 may further comprise processing the first data set, the second data set, the third data set, or any combination thereof, with one or more predictive models and / or one or more machine learning models, described elsewhere herein, for example as shown in FIGS. 23A-23C to provide a classification of the particle. In some embodiments, the one or more predictive models and / or the one or more machine learning models may comprise clustering, classifying, or a combination thereof, of predictive models and / or machine learning models. In some embodiments, the clustering and / or dimensionality reduction may be conducted with a t-distributed stochastic neighbor embedding approach (t-SNE), as shown in FIG. 28A. In some embodiments, the one or more predictive models and / or one or more machine learning models may cluster the first data set, the second data set, the third data set, or any combination thereof, based on at least one or more morphologic features of the first data set, the second data set, the third data set, or any combination thereof. In some embodiments, the one or more predictive models may comprise a convolutional neural network. In some embodiments, the convolutional neural network may comprise a structure of a UNet predictive model, a convolutional autoencoder, a fused UNet predictive model, or any combination thereof. In some embodiments, the classification of the particle may comprise a classification of one or more regions and / or one or more segments of the particle. In some embodiments, the particle may comprise a cell, and where the one or more regions and / or the one or more segments of the particle may comprise one or more subcellular components and / or one or more subcellular regions of the cell.

[0089] In some embodiments, the method 1000 may comprise directing the first scanned beam, the second scanned beam, the light stripe, or any combination thereof, to the particle with a mirror, where the mirror may comprise an annular reflective region and a transmissive region. In some embodiments, the annular reflective region and the transmission region may be spatially separated. In some embodiments, the one or more optical signals emitted and / or scattered by the particle in response to the illumination of the particle with the first scanned beam, the second scanned beam, or a combination thereof, may transmit through the transmission region of the mirror. In some embodiments, the transmission region may comprise an anti -reflective coating.

[0090] In some embodiments, the light stripe of the third light source may be coupled to an optical axis of the first scanned beam, the second scanned beam, or any combination thereof, with a polarization beam splitter. In some embodiments, the first data set, the second data set, the third data set, or any combination thereof, may be processed by one or more processors 1102 of a computer system 1100, described elsewhere herein, and as shown in FIG. 31. In someembodiments, the one or more processors 1102 may comprise a FPGA, GPU, NPU, or any combination thereof.

[0091] Although the above steps of the methods described elsewhere herein, show and / or describe each of the methods or sets of operations in accordance with embodiments described elsewhere herein, a person of ordinary skill in the art will recognize many variations based on the teaching described herein. The steps may be completed in a different order. Steps may be added or omitted. Some of the steps may comprise sub-steps. Many of the steps may be repeated as often as beneficial. One or more of the steps of each of the methods or sets of operations may be performed with circuitry as described herein, for example, one or more of the processor or logic circuitry such as programmable array logic for a field programmable gate array. The circuitry may be programmed to provide one or more of the steps of each of the methods or sets of operations, and the program may comprise program instructions stored on a computer readable memory or programmed steps of the logic circuitry such as the programmable array logic or the field programmable gate array, for example.Predictive Models

[0092] In some embodiments, the one or more processors 1102 of the processing device 1100 may be in electrical communication with memory (1104, 1110), where the memory may store one or more predictive models and / or one or more machine learning models as shown in FIGS.23A-23C, described elsewhere herein. In some embodiments, the one or more predictive models and / or one or more machine learning models may process the first data set, the second data set, the third data set, or a combination thereof to provide a classification of one or more particles. In some embodiments, the one or more predictive models and / or the one or more machine learning models may comprise a clustering, classifying, and / or a combination of one or more predictive models and / or machine learning models thereof. In some embodiments the one or more predictive models and / or one or more machine learning models may cluster the first data set, the second data set, the third data set, or any combination thereof, based on one or more properties of the first data set, the second data set, the third data set, or any combination thereof, for example, as shown in FIG. 28A. In some cases, the one or more properties may comprise morphologic features of the first data set, the second data set, the third data set, or any combination thereof. In some embodiments, the classification performed and / or conducted by the one or more predictive models and / or the one or more machine learning models may comprise a classification of one or more regions and / or one or more segments of a particle imaged by the imaging system and / or devices, described elsewhere herein. In some embodiments, the one or more regions and / or the one or more segments of the particle may comprise one or more subcellular components of a cell.

[0093] In some embodiments, the one or more predictive models may comprise a convolutional neural network, a logistic regression model, a sequential perception model, a k-nearest neighboring model, a support vector machine model, an adaboost model, a random forest model, or any combination thereof. In some embodiments, the convolutional neural network model may comprise a structure of a UNet predictive model, a convolutional autoencoder, a fused UNet predictive model, or any combination thereof.

[0094] Aspects of the disclosure describe a method of training one or more predictive models and / or one or more machine learning models 1008, as shown in FIG. 30. In some embodiments, the method 1008 may comprise: receiving and / or obtaining a first particle image data set and a second particle image data set 1010; processing the first particle image data set and / or the second particle image data set to obtain a first one or more properties of the first particle image data set and / or a second one or more properties of the second particle image data set 1012; gating the first particle image data set and / or the second particle image data set to separate the first one or more properties of the first particle image data set from the second one or more properties of the second particle image data set, thereby indicating a gated first one or more properties of the first particle image data, and / or a gated second one or more properties of the second particle image data set 1014; and training one or more predictive model and / or one or more machine learning models, wherein the one or more predictive models and / or the one or more machine learning models are trained with the gated first one or more properties of the first particle image data set and / or the gated second one or more properties of the second particle image data set 1016. In some embodiments, the first one or more properties of the first particle may comprise morphological features of the first particle. In some embodiments, the second one or more properties of the second particle may comprise morphological features of the second particle. In some embodiments, the morphologic feature(s) of the first particle may be determined from a speed of the first particle. In some embodiments, the morphologic feature of the second particle may be determined from a speed of the second particle. In some embodiments, the first one or more properties, the second one or more properties, or a combination thereof, may comprise a gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof. In some embodiments, the method 1008 may comprise displaying a feature importance rank (FIGS. 28B-28C), feature heatmap (FIG. 28D), or a combination thereof, of the gated first one or moreproperties or the gated second one or more properties that separate or distinguish the first particle from the second particle.

[0095] In some embodiments, the one or more predictive models and / or the one or more machine learning models, as shown in FIGS. 23A-23C, may comprise one or more layers and / or operations. In some cases, one or more input data set(s) and / or one or more images may be processed by the one or more predictive models and / or the one or more machine learning models by one or more convolution, pooling (e.g., max pooling), up-convolution, batch normalization, or any combination thereof, operations and / or steps. In some embodiments, the one or more input data set(s) and / or the one or more images processed by the one or more predictive models and / or one or more machine learning models may comprise two-dimensional (FIGS. 23A-23B) and / or three-dimensional data sets (FIG. 23C). In some embodiments, the one or more predictive models and / or one or more machine learning models may process the two-dimensional data set, the three-dimensional data set, or a combination thereof. In some cases, the one or more predictive models and / or the one or more machine learning models may combine a processed output of the two-dimensional data set(s) and the three-dimensional data set(s). In some embodiments, the result of the processed two-dimensional and / or three-dimensional data set(s) may comprise a characterization, identification, and / or a prediction of one or more properties and / or one or more parameters of one or more particles, described elsewhere herein.

[0096] In some embodiments, one or more layers of the predictive model may comprise a copy operation and / or step that may copy the result of one or more convolution, one or more pooling, or a combination thereof, operations and / or steps to append the copied processed data to one or more datasets processed by other one or more convolution, max pooling, and / or up-convolution steps and / or operations of the predictive model and / or machine learning model, as shown in FIGS. 23A-23C. In some embodiments, the one or more convolution steps and / or operations may comprise one or more sizes of the convolution filter utilized in the one or more convolution steps, as indicated and shown by labeled numbers of FIGS. 23A-23C adjacent and / or next to the one or more convolution steps.

[0097] Aspects of the disclosure describe a method (800, 900) of sorting one or more particles based on processing one or more parameters of an image of particles, as shown in FIGS. 24-25.In some embodiments, the image may comprise data from the first data set, the second data set, the third data set, or any combination thereof, described elsewhere herein. In some embodiments, the image may be generated from a corrected and / or reconstructed data set, for example, a speed corrected and / or reconstructed first data set and / or second data set of one or more particles 902, described elsewhere here. In some embodiments, the methods (800, 900) may comprise one or more analysis, processing, and / or data setup steps and / or operations 801 and / or one or moresorting steps and / or operations 808. In some embodiments, the one or more analysis, processing, and / or data setup steps 801 may comprise receiving and / or providing one or more image data sets of a particle and / or cell 802 and / or reconstructing an image 902 from the first data set, the second data set, the third data set, or any combination thereof, described elsewhere herein. The one or more analysis, processing, and / or data setup steps and / or operations 801 may comprise a user selection of one or more features and / or one or more gates of the one or more image data sets of the particle and / or cell 803; generating and / or providing, by one or more predictive models and / or one or more machine learning models, one or more features and / or one or more gates of the one or more image data sets of the particle and / or cell 804; and selecting, by the one or more predictive models and / or one or more machine learning models, one or more features and / or one or more gates of the one or more image data sets of the particle and / or cell 805. In some cases, the one or more analysis, processing, and / or data setup steps 801 may comprise training one or more predictive models and / or one or more machine learning models with the one or more image data sets of the particle and / or cell 806. In some cases, the one or more analysis, processing, and / or data setup steps and / or operations may comprise image feature identification (e.g., the one or more label-free information and / or cell and / or particle morphologic features, described elsewhere herein) and extraction 904. In some cases, the image feature identification step and / or operation may comprise visualizing and / or displaying one or more scatter plots 906 (for example, as shown in FIGS. 26A-26F) of the one or more particle parameter(s) and / or properties, described elsewhere herein. In some embodiments, the user selected features and / or gates; predictive model and / or machine learning model generated features, weights, and / or gates; and / or predictive model and / or machine learning model selected features and / or gates may be provided to one or more processors of a system 810, described herein. The one or more processors of the system, described herein, may extract, process, and determine if an event based at least on the one or more user selected features and / or gates, the predictive model and / or machine learning model selected and / or generated features and / or gates, is identified in a particle and / or cell image data set 807. In some cases, the trained predictive model and / or machine learning model weights may be provided to a predictive model and / or machine learning model inference module 809 to process the particle and / or cell image data set 807. In some cases, the particle may be sorted by a sorting actuation 811, described herein, based at least on the output of the trained predictive model and / or machine learning model inference module 809 and the output of the one or more processors of the system that have extracted, processed, and / or determined whether the features and / or gates of the particle and / or cell image data have been identified. In some embodiments, the user defined gating criterion of the one or more properties may be provided to one or more predictive models and / or one or more machine learning models, described elsewhere herein, thathave been trained 908 on the one or more particle and / or one more cell user defined gating criterion. Based on the characterization, identification and / or prediction of the one or more predictive models and / or one or more machine learning models, described elsewhere herein, the one or more particles and / or one or more cells may be sorted 811 by a sorting actuator, described herein. In some embodiments, the one or more properties utilized by the one or more predictive models and / or the one or more machine learning models to identify, characterize, and / or sort the one or more particles may be ranked in importance and / or analyzed 910 to determine if a correlation between particle and / or cell properties and groups and / or types of particles and / or cells may be present. In some embodiments, one or more ranked feature importance and / or heatmaps, as shown in FIGS. 28B-28D, may be displayed and / or analyzed by a user and / or the computer system described elsewhere herein.Computer systems

[0098] The present disclosure describes a computer system 1100, shown in FIG. 31, that may be electrically coupled to one or more components of the imaging devices and / or systems, described elsewhere herein, to control and / or actuate one or more components of the imaging system and / or device. In some embodiments, the computer system 1100 may implement methods of the disclosure, described elsewhere herein. In some embodiments, the computer system 1100 can regulate various aspects of, for example, detecting a first data set, a second data set, a third data set, or a combination thereof, with one or more detectors, described elsewhere herein. In some embodiments, the computer system 1100 may control, actuate, and / or provide a scanning waveform to a first light source scanner, a second light source scanner, or a combination thereof, described elsewhere herein. In some embodiments, the computer system may record and / or detect a first data set, a second data set, and / or a third data set, comprising fluorescent and / or label-free image information of one or more particles. In some embodiments, the computer system may process and / or determine one or more parameters of one or more particles from the first data set, the second data set, and / or the third dataset. In some embodiments, the computer system may identify one or more particles based on the one or more parameters of the one or more parameters and / or sort the one or more particles based at least on the identity of the one or more particles. In some embodiments, the computer system may actuate and / or control a sorting apparatus to control the sorting of the one or more identified particles into one or more wells (e.g., of a multi-well plate), one or more vessels, one or more reservoirs, or any combination thereof.

[0099] In some embodiments, the disclosure describes a system (e.g., a computer system 1100) that may adjust the one or more properties of a particle. In some embodiments, the system may comprise: one or more processors 1102, and memory (1104, 1110) storing one or more programsfor execution by the one or more processors 1102, the one or more programs comprising instructions to: receive or obtain a first data set of a particle generated by scanning the particle with a first light source optically coupled to a first beam resizing optical element and a first light source scanner, and / or a second data set of the particle generated by scanning the particle with a second light source optically coupled to a second beam resizing optical element and a second light source scanner; process the first data set and the second data set with one or more processing parameters; and adjusting a display of the particle (e.g., by a user interface on a display, described elsewhere herein) when the one or more processing parameters are changed. In some embodiments, the first beam resizing optical element may comprise a first anamorphic prism pair. In some embodiments, the second beam resizing optical element may comprise a second anamorphic prism pair. In some embodiments, the one or more processing parameters may comprise compensation, ellipse fit based aspect ratio, or a combination thereof. In some embodiments, a user of the system may change the one or more processing parameters with a user interface of the system. In some embodiments, the one or more processing parameters may be changed in real-time when the first data set, the second data set, or a combination thereof, is received and / or obtained. In some embodiments, real-time processing of parameters may comprise a rate of at least about 30 events per second, where each event may comprise receiving and / or obtaining and processing the first data set, the second dataset, or a combination thereof. In some embodiments, the particle may comprise a plurality of particles. In some embodiments, the plurality of particles may comprise cell clusters, spheroids, organelles, or a combination thereof. In some embodiments, the display of the particle may comprise a scatter plot of one or more properties of the particle, where the one or more properties of the particle may be determined from the first data set, the second data set, or a combination thereof. In some embodiments, the display of the particle may comprise an image of the particle. In some embodiments, the particle may comprise a cell.

[0100] The computer system 1100 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. The computer system 1100 may comprise a central processing unit (CPU, also “processor”, “computer processor”, and / or “one or more processors”, described elsewhere herein) 1102, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1100 may comprise memory and / or memory location 1110 (e.g., random-access memory, read-only memory, and / or flash memory), electronic storage unit 1104 (e.g., hard disk), communication interface 1106 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1108, such as cache, other memory, data storage and / or electronic display adapters. The memory 1110,storage unit 1104, interface 1106 and peripheral devices 1108 may be in communication with the CPU 1102 through a communication bus (solid lines), such as a motherboard. The storage unit 1104 can be a data storage unit (or data repository) for storing data. The computer system 1100 can be operatively coupled to a computer network (“network”) 1116 with the aid of the communication interface 1106. The network 1116 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1116 in some cases may be a telecommunication and / or data network. The network 1116 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1116, in some cases with the aid of the computer system 1100, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1100 to behave as a client or a server.

[0101] The CPU 1102 can execute a sequence of machine-readable instructions, which can be embodied in a program or software, described elsewhere herein. The instructions may be stored in a memory location, such as the memory (1104, 1110). The instructions can be directed to the CPU 1102, which can subsequently program or otherwise configure the CPU 1102 to implement methods of the present disclosure, described elsewhere herein. Examples of operations performed by the CPU 1102 can include fetch, decode, execute, and writeback.

[0102] The CPU 1102 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1100 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC), a field programmable array (FPA), or a combination thereof.

[0103] The storage unit 1104 can store files, such as drivers, libraries and saved programs. The storage unit 1104 can store one or more particles’ first imaging data set, second imaging data set, and / or third imaging data set. In some embodiments, the storage unit 1104 may store one or more parameters and / or properties of one or more particles imaged by the imaging systems and / or devices described elsewhere herein. In some embodiments, the storage unit may store user preferences and / or user programs used to detect, acquire, and / or visualize the first data set, the second data set, the third data set, or any combination thereof, and / or one or more parameters and / or one or more properties of the one or more particles. The computer system 1100 in some cases can include one or more additional data storage units that are external to the computer system 1100, such as located on a remote server that is in communication with the computer system 1100 through an intranet or the Internet 1116.

[0104] The computer system 1100 can communicate with one or more remote computer systems through the network 1116. For instance, the computer system 1100 can communicate with a remote computer system of a user. The remote computer systems may comprise, for example,personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), personal digital assistants, or any combination thereof. The user can access the computer system 1100 via the network 1116.

[0105] Methods, as described elsewhere herein, can be implemented by way of machine (e.g., computer processor) executable code (i.e., software and / or instructions) stored on an electronic storage location of the computer system 1100, such as, for example, on the memory 1110 or electronic storage unit 1104. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 1102. In some cases, the code can be retrieved from the storage unit 1104 and stored on the memory 1110 for ready access by the processor 1102. In some situations, the electronic storage unit 1104 can be precluded, and machine-executable instructions are stored on memory 1110.

[0106] The code can be pre-compiled and / or configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.

[0107] Aspects of the systems and methods provided herein, such as the computer system 1100, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.“Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer and / or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and / or electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0108] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement databases. Volatile storage media may comprise dynamic memory, such as main memory of such a computer platform. Tangible transmission media may comprise coaxial cables, copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore may comprise, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Any of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0109] The computer system 1100 can include or be in communication with an electronic display 1112 that comprises a user interface (UI) 1114 for providing, for example, a display of one or more properties and / or parameters of one or more particles and / or one or more population of particles; a display of a first data set, a second data set, and / or third data set; or any combination thereof. Examples of UFs include, without limitation, a graphical user interface (GUI) of software displayed on a display 1112 of the computer system and / or web-based user interface.

[0110] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm, as described by the methods described elsewhere herein, can be implemented by way of software upon execution by the central processing unit 1102. The algorithm can, for example, process a first data set, a second data set, a third data set, or a combination thereof to determine one or more parameters and / or one or more properties of one or more particles, described elsewhere herein.DEFINITIONS[OHl] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greaterthan” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0112] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0113] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art.Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

[0114] The term “label-free” or “label-free information” described herein may mean information and / or optical signal determined and / or detected from one or more particles and / or one or more cells that does not result from an added fluorescent molecule that interacts with and / or binds to the one or more particles and / or the one or more cells.

[0115] The term “gating” or to “gate” described herein may mean a process and / or an operation conducted by one or more computer processors and / or a human user, to separate and / or classify one or more datasets of a plurality of datasets. In some cases, the one or more datasets may be displayed overlaid in a graphical representation. The term “gating” may mean indicating, selecting, providing, and / or drawing a separating feature, e.g., a curved and / or straight line feature on the plurality of datasets displayed overlaid in the graphical representation. The term “gate line” may indicate a linear and / or curved line segment that may divided and / or separate one or more data samples plotted in a graph into one or more data sets and / or one or more clusters of data sets.

[0116] The term “morphological feature” described herein may mean one or more physical characteristic features of one or more particles, and / or one or more cells, and / or one or more subcellular components described elsewhere herein. For example, the term “morphologicalfeature” may mean the size, e.g., diameter, eccentricity, area , granularity, texture, or the like, of the one or more particles, and / or one or more cells, and / or one or more subcellular components.EXAMPLESExample 1: Image Feature Extraction and Training A Predictive Model

[0117] The imaging system, devices, and methods, described elsewhere herein, were used to collect data of two cell populations, that was used to train a predictive model and / or a machine learning model. A population of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells were provided to the imaging system and / or device, described elsewhere herein, and one or more properties of a first data set, second data set, and / or third data of the CHO and HEK cells populations was determined with the imaging system (scatter plot data for each of the cell properties shown in FIGS. 26A-26F).

[0118] The data of the one or more scatter plots was then gated by a user to separate the two populations of cells in each of the scatter plots. The resulting multi-dimensional data set was then cluster and / or reduced in dimensionality into a two-dimensional t-distributed stochastic neighbor data set, as shown in FIG. 28A. The predictive model and / or machine learning model were then trained on the one or more properties of the two populations that contributed the highest component of data dimensionality reduction and clustering. Through the training process of the predictive model and / or machine learning model, 5 total folds were implemented during the training and validation training process, where the combined accuracy and loss curves are shown in FIG. 27A, combined training accuracy and loss are shown in FIG. 27B-27C, and validation accuracy and loss are shown in FIG. 27D-27E.

[0119] Model property and / or image feature analysis were conducted, and the resulting feature importance is shown in FIGS. 28B-28C, corresponding to feature importance using MDI model and a full model, respectively, and a correlation heat map between the CHO and HEK cell lines shown in FIG. 28D. From this study, the properties of the first, second, and / or third data sets of CHO and HEK cells that lead to the greatest positive and / or correct identification and sorting of the CHO and HEK cell was contrast, maximum intensity across the cell, integrated intensity across the cell, and the area of the cell, with lesser contributions from the gradient RMS of the cell and aspect ratio.Example 2: Determining a Speed of a Particle and Correcting a Particle Image Dataset

[0120] The imaging systems, devices, and methods, described elsewhere herein, were utilized to obtain transmission images of bead particles with a diameter of 16 pm (FIGS. 9A, 12A-12B), 15pm (FIG. 8A), 10 pm (FIGS. 10A), 7 pm (FIG. 8F), and 3 pun (FIGS. 11 A, 15A-15B). As can be seen from FIGS. 8A, 8F, 9A-9B, 10A-10B, 11A-11B, 12A-12B, 13A-13B, 14A-14B, and 16A-16B, the image(s) of the particle(s) are distorted due to the particle variations in flow rate between different size and shaped particles as the particle flows and / or travels through a fluidic channel and is imaged. Ahead of further processing of the temporal light transmission intensity of each of the particles, a background light intensity was subtracted from the distorted images of the particles, as shown in FIGS. 9B, 10B, 11B, 12B, 13B, 14B, and 15B.

[0121] To correct for the non-uniform traveling speed, the speed of the particle was measured for each of the aforementioned particles by the methods described elsewhere herein. The temporal light transmission intensity of each of the particles (FIGS. 8B-8C, 8G-8H, 9C, IOC, 11C, 12C, 13C, 14C, and 15C) was measured and / or detected as the particles traversed and / or flowed through the light stripe of the third light source, the first scanned beam of the first light source, and the second scanned beam of the second light sources.

[0122] Ahead of performing cross correlation on the temporal light transmission intensity for each of the particles to determine speed, the temporal light transmission intensity was inverted, as shown in FIGS. 9D, 10D, and 11D.

[0123] Autocorrelation for each of the particles’ inverted temporal light transmission intensity curves was then conducted to determine each particles’ speed (shown in FIGS. 81, 9E, 10E, HE, 12D, 13D, 14D, and 15D), which was then used to correct and / or resize the image of the particles, shown in FIGS. 8E, 8J, 9F-9G, 10F-10G, 11F-11G, 12E-12F, 13E-13F, 14E-14F, and 15E-15F.Example 3: Sample and Sheath Flow Rates and Measured Travel Speed of Particles

[0124] The imaging systems, devices, and methods, described elsewhere herein, were used to analyze the variability of particle travel speeds when sample and fluidic sheath flow rates are varied. The findings from such an experiment can inform sample and fluidic sheath flow rates that minimize the variability and / or non-uniformity of particle travel speeds to reduce particle and / or cell imaging distortion and / or correction needed.

[0125] Particle speed was measured by the imaging systems, devices, and methods, described elsewhere herein, for sample and sheath flow rates of: sample flow rate of 12 pL / min and sheath flow rate of 120 pL / min (FIG. 16A); sample flow rate of 16 pL / min and sheath flow rate of 120 pL / min (FIG. 16B); sample flow rate of 24 pL / min and sheath flow rate of 120 pL / min (FIG. 16C); sample flow rate of 12 pL / min and sheath flow rate of 100 pL / min (FIG. 16D); sample flow rate of 16 pL / min and sheath flow rate of 100 pL / min (FIG. 16E); and sample flow rate of 24 pL / min and sheath flow rate of 160 pL / min (FIG. 16F).

[0126] From this study, it can be understood that the distribution of particle and / or cell speed decreases as the flow ratio between sample flow rate and sheath flow rate decreases.Example 4: Empirical Determination of Particle Speed

[0127] By imaging a particle and / or cell with a known diameter, shape, and / or size with the imaging systems, devices, and / or methods, described elsewhere herein, a speed correction factor may be determined empirically. A plurality of spherical particles of the same diameter were imaged with the imaging systems, devices, and / or methods, described elsewhere herein, and the particles width and height ratio and the cross-correlation delay of the temporal light transmission intensity were determined, as shown in FIG. 17A. An example distorted image of a particle from the plurality of particles acquired is shown in FIG. 17B. Speed of a particle was determined from the linear slope of the relationship of measured width to height ratio of the particles and crosscorrelation delay of the temporal light transmission intensity profiles of the particle shown in FIG. 17A. Since the particles share the same diameter, such a slope represents the non-uniform travel speed for the particle. Thus, shifting and / or correcting the originally acquired particle data by the speed correction would correct distortions caused by non-uniform travel and / or flow speed of the particles. FIG. 17C shows a shifted intensity segment of an originally acquired data set shifted by the speed correction determined from calculating the cross-correlation delay. From FIG. 17C, it can be seen that the two temporal light transmission intensity profiles are similar in shape that the cross correlation delay provides good profile alignment. FIG. 17D shows the speed correction applied to the original distorted particle images shown in FIG. 17B, results in an image that is no longer distorted as shown in the original image.Example 5: Imaging Resolution Improvements by Corrective Optical Element

[0128] The imaging systems, devices, and / or methods, described elsewhere herein, use one or more beam size correction optical element(s) to improve the imaging resolution and ability of the imaging systems, device, and / or methods to image particles and / or cells of a plurality of sizes and shapes.

[0129] A series of fluorescence and transmission bright field images of particles with a diameter of 15 pm (FIGS. 18A-18B, 19A-19B), 7 pm (FIGS. 18C-18D, 19C-19D), and 1 pm (FIGS. 18E-18F, 19E-19F) were acquired at 200kHz (FIGS. 18A-18F) and 400kHz (FIGS. 19A-19F) scan rates with the imaging systems, devices, and / or methods described elsewhere herein, with (FIGS. 18B, 18D, 18F, 19B, 19D, 19F) and without (FIGS. 18A, 18C, 18E, 19A, 19C, 19E) beam size correction optical elements (e.g., a cylindrical lens). From the observable increase in sharpness of the images of the particles acquired and / or detected with the beam size correctionoptical element(s) (FIGS. 18B, 18D, 18F, 19B, 19D, 19F), compared with the image of the particles taken without the beam size correction optical elements (FIGS. 18A, 18C, 18E, 19A, 19C, 19E), it can be seen that the beam size correction optical element(s) provide an improvement in the imaging systems, devices, and / or methods ability to visualize and render images of particles of all sizes.

[0130] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0131] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A particle imaging system, comprising:(a) an imaging device comprising:(i) a first light source optically coupled to a first beam resizing optical element, wherein an output of the first beam resizing optical element is optically coupled to a first light source scanner configured to scan a first beam of the first light source along an axis of a particle to obtain a first data set comprising fluorescent information or label-free image information of the particle,(ii) a second light source optically coupled to a second beam resizing optical element, wherein an output of the second beam resizing optical element is optically coupled to a second light source scanner configured to scan a second beam of the second light source along the axis of the particle to obtain a second data set comprising fluorescent information or label-free image information of the particle, wherein the particle is flowing in a channel at an angle to an optical axis of the first beam of the first light source and an optical axis of the second beam of the second light source, and wherein the first beam resizing optical element and the second beam resizing optical element differ; and(b) a data processing device in communication with the imaging device, wherein the data processing device comprises a processor configured to process the first data set, the second data set, or a combination thereof, obtained by the imaging device to determine one or more properties associated with the particle.

2. The particle imaging system of claim 1, wherein the first beam resizing optical element comprises a first anamorphic prism pair or a first cylindrical lens, and wherein the second beam resizing optical element comprises a second anamorphic prism pair or a second cylindrical lens.

3. The particle imaging system of claims 1 or 2, wherein the first data set, the second data set, or a combination thereof, is generated by a detector configured to detect one or more optical signals emitted, attenuated, or scattered by the particle in response to the illumination of particle by the first scanned beam, the second scanned beam, or a combination thereof, and wherein the one or more optical signals emitted or scattered by the particle are emitted, attenuated, or scattered along an optical axis towards an emission of the first light source, the second light source, or a combination thereof.

4. The particle imaging system of claim 3, wherein the one or more optical signals emitted or scattered by the particle along the optical axis towards the emission of the first light source, the second light source, or a combination thereof, comprise, darkfield optical signals, back scattered optical signals, epifluorescent optical signals, or a combination thereof.

5. The particle imaging system of any one of claims 1-4, wherein the detector comprises a camera, one or more photomultiplier tubes, one or more photodiodes, avalanche photodetector, array of photomultiplier tubes, a fiber coupled detector, or a combination thereof.

6. The particle imaging system of claim 5, wherein the one or more photomultiplier tubes are optically coupled to the one or more optical signals emitted, or scattered by the particle through a beam splitter.

7. The particle imaging system of any one of claims 2-6, wherein the first scanned beam of light is coupled to a third anamorphic prism pair, and wherein the second scanned beam of light is coupled to a fourth anamorphic prism pair.

8. The particle imaging system of claim 7, wherein the first anamorphic prism pair and the third anamorphic prism pair magnify a scanned field of view of the first scanned beam provided by the first light source scanner, and wherein the second anamorphic prism pair and the fourth anamorphic prism pair magnify a scanned field of view of the second scanned beam provided by the second light source scanner.

9. The particle imaging system of claims 7 or 8, wherein the first anamorphic prism pair, the second anamorphic prism pair, or a combination thereof, comprise a reversed optical orientation compared with the third anamorphic prism pair, the fourth anamorphic prism pair, or a combination thereof.

10. The particle imaging system of any one of claims 1-9, wherein the first light source scanner, the second light source scanner, or a combination thereof, comprise an Acousto-Optic Deflector (AOD).

11. The particle imaging system of any one of claims 1-10, wherein the first scanned beam of light is coupled to a first cylindrical lens, wherein the second scanned beam of light is coupled to a second cylindrical lens, and wherein the first cylindrical lens and the second cylindrical lens differ.

12. The particle imaging system of claim 11, wherein the first cylindrical lens compensates for a first astigmatism of the first scanned beam of light, and wherein the second cylindrical lens compensates for a second astigmatism of the second scanned beam of light.

13. The particle imaging system of any one of claims 2-12, comprising a third light source optically coupled to a fifth anamorphic prism pair and a third cylindrical lens, wherein a light stripe of the third light source outputted by the third cylindrical lens illuminates the particle thereby producing a third data set comprising fluorescent information or label-free image information of the particle.

14. The particle imaging system of claim 13, wherein the light stripe of the third light source does not spatially overlap with the first scanned beam of the first light source, the second scanned beam of the second light source, or a combination thereof.

15. The particle imaging system of claims 13 or 14, wherein the data processing device processes the third data set in comparison to the first data set, the second data set, or a combination thereof, to determine a speed of the particle flowing in the channel.

16. The particle imaging system of claim 15, wherein the speed of the particle flowing in the channel is determined by conducting autocorrelation, thresholding, or a combination thereof, with the first data set, the second data set, the third data set, or a combination thereof.

17. The particle imaging system of any one of claims 13-16, wherein the third light source comprises a light emitting diode, a laser, super luminescent diode, white light source, or any combination thereof.

18. The particle imaging system of any one of claims 13-17, comprising a mirror, wherein the mirror comprises an annular reflective region and a transmissive region, wherein the annular reflective region and the transmission region are spatially separated, wherein the first scanned beam, the second scanned beam, the light stripe, or a combination thereof, are reflected from the annular reflective region toward the particle, and wherein the one or more optical signals emitted or scattered by the particle transmit through the transmission region.

19. The particle imaging system of claim 18, wherein the transmission region comprises an anti- reflective coating.

20. The particle imaging system of any one of claims 13-19, wherein the light stripe of the third light source is coupled to an optical axis of the first scanned beam, the second scanned beam, or a combination thereof, with a polarization beam splitter.

21. The particle imaging system of any one of claims 1-20, wherein the imaging device comprises a bright field detector, a dark field detector, a speed detector, or any combination thereof, optically coupled to the particle, wherein the bright field detector, the dark field detector, the speed detector, or any combination thereof, is configured to detect one or more scattered or attenuated optical signals of the particle in response to the illumination of the particle by the first scanned beam, the second scanned beam, or a combination thereof, and wherein the one or more optical signals emitted or scattered by the particle are emitted or scattered along an optical axis away from an emission of the first light source, the second light source, or a combination thereof.

22. The particle imaging system of any one of claims 13-20, wherein the imaging device comprises a bright field detector, a dark field detector, or a combination thereof, optically coupled to the particle, wherein the bright field detector, the dark field detector, or a combination thereof, is configured to detect one or more scattered or attenuated optical signals of the particle in response to the illumination of the particle by the first scanned beam, the second scanned beam, the light stripe, or any combination thereof, and wherein the one or more optical signals emitted or scattered by the particle are emitted or scattered along an optical axis away from an emission of the first light source, the second light source, the third light source, or any combination thereof.

23. The particle imaging system of claims 21 or 22, wherein the one or more optical signals emitted or scattered by the particle along the optical axis away from the emission of the first light source, the second light source, third light source, or any combination thereof, comprise brightfield microscopy optical signals, forward scattered optical signals, or a combination thereof.

24. The particle imaging system of any one of claims 13-23, wherein the processor is in electrical communication with memory storing one or more predictive models configured to process the first data set, the second data set, the third data set, or a combination thereof, to provide a classification of the particle.

25. The particle imaging system of claim 24, wherein the one or more predictive models comprise clustering, classifying, or a combination thereof predictive models.

26. The particle imaging system of claims 24 or 25, wherein the one or more predictive models cluster the first data set, the second data set, the third data set, or any combination thereof, based on one or more morphologic features of the first data set, the second data set, the third data set, or a combination thereof.

27. The particle imaging system of any one of claims 24-26, wherein the classification of the particle comprises a classification of one or more regions or one or more segments of the particle.

28. The particle imaging system of claim 27, wherein the particle comprises a cell, and wherein the one or more regions or the one or more segments of the particle comprise one or more subcellular components of the cell.

29. The particle imaging system of any one of claims 24-27, wherein the one or more predictive models comprise a convolutional neural network, a logistic regression model, sequential perceptron model, k-nearest neighboring model, support vector machine model, adaboost model, random forest model, or any combination thereof.

30. The particle imaging system of claim 29, wherein the convolutional neural network comprises a structure of a UNet predictive model, a convolutional autoencoder, a fused UNet predictive model, or any combination thereof.

31. The particle imaging system of any one of claims 1-30, wherein the first scanned beam of the first light source and the second scanned beam of the second light source spatially overlap.

32. The particle imaging system of any one of claims 1-31, wherein the first scanned beam of the first light source and the second scanned beam of the second light source do not spatially overlap.

33. The particle imaging system of any one of claims 2-32, wherein an intensity of the first light source comprises a first intensity modulation frequency 180 degrees out of phase with a second intensity modulation frequency of the second light source.

34. The particle imaging system of any one of claims 1-33, wherein the first light source or the second light source comprises a center wavelength of about 355nm to about 785 nm.

35. The particle imaging system of any one of claims 1-34, wherein the first light source scanner, the second light source scanner, or a combination thereof, is provided an amplitude modulated chirp driving signal to scan the first beam, the second beam, or a combination thereof.

36. The particle imaging system of claim 35, wherein the amplitude modulated chirp driving signal provides an output power of at least 5 mW of the first scanned beam, the second scanned beam, or a combination thereof, across a field of view of the first light source scanner, the second light source scanner, or a combination thereof.

37. The particle imaging system of any one of claims 1-36, wherein the data processing device comprises a field programmable gate array (FPGA), graphical processing unit (GPU), neural processing unit (NPU), or any combination thereof.

38. The particle imaging system of any one of claims 1-37, wherein the first light source, the second light source, or a combination thereof, comprise fiber coupled lasers.

39. The particle imaging system of any one of claims 1-38, wherein the particle comprises one or more cells.

40. The particle imaging system of any one of claims 1-39, wherein the one or more properties associated with the particle comprise a gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof.

41. The particle imaging system of any one of claims 1-40, wherein the one or more properties are used to align the fluidic channel and the imaging device.

42. The particle imaging system of any one of claims 13-41, wherein the one or more properties are determined by cross-correlation of the first data set and the second data set, the first data set and the third data set, the second data set and the third data set, or a combination thereof.

43. The particle imaging system of any one of claims 13-42, wherein the first data set, the second data set, the third data set, or a combination thereof, comprise an image data set of the particle.

44. The particle imaging system of any one of claims 1-43, wherein the first scanned beam and the second scanned beam are sequentially scanned along the axis of the particle.

45. The particle imaging system of any one of claims 1-44, wherein when the first scanned beam is scanned along the axis of the particle, the second scanned beam is directed to a first beam capturing optical component, and wherein when the second scanned beam is scanned along the axis of the particle, the first scanned beam is directed to a second beam capturing optical component.

46. The particle imaging system of claim 45, wherein the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, comprise a beam dump.

47. The particle imaging system of any one of claims 1-46, wherein a background signal is removed or subtracted from the first data set, the second data set, or a combination thereof, wherein the background signal comprises one or more optical signals detected, obtained, and / or collected when the channel is illuminated with the first scanned beam, the second scanned beam, or a combination thereof.

48. The particle imaging system of any one of claims 1-47, wherein the first data set, the second data set, or a combination thereof, is phase shifted by one or more obtained data samples to synchronize the scanning of the first light source scanner and obtaining the first data set and to synchronize the scanning of the second light source scanner and obtaining the second data set.

49. The particle imaging system of any one of claims 1-48, wherein the first light source scanner and the second light scanner differ.

50. The particle imaging system of any one of claims 1-49, wherein the data processing device sorts the particle based at least on the one or more properties associated with the particle.

51. The particle imaging system of any one of claims 1-50, wherein the data processing device sorts the particle with a piezo electric sorting apparatus.

52. The particle imaging system of any one of claims 1-51, wherein the data processing device sorts the particle based at least on the one or more properties associated with the particle determined by gating the first data set or the second data set.

53. The particle imaging system of any one of claims 1-52, wherein the label-free image information comprises non-fluorescent image information of the particle.

54. A method for scanning a particle, comprising:(a) moving a particle along a first direction;(b) generating a first scanned beam of a first light source with a first light source scanner and a second scanned beam of a second light source with a second light source scanner, wherein an output of the first light source is optically coupled to a first beam resizing optical element and an output of the second light source is optically coupled to a second beam resizing optical element, wherein an output of the first beam resizing optical element is optically coupled to the first light source scanner and an output of the second beam resizing optical element is optically coupled to the second light source scanner, and wherein the first beam resizing optical element and the second beam resizing optical element differ; and(c) scanning the particle with the first scanned beam of the first light source and the second scanned beam of the second light source in a second direction at an angle to the first direction.

55. The method of claim 54, wherein the first beam resizing optical element comprises a first anamorphic prism pair or a first cylindrical lens, and wherein the second beam resizing optical element comprises a second anamorphic prism pair or a second cylindrical lens.

56. The method of claims 54 or 55, further comprising detecting a first data set of the particle from one or more optical signals emitted, attenuated, or scattered by the particle in response to illumination of the particle with the first scanned beam, and detecting a second data set of the particle from one or more optical signals emitted or scattered by the particle in response to illumination of the particle with the second scanned beam.

57. The method of any one of claims 54-56, wherein the first data set, the second data set, or a combination thereof comprise an image data set of the particle.

58. The method of any one of claims 5554-57, comprising processing the first data set and the second data set to determine one or more properties associated with the particle.

59. The method of claim 58, wherein the one or more properties of the particle comprise speed, fluorescent signal, morphologic feature, or a combination thereof.

60. The method of claim 59, wherein the morphologic feature data is adjusted based on the particle speed.

61. The method of any one of claims 55-60, wherein the one or more optical signals emitted or scattered by the particle in response to illumination of the particle by the first scanned beam, the second scanned beam, or a combination thereof, are emitted or scattered along an optical axis towards an emission of the first light source, the second light source, or a combination thereof.

62. The method of claim 61, wherein the one or more optical signals emitted, attenuated, or scattered by the particle in response to the illumination along the optical axis towards the emission of the first light source, the second light source, or a combination thereof, comprise darkfield optical signals, back scattered optical signals, epifluorescent optical signals, or a combination thereof.

63. The method of claims 55-62, wherein the one or more optical signals emitted or scattered by the particle in response to the illumination of the particle with the first scanned beam, the second scanned beam, or a combination thereof, is detected by a camera, one or more photomultiplier tubes, one or more photodiodes, a fiber coupled detector, or any combination thereof.

64. The method of claims 55-63, wherein the first scanned beam is coupled to a third anamorphic prism pair, and wherein the second scanned beam is coupled to a fourth anamorphic prism pair.

65. The method of claim 64, wherein the first anamorphic prism pair and the third anamorphic prism pair magnify a scanned field of view of the first scanned beam provided by the first light source scanner, and wherein the second anamorphic prism pair and the fourthanamorphic prism pair magnify a scanned field of view of the second beam provided by the second light source scanner.

66. The method of claims 64 or 65, wherein the first anamorphic prism pair, the second anamorphic prism pair, or a combination thereof, comprise a reversed optical orientation compared with the third anamorphic prism pair, the fourth anamorphic prism pair, or a combination thereof.

67. The method of claims 54-66, wherein the first light source scanner, the second light source scanner, or a combination thereof, comprise an Acousto-Optical Deflector (AOD).

68. The method of claims 54-67, wherein the first scanned beam of light is coupled to a first cylindrical lens, wherein the second scanned beam of light is coupled to a second cylindrical lens, and wherein the first cylindrical lens and the second cylindrical lens differ.

69. The method of claim 68, wherein the first cylindrical lens compensates for a first astigmatism of the first scanned beam of light, and wherein the second cylindrical lens compensates for a second astigmatism of the second scanned beam of light.

70. The method of any one of claims 55-69, wherein a third light source is optically coupled to a fifth anamorphic prism pair and a third cylindrical lens, wherein a light stripe of the third light source outputted by the third cylindrical lens illuminates the particle thereby producing a third data set of the particle.

71. The method of claim 70, wherein the light stripe of the third light source does not spatially overlap with the first scanned beam of the first light source, the second scanned beam of the second light source, or a combination thereof.

72. The method of claims 70 or 71, comprising processing the third data set of the particle in comparison to the first data set, the second data set, or a combination thereof, to determine a speed of the particle flowing in the first direction.

73. The method of claim 72, wherein the speed of the particle flowing in the first direction is determined with a processing latency of less than about 100 milliseconds.

74. The method of claims 72 or 73, wherein the speed of the particle flowing in the first direction is determined by conducting autocorrelation, cross-correlation, thresholding, or a combination thereof, with the first data set, second data set, third data set, or any combination thereof.

75. The method of any one of claims 70-74, further comprising detecting one or more optical signals scattered, attenuated, or emitted from the particle with a bright field detector, dark field detector, a speed detector, or a combination thereof, in response to the illumination of the particle by the first scanned beam, the second scanned beam, the light stripe, or a combination thereof, wherein the bright field detector, the dark field detector, the speed detector, or any combination thereof, detects the one or more optical signals emitted, attenuated, or scattered by the particle along an optical axis away from an emission of the first light source, the second light source, the third light source, or any combination thereof.

76. The method of claim 75, wherein the one or more optical signals emitted, attenuated, or scattered by the particle along the optical axis away from the emission of the first light source, the second light source, the third light source, or any combination thereof, comprise brightfield microscopy optical signals, forward scatter optical signals, or a combination thereof.

77. The method of any one of claims 70-76, comprising processing the first data set, the second data set, the third data set, or a combination thereof, with one or more predictive models to provide a classification of the particle.

78. The method of claim 77, wherein the one or more predictive models comprise clustering, classifying, or a combination thereof predictive models.

79. The method of claims 77 or 78, wherein the one or more predictive models cluster the first data set, the second data set, the third data set, or a combination thereof, based on one or more morphologic features of the first data set, the second data set, the third data set, or a combination thereof.

80. The method of any one of claims 77-79, wherein the classification of the particle comprises a classification of one or more regions or one or more segments of the particle.

81. The method of claim 80, wherein the particle comprises a cell, and wherein the one or more regions or the one or more segments of the particle comprise one or more subcellular components of the cell.

82. The method of any one of claims 77-80, wherein the one or more predictive models comprise a convolutional neural network.

83. The method of claim 82, wherein the convolutional neural network comprises a structure of a UNet predictive model, a convolutional autoencoder, a fused UNet predictive model, or any combination thereof.

84. The method of any one of claims 70-83, comprising directing the first scanned beam, the second scanned beam, the light stripe, or a combination thereof, to the particle with a mirror, wherein the mirror comprises an annular reflective region and a transmissive region, wherein the annular reflective region and the transmission region are spatially separated, and wherein the one or more optical signals emitted or scattered by the particle in response to the illumination of the particle with the first scanned beam, the second scanned beam, or a combination thereof, transmit through the transmission region.

85. The method of claim 84, wherein the transmission region comprises an anti -reflective coating.

86. The method of any one of claims 70-85, wherein the light stripe of the third light source is coupled to an optical axis of the first scanned beam, the second scanned beam, or a combination thereof, with a polarization beam splitter87. The method of any one of claims 54-86, wherein the first scanned beam of the first light source and the second scanned beam of the second light source spatially overlap.

88. The method of any one of claims 54-87, wherein the first scanned beam of the first light source and the second scanned beam of the second light source do not spatially overlap.

89. The method of any one of claims 70-88, wherein the first data set, the second data set, the third data set, or any combination thereof, is processed by one or more processors.

90. The method of claim 89, wherein the one or more processors comprises a field programmable gate array (FPGA), graphic processing unit (GPU), neural processing unit (NPU), or any combination thereof.

91. The method of any one of claims 54-90, wherein the first light source or the second light source comprises a center wavelength of about 355nm to about 785nm.

92. The method of any one of claims 54-91, wherein the first light source scanner, the second light source scanner, or a combination thereof, is provided an amplitude modulated chirp driving signal to generate the first scanned beam, the second scanned beam, or a combination thereof.

93. The method of claim 92, wherein the amplitude modulated chirp driving signal provides an output power of at least about 5mW of the first scanned beam, the second scanned beam, or a combination thereof, across a field of view of the first light source scanner, the second light source scanner, or a combination thereof.

94. The method of any one of claims 54-93, wherein the first light source, the second light source, or a combination thereof, comprise fiber coupled lasers.

95. The method of any one of claims 54-94, wherein the particle comprises one or more cells.

96. The method of any one of claims 58-95, wherein the one or more properties associated with the particle comprise gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof.

97. The method of any one of claims 58-96, wherein the one or more properties associated with the particle are used to align a channel the particle is moving through along the first direction with the first scanned beam, the second scanned beam, or a combination thereof.

98. The method of claim 97, wherein a background signal is removed or subtracted from the first data set, the second data set, or a combination thereof, wherein the background signalcomprises one or more optical signals detected, obtained, and / or collected when the channel is illuminated with the first scanned beam, the second scanned beam, or a combination thereof.

99. The method of any one of claims 54-98, wherein the first data set, the second data set, or a combination thereof, is phase shifted by one or more detected data samples to synchronize the scanning of the first light source scanner and detecting the first data set and to synchronize the scanning of the second light source scanner and detecting the second data set.

100. The method of any one of claims 70-99, wherein the one or more properties associated with the particle are determined by cross-correlation of the first data set and the second data set, the first data set and the third data set, the second data set and the third data set, or any combination thereof.

101. The method of any one of claims 70-100, wherein the first data set, the second data set, the third data set, or a combination thereof, comprise an image data set of the particle.

102. The method of any one of claims 54-101, wherein the first scanned beam and second scanned beam are sequentially scanned in the second direction.

103. The method of any one of claims 54-102, wherein when the first scanned beam is scanned in the second direction, the second scanned beam is directed to a first beam capturing optical component, and wherein when the second scanned beam is scanned in the second direction, the first scanned beam is directed to a second beam capturing optical component.

104. The method of claim 103, wherein the first beam capturing optical component, the second beam capturing optical component, or a combination thereof, comprises a beam dump.

105. The method of any one of claims 54-104, wherein the first light source scanner and the second light source scanner differ.

106. The method of any one of claims 58-105, comprising sorting the particle based at least on the one or more properties associated with the particle.

107. A system for adjusting a display of a particle, or the particle properties comprising:one or more processors, and memory storing one or more programs for execution by the one or more processors, the one or more programs comprising instructions to:receive or obtain a first data set of a particle generated by scanning the particle with a first light source optically coupled to a first beam resizing optical element and a first light source scanner, or a second data set of the particle generated by scanning the particle with a second light source optically coupled to a second beam resizing optical element and a second light source scanner;process the first data set and the second data set with one or more processing parameters; and adjust the display of the particle or the particle’s features, when the one or more processing parameters are changed.

108. The system of claim 107, wherein the first beam resizing optical element comprises a first anamorphic prism pair, and wherein the second beam resizing optical element comprises a second anamorphic prism pair.

109. The system of claims 107 or 108, wherein the one or more processing parameters comprise compensation, ellipse fit based aspect ratio, or a combination thereof.

110. The system of any one of claims 107-109, wherein a user of the system changes the one or more processing parameters with a user interface of the system.

111. The system of any one of claims 107-110, wherein the one or more processing parameters are changed in real-time when the first data set, the second data set, or a combination thereof, is received or obtained.

112. The system of claim 111, wherein real-time comprises a rate of at least about 30 events per second, wherein each event comprises receiving or obtaining and processing the first data set, the second data set, or a combination thereof.

113. The system of any one of claims 107-112, wherein the particle comprises a plurality of particles.

114. The system of claim 113, wherein the plurality of particles comprise cell clusters, spheroids, organelles, or a combination thereof.

115. The system of any one of claims 107-113, wherein the display of the particle comprises a scatter plot of one or more properties of the particle, wherein the one or more properties of the particle are determined from the first data set, the second data set, or a combination thereof.

116. The system of any one of claims 107-115, wherein the display of the particle comprises an image of the particle.

117. The system of any one of claims 107-116, wherein the particle comprises a cell.

118. A method of training a predictive model to sort a particle, comprising:(a) receiving or obtaining a first particle image data set of a first particle and a second particle image data set of a second particle;(b) processing the first particle image data set or the second particle image data set to obtain a first one or more properties of the first particle image data set or a second one or more properties of the second particle image data set;(c) gating the first particle image data set or the second particle image data set to separate the first one or more properties of the first particle image data set from the second one or more properties of the second particle image data set, thereby indicating a gated first one or more properties of the first particle image data, and a gated second one or more properties of the second particle image data set; and(d) training the predictive model to sort the first particle and the second particle, wherein the predictive model is trained with the gated first one or more properties of the first particle image data set and the gated second one or more properties of the second particle image data set.

119. The method of claim 118, wherein the gating the first particle image data set or the second particle image data set comprises displaying the first one or more properties of the first particle image data set and the second one or more properties of the second particle image data set on a plot and selecting or providing a gate line by the user on the plot to separate the first one or more properties of the first particle image data and the second one or more properties of the second particle image data set.

120. The method of claims 118 or 119, wherein the first one or more properties comprise morphological features of the first particle, wherein the second one or more properties comprise morphological features of the second particle.

121. The method of claim 120, wherein the morphologic feature of the particles is adjusted by the speed of the particles.

122. The method of any one of claims 118-121, wherein the first one or more properties, the second one or more properties, or a combination thereof, comprise gradient root mean square, contrast, particle centroid location, particle area, particle aspect ratio, particle integrated intensity, particle average intensity, particle maximum intensity, length of particle major axis, length of particle minor axis, particle eccentricity, particle perimeter, particle delta center of mass, particle spot count, speed of the particle, or any combination thereof properties of the first data set, the second data set, or a combination thereof.

123. The method of any one of claims 118-122, further comprising displaying a feature importance rank, feature heatmap, or a combination thereof, of the gated first one or more properties or the gated second one or more properties that separate or distinguish the first particle from the second particle.

124. The method of any one of claims 118-123, wherein the predictive model comprises a convolutional neural network, a logistic regression model, sequential perceptron model, k- nearest neighboring model, support vector machine model, adaboost model, random forest model, or any combination thereof.

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