Multidirectional light-based cell differentiation from a microfluidic chip

Abstract

Techniques are disclosed for distinguishing between different particle types in a sample flowing through a microfluidic chip. The sample may have an initial ratio of a first cell type to a second cell type. An illumination source may emit coherent light into the sample, and light exiting the chip in a first direction may be detected using a first photodetector, and light exiting the chip in a second direction (e.g., orthogonal to the first direction) may be detected using a second photodetector. The detected light may be fluorescent light. An orientation characteristic of a plurality of cells in the sample may be determined based on the light detected by the detector. Based on the orientation characteristic and the detected light, a biasing operation may be performed on each cell in the sample to obtain a corrected ratio of the cell type in the sample.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. patent application Ser. No. 63 / 162,138, filed March 17, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Standard flow cytometry techniques are insufficient to effectively and specifically differentiate a particular particle or cell type in a sample. When differentiating between different cell types, one differentiation characteristic or factor can be the amount of nucleic acid found in the different cell types. However, if the cells or particles have asymmetric shapes, the apparent characteristic or factor (e.g., the amount of DNA detectable using fluorescence in different cells) can vary depending on the position or orientation of the cells or particles, distorting the results and making the comparison less reliable. For example, the amount of DNA detectable in sperm cells with an X chromosome ("female" sperm cells) is about 4 percent more than the amount of DNA detectable in sperm cells with a Y chromosome ("male" sperm cells). However, male sperm cells may appear to have as much DNA as female sperm cells, more DNA than female sperm cells, or otherwise slightly different amounts of DNA, depending on the position and / or orientation of the male sperm cells when the fluorescence is observed. Thus, male sperm cells may appear indistinguishable from female sperm cells, male sperm cells may be misclassified as female sperm cells, and female sperm cells may be misclassified as male sperm cells. Summary of the Invention [Means for solving the problem]

[0003] Various embodiments relate to a method that includes providing a sample including a particle population having a ratio of a first particle type to a second particle type to a microfluidic chip having a sample channel; emitting illumination light from a light source along a first axis as the sample passes through the channel such that the illumination light coincides with particles in the sample; detecting the first light emitted from the sample along the first axis using a first light detector; detecting the second light emitted from the sample along the second axis using the first light detector or a second light detector; and performing a biasing operation to modify the ratio of the first particle type to the second particle type based on the detected first light emitted along the first axis and the detected second light emitted along the second axis.

[0004] In various embodiments, the first light and the second light (i) result from the incidence of illumination light on particles in the sample, and (ii) are fluorescent light traveling in multiple different directions from fluorescent particles in the sample. In various embodiments, the method further includes generating an intensity map of peak fluorescence associated with each particle type based on light emitted from the sample along the first axis and the second axis; detecting a first population center associated with the first particle type and a second population center associated with the second particle type based on the intensity map; calculating a relative intensity along a first line passing through the first population center; calculating a slope of a second line connecting the first population center and the second population center; locating a first saddle point along a third line connecting the first population associated with the first particle type and the second population associated with the second particle type; locating a second saddle point along a fourth line connecting the first population and the second population; and generating a gate by bounding the first population or the second population, the gate being bounded based on the first saddle point, the second saddle point, and the additional boundary point. In various embodiments, the method further includes calculating a kernel in the pixel map, adding a first instance of the kernel to the pixel map based on light emitted from the sample along a first axis and a second axis, adding a second instance of the kernel to the pixel map based on light emitted from the sample along the first axis and a second axis, deriving a first histogram from the pixel map, removing data from the pixel map, deriving a second histogram from a first row of the pixel map, generating a first trough point from the second histogram, deriving a third histogram from a second row of the pixel map, generating a second trough point from the third histogram, generating a polynomial function based on the first trough point and the second trough point, and bounding a population based on the polynomial function and additional bounding points.In various embodiments, the method further includes calculating a kernel in the pixel map, adding a predefined number of instances of the kernel to the pixel map based on light emitted from the plurality of samples along a first axis and a second axis, deriving a first histogram from the pixel map along the first axis, removing data from the pixel map based on the first histogram, generating a second histogram from the pixel map along the second axis, determining a region of interest in the pixel map based on the second histogram, generating an additional plurality of histograms along the second axis of the region of interest of the pixel map, determining a plurality of trough points based on the additional plurality of histograms, generating a polynomial function based on the multiple trough points, and bounding the population based on the polynomial function and the additional boundary points. In various embodiments, the first axis is at an angle to the second axis, the angle being between 45 degrees and 135 degrees. In various embodiments, the angle being between 75 degrees and 105 degrees. In various embodiments, the angle is between 85 degrees and 95 degrees. In various embodiments, the first axis is orthogonal to the second axis. In various embodiments, the first axis and the second axis are along the same direction, and a beam splitter or dichroic mirror is used to observe the first light and the second light based on the difference in wavelength using a first detector. In various embodiments, the first light is detected using a first detector positioned at a first position along the first axis, and the second light is detected using a second detector positioned at a second position along the second axis. In various embodiments, no more than two detectors are used to detect the emission from the sample. In various embodiments, (i) the first light is detected using a first detector positioned at a first position along the first axis, and (ii) the second light is detected using a first detector via a fiber optic element that transmits light from a second position along the second axis to a first position along the first axis.In various embodiments, the first detector comprises an avalanche photodiode having a first pixel detection area and a second pixel detection area, and the method further includes determining a physical location characteristic of each particle based on the intensity of the first light and the second light at the first pixel detection area and the second pixel detection area, respectively. In various embodiments, the biasing operation includes emitting a third light into the sample using the second light source to change a state of individual particles in the sample. In various embodiments, the emitting the third light activates particles in the sample. In various embodiments, the particles are a first particle type and activating the particles using the third light converts the particles to a second particle type. In various embodiments, the emitting the second light inactivates particles in the sample. In various embodiments, the particles in the particle population are cells and inactivating the cells includes ablatating the cells. In various embodiments, the illumination light is coincident with the sample at a first location in the path of the sample flowing through the sample channel, and a third light is emitted into the sample to be coincident with the sample at a second location in the path of the sample, the second location being downstream of the first location. In various embodiments, the method further comprises determining an orientation characteristic of each particle in the set of one or more particles in the sample. In various embodiments, the orientation characteristic is determined based on the detected first light and the detected second light. In various embodiments, a biasing action is performed on each particle based on the orientation characteristic of each particle. In various embodiments, the particle population is a cell population, the first particle type is a first cell type, and the second particle type is a second cell type. In various embodiments, the method further comprises determining an orientation characteristic of each cell in the sample based on the detected first light and the detected second light. In various embodiments, the orientation characteristic comprises a relative orientation of a primary surface of each cell with respect to a second axis. In various embodiments, the primary surface of each cell is a flat side of the cell or an edge side of the cell. In various embodiments, the first cell type is a male sperm cell that has a Y chromosome and the second cell type is a female sperm cell that does not have a Y chromosome. In various embodiments, the second cell type is an "XY" sperm cell that has an X and a Y chromosome, which will produce XXY offspring.In various embodiments, the first cell type may be the desired ("target") cell type (e.g., "male" sperm cells having one Y chromosome in each cell, or "female" sperm cells having one X chromosome in each cell), and the second cell type may be all other cell types that are undesired and are to be proportionately eliminated or otherwise proportionately reduced or limited (e.g., "non-male" or "non-female" cell types having multiple chromosomes). In various embodiments, the ratio corrected by the biasing operation includes more male sperm cells relative to female sperm cells. In various embodiments, performing the biasing operation includes inactivating female sperm cells in the sample. In various embodiments, the light source is a laser having a first wavelength, and the first light and the second light are fluorescent lights having a second wavelength different from the first wavelength. In various embodiments, the light source is a first light source, and performing the biasing operation includes emitting light into the sample using a second light source to change a state of either the first particle type or the second particle type in the sample. In various embodiments, the biasing operation is performed based on the fluorescence detected along the first axis in combination with the fluorescence detected along the second axis. In various embodiments, the method further includes generating a histogram of intensities along the first axis and the second axis, and identifying regions in the histogram that correspond to the first type of particles or the second type of particles. In various embodiments, the first light and the second light are fluorescence of particles in the sample, and the histogram corresponds to fluorescence intensity along the first axis and the second axis. In various embodiments, the first light and the second light are fluorescent light, and the method further includes (i) determining an orientation characteristic of each particle in the set particles in the sample based on the detected first light and the detected second light, (ii) generating a histogram of fluorescence intensity in a first axis and a second axis, and (iii) identifying regions in the histogram corresponding to particles having the first orientation characteristic and particles having the second orientation characteristic. In various embodiments, the method is implemented on a first microfluidic system in which light is detected in multiple directions, and the method further includes performing a second biasing operation based on the identified regions for a second microfluidic system that does not have multi-directional light detection.In various embodiments, the microfluidic chip further comprises an access port, through which the second light emitted along the second axis is collected. In various embodiments, the access port of the microfluidic chip comprises a fiber optic element for transmitting the second set of light. In various embodiments, the fiber optic element terminates in a beam splitting element configured to separate the fluorescent wavelengths from the wavelengths corresponding to the illumination light source. In various embodiments, the method further comprises performing a gating operation based on the detected first light emitted along the first axis and the detected second light emitted along the second axis. In various embodiments, the gating operation affects a hydrodynamic flow of the sample in the microfluidic chip. In various embodiments, the biasing operation requires both the detected first light and the detected second light to modify the ratio of the first particle type to the second particle type. Various embodiments relate to a product comprising a particle population having a modified ratio of the first particle type to the second particle type produced according to the method.

[0005] Various embodiments relate to a method including providing a sample in a microfluidic chip having a sample channel, the sample including a particle population having a first ratio of a first particle type to a second particle type; emitting coherent light using an illumination source along an illumination axis such that the coherent light coincides with the sample as it passes through the channel; detecting a first set of one or more light of one or more wavelengths traveling in a first direction from the sample using a first photodetector at a first location along a first direction from the microfluidic chip; detecting a second set of one or more light of one or more wavelengths traveling in a second direction from the sample using a second photodetector at the first location or at a second location along a second direction from the microfluidic chip; and performing a biasing operation to obtain a modified particle population having a second ratio of the first particle type to the second particle type based on the first set of light and the second set of light.

[0006] In various embodiments, the method further includes generating an intensity map of peak fluorescence associated with each particle type based on light emitted from the sample along the first axis and the second axis; detecting a first population center associated with the first particle type and a second population center associated with the second particle type based on the intensity map; calculating a relative intensity along a first line passing through the first population center; calculating a slope of a second line connecting the first population center and the second population center; locating a first saddle point along a third line connecting the first population associated with the first particle type and the second population associated with the second particle type; locating a second saddle point along a fourth line connecting the first population and the second population; and generating a gate by bounding the first population or the second population, wherein the gate is bounded based on the first saddle point, the second saddle point, and the additional boundary point. In various embodiments, the method further includes calculating a kernel in the pixel map, adding a first instance of the kernel to the pixel map based on light emitted from the sample along a first axis and a second axis, adding a second instance of the kernel to the pixel map based on light emitted from the sample along the first axis and a second axis, deriving a first histogram from the pixel map, removing data from the pixel map, deriving a second histogram from a first row of the pixel map, generating a first trough point from the second histogram, deriving a third histogram from a second row of the pixel map, generating a second trough point from the third histogram, generating a polynomial function based on the first trough point and the second trough point, and bounding the population based on the polynomial function and additional bounding points. In various embodiments, the first set of lights includes fluorescence from particles in the sample in a first direction and the second set of lights includes fluorescence from particles in the sample in a second direction. In various embodiments, the illumination light incident on the particles in the sample causes the particles in the sample to fluoresce. In various embodiments, the biasing action includes a gating action that affects the sample flow in the microfluidic chip.In various embodiments, the biasing operation includes emitting a second coherent light to the sample. In various embodiments, the second coherent light is emitted using a second light source. In various embodiments, the second coherent light has a wavelength lower than a wavelength of the first coherent light. In various embodiments, the first coherent light coincides with the sample at a first location along the path of the sample flow, and the second coherent light is emitted to coincide with the sample at a second location along the path downstream from the first location. In various embodiments, the second coherent light activates particles in the sample. In various embodiments, the second coherent light deactivates particles in the sample. In various embodiments, the first light detector is a first detection region of the detector module, and the second detector is a second detection region of the detector module. In various embodiments, the method further includes determining a physical location characteristic of each particle based on the intensities of the first light and the second light in the first detection region and the second detection region. In various embodiments, the particle population is a cell population, the first particle type is a first cell type, and the second particle type is a second cell type. In various embodiments, the method further includes determining an orientation characteristic of a plurality of cells in the sample. In various embodiments, the orientation characteristic is determined based on the first set of detected light and the second set of detected light. In various embodiments, a biasing operation is performed (i) for each of the plurality of cells, and (ii) additionally based on the orientation characteristic of each of the plurality of cells. In various embodiments, the first cell type is male sperm cells having a Y chromosome, and the second cell type is female sperm cells not having a Y chromosome (e.g., having an X chromosome). In various embodiments, the orientation characteristic of the plurality of cells includes (i) a first orientation corresponding to a primary surface of the cells facing an illumination access, and (ii) a second orientation corresponding to a secondary surface of the cells oriented facing an illumination axis. In various embodiments, the orientation characteristics of the plurality of cells include (i) a first orientation corresponding to a cell oriented flat side relative to at least one of the first detector or the second detector, and (ii) a second orientation corresponding to a cell oriented edge side relative to at least one of the first detector or the second detector.In various embodiments, the orientation characteristics of the plurality of cells further include a third orientation corresponding to cells transitionally oriented between the edge side and the flat side. In various embodiments, the second proportion is greater than the first proportion such that as a result of the biasing operation, the modified cell population has more male sperm cells relative to female sperm cells. In various embodiments, performing the biasing operation includes inactivating one or more cells of the first cell type or the second cell type in the sample. In various embodiments, the first set of lights includes at least one of light scattering or fluorescence in a first direction and the second set of lights includes at least one of light scattering or fluorescence in a second direction. In various embodiments, the illumination source is a laser having a first wavelength and the fluorescence has a second wavelength different from the first wavelength. In various embodiments, performing the biasing operation includes changing a state of one or more particles of the first particle type or the second particle type in the sample. In various embodiments, the biasing operation is performed based on the fluorescence detected in the first direction combined with the fluorescence detected in the second direction. In various embodiments, the biasing operation is performed based on (i) the fluorescence in the first direction, and (ii) both light scattering and fluorescence in the second direction. In various embodiments, the method further includes generating a histogram of the fluorescence intensity in the first direction and the second direction, and identifying regions in the histogram corresponding to particles of the first cell type or particles of the second cell type. In various embodiments, the method further includes generating a histogram of the fluorescence intensity in the first direction and the second direction, and identifying regions in the histogram corresponding to particles having a first orientation characteristic and particles having a second orientation characteristic. In various embodiments, the method further includes generating a histogram of fluorescence intensity in the first direction and the second direction, and identifying regions in the histogram that correspond to at least one of: (i) particles of the first cell type, (ii) particles of the second cell type, (iii) particles having a first orientation characteristic, or (iv) particles having a second orientation characteristic.In various embodiments, the method is implemented on a first microfluidic system where light is detected in multiple directions, and the method further includes performing a second biasing operation based on the identified region for a second microfluidic system that does not have multi-directional light detection. In various embodiments, the first direction is along an illumination axis and the second direction is along a second axis angled relative to the illumination axis. In various embodiments, the second direction is orthogonal to the illumination axis. In various embodiments, the second direction is at an angle relative to the illumination axis, the angle being between 45 degrees and 135 degrees. In various embodiments, the microfluidic chip further comprises an access port through which the second set of light is collected. In various embodiments, the access port of the microfluidic chip comprises a fiber optic element for transmitting the second set of light. In various embodiments, the fiber optic element terminates in a beam splitting element configured to separate the fluorescent wavelengths from the wavelengths corresponding to the illumination source. In various embodiments, the method further includes filtering a third set of light from the second illumination source. In various embodiments, the filtering further comprises filtering with an optical fiber defined by a length having a curved shape. In various embodiments, the filtering can be wavelength-based (e.g., by use of an embedded Bragg grating) and / or spatial-based (e.g., based on position and / or numerical aperture (NA)) of the fiber. In various embodiments, the method further comprises determining inactivation of particles in the sample based on detecting a third set of characteristics of the light. In various embodiments, the method further comprises altering at least one of (i) the microfluidic chip, or (ii) the manner in which the sample is provided to the microfluidic chip, to obtain a third ratio of the first particle type to the second particle type based on the modified cell population. In various embodiments, the method further comprises collecting the sample via the channel following the biasing operation. Various embodiments relate to a product comprising a modified particle population having a second ratio of the first particle type to the second particle type produced according to this method.

[0007] Various embodiments relate to a flow cytometry system comprising: an illumination source configured to emit coherent light having a first wavelength and coinciding with a sample channel of a microfluidic chip; a first photodetector configured to detect light traveling in a first direction from the sample channel; and a second photodetector configured to detect light traveling in a second direction from the sample channel; and a control unit configured to: (i) illuminate a sample passing through the sample channel of the microfluidic chip, the sample including a cell population having a first ratio of a first cell type to a second cell type; (ii) detect the first light using the first photodetector and detect the second light using the second photodetector; and (iii) control one or more biasing mechanisms to perform a biasing operation to modify the cell population to obtain a second ratio of the first cell type to the second cell type based on both the first light detected using the first photodetector and the second light detected using the second photodetector.

[0008] In various embodiments, the system further includes a microfluidic chip, the microfluidic chip comprising an access port having a fiber optic element through which the second light travels to reach the second detector. In various embodiments, the first direction is an illumination axis of the illumination source, and the second detector is positioned to detect light at an angle to the illumination axis. In various embodiments, the angle is between 45 degrees and 135 degrees. In various embodiments, the first direction is an illumination axis of the illumination source, and the second detector is positioned to detect light traveling orthogonally to the illumination axis. In various embodiments, the one or more biasing mechanisms include a second laser having a higher intensity or lower wavelength relative to the illumination laser, and the control unit is further configured to inactivate one or more cells of the first cell type or the second cell type in the sample using the second laser. In various embodiments, the one or more biasing mechanisms include an actuator that affects sample flow in the microfluidic chip. In various embodiments, the control unit is configured to perform a biasing operation based on the forward fluorescence detected using the first detector and the side fluorescence detected using the second detector. In various embodiments, the control unit is further configured to generate a histogram of the fluorescence intensity in the first direction and the second direction. In various embodiments, the control unit is further configured to identify a region in the histogram that corresponds to at least one of: (i) particles of the first cell type, (ii) particles of the second cell type, (iii) particles having a first orientation characteristic, or (iv) particles having a second orientation characteristic. In various embodiments, the control unit is further configured to control the one or more biasing mechanisms to perform a second biasing operation on the second sample using only the detected light traveling in the first direction and not in the second direction based on the identified region.

[0009] Various embodiments relate to a method including providing a sample to a microfluidic chip having a sample channel. The sample may include a population having a first ratio of a first particle type to a second particle type. For example, the sample may include a cell population having a first ratio of a first cell type to a second cell type. An illumination source may be used to provide coherent light along an illumination axis. The coherent light may be provided such that the coherent light coincides with the sample as it passes through the channel. A first light detector may be used to detect a first set of one or more lights at one or more wavelengths. The first set of lights may be detected in a first direction (e.g., along an illumination axis) from the microfluidic chip. The first light detector may be positioned or oriented at a first location, e.g., relative to the microfluidic chip or the illumination source. A second light detector may be used to detect a second set of one or more lights at one or more wavelengths. The second set of lights may be detected in a second direction (angled with respect to the first direction) from the microfluidic chip. A second light detector may be positioned or oriented at a second location, e.g., relative to the microfluidic chip, or relative to the microfluidic chip or the illumination light source. A biasing operation may be performed. The biasing operation may be performed based at least in part on the first set of lights and the second set of lights. The biasing operation may be performed, e.g., to obtain a modified cell population. The modified cell population may have a second ratio of the first cell type to the second cell type. The second ratio may be higher or lower than the first population.

[0010] In various embodiments, one or more orientation characteristics and / or one or more position characteristics of multiple cells or other particles in a sample (eg, each cell traveling within a sample channel) may be determined.

[0011] In various embodiments, the orientation feature and / or the position feature may be determined based on, for example, the first set of detected light and the second set of detected light.

[0012] In various embodiments, a biasing action may be performed on each of the plurality of cells. The biasing action may be based on one or more orientation and / or position characteristics of each of the plurality of cells.

[0013] In various embodiments, the first cell type may be a male sperm cell having a Y chromosome. The second cell type may be, for example, a female sperm cell having an X chromosome or otherwise having no single Y chromosome. The second cell type may be, for example, a sperm cell having an X and a Y chromosome, which may be an "XY" sperm cell.

[0014] In various embodiments, the orientation feature of the plurality of cells may include a first orientation corresponding to a cell oriented flat side relative to at least one of the first detector or the second detector. The orientation feature may include a second orientation corresponding to a cell oriented edge side relative to at least one of the first detector or the second detector. The orientation feature may include a third orientation corresponding to a cell oriented transitionally between the edge side and the flat side.

[0015] In various embodiments, the second proportion may be greater than the first proportion, such that as a result of the biasing operation, the modified cell population has more male sperm cells relative to female sperm cells.

[0016] In various embodiments, the first set of light may include light scattering and / or fluorescence in a first direction. The second set of light may include light scattering and / or fluorescence in a second direction. In certain embodiments, the first and second sets of light are fluorescence emitted in two different directions.

[0017] In various embodiments, the illumination source may be a laser having a first wavelength, and the fluorescent light may have a second wavelength different from the first wavelength.

[0018] In various embodiments, performing the biasing operation may include ablating, inactivating, or otherwise changing the state of one or more cells (or other particles) of a first cell type and / or a second cell type in the sample. The particle state may be changed using emission from a light source. For example, cells may be ablated using emission from an ablation light source, which may have a higher intensity and / or a different wavelength (e.g., lower wavelength) than the illumination light source. The ablation light source may have a different fluence (e.g., intensity / area) than the detection light source or the non-ablation light source. The light source may inactivate or "kill" cells or other particles in the sample.

[0019] In various embodiments, the biasing action may be performed based on the fluorescence detected in a first direction in combination with the fluorescence detected in a second direction.

[0020] In various embodiments, the biasing action may be performed based on (i) fluorescence in a first direction, and (ii) both light scatter and fluorescence in a second direction.

[0021] In various embodiments, a histogram of the fluorescence intensity in the first direction and the second direction may be generated, and regions in the histogram that correspond to particles (e.g., cells) of a first particle type (e.g., a first cell type) or particles (e.g., cells) of a second particle type (e.g., a second cell type) may be identified.

[0022] In various embodiments, a histogram of the fluorescence intensity in the first direction and the second direction may be generated, and regions in the histogram corresponding to particles (e.g., cells) having the first orientation characteristic and particles (e.g., cells) having the second orientation characteristic may be identified.

[0023] In various embodiments, the method may be implemented on a first microfluidic system where light is detected in multiple directions (e.g., using a first detector and a second detector at a first location and a second location, respectively). What is learned may then be applied to a microfluidic system without multi-directional light detection. In various embodiments, a second biasing operation may be performed based on the identified region on the second microfluidic system without multi-directional light detection. The identified region may serve as a guide indicating the expected particle or cell type based on light from a single detector.

[0024] In various embodiments, the first direction may be along an illumination axis and the second direction may be along a second axis that is angled relative to the illumination axis.

[0025] In various embodiments, the second direction may be orthogonal to the illumination axis.

[0026] In various embodiments, the second direction may be at an angle to the illumination axis, for example, between 85 degrees and 95 degrees, or between 80 degrees and 100 degrees, or between 75 degrees and 105 degrees, or between 70 degrees and 110 degrees, or between 65 degrees and 115 degrees, or between 60 degrees and 120 degrees, or between 55 degrees and 125 degrees, or between 50 degrees and 130 degrees, or between 45 degrees and 135 degrees, or between 30 degrees and 150 degrees.

[0027] In various embodiments, the microfluidic chip may further comprise an access port through which the second set of light is collected.

[0028] In various embodiments, the access port of the microfluidic chip may include one or more fiber optic elements that may transmit a second set of light, for example to a second detector.

[0029] In various embodiments, the fiber optic element may terminate in a beam splitting element that may be configured to separate the fluorescent wavelengths from wavelengths corresponding to the illumination source.

[0030] In various embodiments, a third set of light may be filtered from a second illumination source.

[0031] In various embodiments, filtering may include filtering with an optical fiber defined by a length having a curved shape.

[0032] In various embodiments, the inactivation of cells in the sample may be determined based on the third set of light.

[0033] In various embodiments, based on the modified cell population, at least one of (i) the microfluidic chip, or (ii) the manner in which the sample is provided to the microfluidic chip may be altered to, for example, obtain a third ratio of the first cell type to the second cell type.

[0034] In various embodiments, the sample may be collected through the channel after the biasing action.

[0035] Various embodiments relate to products comprising a modified particle population having a second ratio of the first particle type to the second particle type produced according to the methods discussed above.

[0036] Various embodiments relate to a flow cytometry system. The system may include an illumination light source that may be configured to emit coherent light having a first wavelength and matching a sample channel of a microfluidic chip. The system may include a first photodetector configured to detect light traveling in a first direction from the sample channel. The system may include a second photodetector configured to detect light traveling in a second direction from the sample channel. The system may include a control unit. The control may be configured to illuminate a sample passing through a sample channel of a microfluidic chip. The sample may include a cell population (or other particle population) having a first ratio of a first cell type (or a first particle type) to a second cell type (or a second particle type). The control unit may be configured to detect the first light using the first photodetector and detect the second light using the second photodetector. The control unit may control a biasing mechanism to perform a biasing operation. The biasing operation may be based on both the first light detected using the first photodetector and the second light detected using the second photodetector. A biasing operation may be performed to modify the cell population (or particle population) to obtain a second ratio of the first cell type (or first particle type) to the second cell type (or second particle type).

[0037] In various embodiments, the system may further include a microfluidic chip. The microfluidic chip may include an access port. The access port may include a fiber optic element through which the second light travels to reach the second detector.

[0038] In various embodiments, the first direction may be the illumination axis of the illumination source. The second detector may be positioned to detect light at an angle to the illumination axis. The angle may be between 75 and 105 degrees, or between 60 and 120 degrees, or between 45 and 135 degrees, or between 30 and 150 degrees. The angle may be substantially 90 degrees such that the second detector detects light traveling perpendicular to the illumination axis.

[0039] In various embodiments, the biasing mechanism may include a second light source, which may be an ablation laser or other inactivation light source. The control unit may be configured to use the ablation laser or other inactivation light source to ablate or otherwise inactivate one or more cells of the first cell type or the second cell type in the sample (or otherwise ablate or inactivate one or more particles of the first particle type or the second particle type in the sample). The biasing mechanism may include one or more activation light sources (alternatively or in addition to the ablation laser or inactivation light source) that may be controlled by the control unit to, for example, activate particles in the sample based on the detected light.

[0040] In various embodiments, the control unit may be configured to perform a biasing operation based on the "forward" fluorescence detected using the first detector and the "side" fluorescence detected using the second detector. In various embodiments, the control unit may be configured to perform a biasing operation based on the first fluorescence detected using the first detector and the second fluorescence detected using the second detector. The first fluorescence and the second fluorescence may each be fluorescence detected from one direction. The first fluorescence and the second fluorescence may be fluorescence detected from different directions. In various embodiments, the disclosed axes may be "flipped" with respect to each other, for example, such that either detector may be labeled as the "first" detector or the "second" detector, and similarly, either direction may be the "forward" direction or the "side" direction.

[0041] Various embodiments relate to a microfluidic chip. The chip may include a sample channel extending from a sample inlet to a sample outlet. The chip may be configured to receive a sample including a cell population (or other particle population). The chip may include an access port extending from an opening in the microfluidic chip to the sample channel. The chip may include a fiber optic element positioned at the access port and configured to transmit wavelengths of light having wavelengths corresponding to at least one of light scattering and fluorescence from a sample passing through the sample channel. The microfluidic chip may be configured to illuminate the sample using an illumination laser along an illumination axis. The chip may be configured to collect light emitted coaxially to the illumination axis. A fiber optic element may be configured to collect light emitted orthogonally from cells illuminated using the illumination laser. Orientational features of cells in the cell population of the sample may be derivable from the light collected by the fiber optic element and the light collected coaxially to the illumination axis.

[0042] In various embodiments, the chip may include a beam splitting element configured to separate the fluorescent wavelengths of light from the scattered wavelengths of light transmitted through the fiber optic element.

[0043] In various embodiments, the orientation characteristic of cells in the cell population of the sample is the relative orientation of the primary surface of the particle with respect to the illumination axis.

[0044] Various embodiments relate to products including modified cell populations having a modified ratio of a first cell type to a second cell type generated according to certain methods. The method may include providing a sample to a microfluidic chip having a sample channel. The sample may include a cell population having an initial ratio of a first cell type to a second cell type. The method may include emitting coherent light along an illumination axis using an illumination light source such that the coherent light coincides with the sample as it passes through the channel. The method may include detecting a first set of one or more lights of one or more wavelengths in a first direction from the microfluidic chip and at a first location using a first photodetector at the first location. The method may include detecting a second set of one or more lights of one or more wavelengths in a second direction from the microfluidic chip and at a second location using a second photodetector at the second location. The method may include determining a position and / or orientation for each cell in the sample based on the first set of detected light and the second set of detected light. The method may include performing a biasing operation to obtain a modified cell population having a modified ratio of the first cell type to the second cell type. The biasing operation may be performed based on the first set of lights, the second set of lights, and / or the determined orientation and / or position.

[0045] In various embodiments, the first cell type may be male sperm cells that have a Y chromosome and the second cell type may be female sperm cells that do not have a Y chromosome.

[0046] In various embodiments, as a result of the biasing operation, the modified ratio may be greater than the initial ratio, such that the modified cell population has more male sperm cells relative to female sperm cells.

[0047] In various embodiments, the first set of lights may include at least one of light scattering or fluorescence in a first direction, and the second set of lights may include at least one of light scattering or fluorescence in a second direction.

[0048] In various embodiments, the illumination source may be a laser having a first wavelength and the fluorescent light may have a second wavelength different from the first wavelength.

[0049] In various embodiments, performing the biasing action may include ablating one or more cells of the first cell type or the second cell type in the sample.

[0050] In various embodiments, the biasing action may be performed based on the fluorescence detected in a first direction in combination with the fluorescence detected in a second direction.

[0051] In various embodiments, the biasing action may be performed based on (i) fluorescence in a first direction, and (ii) both light scatter and fluorescence in a second direction.

[0052] In various embodiments, the method may include generating a histogram of fluorescence intensity in a first direction and a second direction, and identifying regions in the histogram that correspond to cells of the first cell type or cells of the second cell type.

[0053] In various embodiments, the first direction may be along an illumination axis and the second direction may be along a second axis that is angled relative to the illumination axis.

[0054] In various embodiments, the second direction may be orthogonal to the illumination axis.

[0055] In various embodiments, the second direction may be at an angle to the illumination axis, which may be between 75 and 105 degrees, or between 60 and 120 degrees, or between 45 and 135 degrees, or between 30 and 150 degrees.

[0056] In various embodiments, the microfluidic chip may include an access port through which the second set of light is collected.

[0057] In various embodiments, the access ports of the microfluidic chip may include fiber optic elements for transmitting the second set of light.

[0058] In various embodiments, the fiber optic element may terminate in a beam splitting element configured to separate the fluorescent wavelengths from wavelengths corresponding to the illumination source.

[0059] In various embodiments, the method may include filtering a third set of light from the second illumination source.

[0060] In various embodiments, filtering may include filtering with an optical fiber defined by a length having a curved shape.

[0061] In various embodiments, the method may further include determining the inactivation of cells in the sample based on the third set of light.

[0062] In various embodiments, the method may further include filtering a third set of light from the second illumination source.

[0063] Various embodiments relate to a method that includes using an illumination light source to emit coherent light along an illumination axis such that the coherent light coincides with the sample as it passes through a sample channel of a microfluidic chip. The sample may include a cell population having a first ratio of a first cell type to a second cell type. A first light detector at a first location may be used to detect a first set of one or more lights of one or more wavelengths in a first direction (e.g., along or parallel to the illumination axis) from the microfluidic chip. A second light detector at a second location may be used to detect a second set of one or more lights of one or more wavelengths in a second direction (e.g., an axis angled relative to the first direction) from the microfluidic chip. An orientation characteristic of a plurality of cells in the sample may be determined based on the first set of lights and the second set of lights. A biasing operation may be performed to obtain a modified cell population having a second ratio of the first cell type to the second cell type. The biasing action may be performed based on (i) the orientation characteristic of the plurality of cells, and (ii) at least one of the first set of lights or the second set of lights.

[0064] In various embodiments, the first cell type may be male sperm cells that have a Y chromosome and the second cell type is female sperm cells that do not have a Y chromosome.

[0065] In various embodiments, the second proportion may be greater than the first proportion, such that as a result of the biasing operation, the modified cell population has more male sperm cells relative to female sperm cells.

[0066] In various embodiments, the orientation characteristics of the plurality of cells may include (i) a first orientation corresponding to a cell oriented flat side relative to at least one of the first detector or the second detector, and (ii) a second orientation corresponding to a cell oriented edge side relative to at least one of the first detector or the second detector.

[0067] In various embodiments, the orientation feature of the plurality of cells may include a third orientation corresponding to cells transitionally oriented between the edge side and the flat side.

[0068] In various embodiments, the first set of lights may include at least one of light scattering or fluorescence in a first direction, and the second set of lights may include at least one of light scattering or fluorescence in a second direction.

[0069] In various embodiments, the illumination source may be a laser having a first wavelength and the fluorescent light may have a second wavelength different from the first wavelength.

[0070] In various embodiments, performing the biasing action may include ablating one or more cells of the first cell type or the second cell type in the sample.

[0071] In various embodiments, the biasing action may be performed based on the fluorescence detected in a first direction and the fluorescence detected in a second direction.

[0072] In various embodiments, a histogram of fluorescence intensity in the first direction and the second direction may be generated. In various embodiments, the histogram is a two-dimensional (2D) histogram combining information from both detectors (as opposed to a pair of one-dimensional (1D) histograms for independent detectors). A first region in the histogram may be identified that corresponds to cells of the first cell type or cells of the second cell type. Alternatively or additionally, a second region in the histogram may be identified that corresponds to cells having the first orientation characteristic, and a third region may be identified that corresponds to cells having the second orientation characteristic.

[0073] In various embodiments, a histogram of fluorescence intensity in a first direction may be generated, and a histogram of fluorescence intensity in a second direction may be generated. The first histogram may be generated based on the fluorescence detected by the first detector, and the second histogram may be generated based on the fluorescence detected by the second detector. In various embodiments, the first and second histograms may be combined or overlaid to generate a combined histogram that includes fluorescence information detected from the first and second directions, or from the first and second detectors.

[0074] In certain exemplary embodiments, the biasing action modifies the ratio of the first particle type to the second particle type. Hypothetically, if a sample contains 1000 cells (or other particles), 450 (45%) of a first type (e.g., "female"), 450 (45%) of a second type (e.g., "male"), and 100 (10%) of a third type (e.g., dead cells), there will be a sample with a ratio of 450 of the first type to 450 of the second type (e.g., male to female), i.e., a 1:1 ratio. Following a biasing action that tends to cauterize, kill, or otherwise inactivate the first type (e.g., female cells) in a sample containing the first and second types (e.g., female and male cells), which may result in, for example, 100 (10%) female cells, 400 (40%) male cells, and 500 (50%) dead cells. The new (corrected) ratio of the first cell type to the second cell type in this hypothetical case would be 100 female to 400 male, i.e., a ratio of 1:4. Another hypothetical biasing operation might leave a yield of 50 female, 500 male, 150 dead, and 300 of a fourth type (e.g., "castration"). Here, the corrected ratio would be 50 female to 500 male, i.e., 1:10. Another assumption would be that if zero female cells were left, the corrected ratio would be 0:1. Thus, in various embodiments, even though there may be different mix ratios of particle types (e.g., cell types), the ratios can be altered so that certain types are proportionally higher or lower after biasing compared to before biasing.

[0075] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the following drawings and detailed description. [Brief description of the drawings]

[0076] [Figure 1] 1 depicts a microfluidic system including a computing device and a sample processing system, according to various potential embodiments. [Diagram 2] 1 depicts an exemplary configuration for components of a sample processing system, according to various potential embodiments. [Diagram 3] 1 depicts another exemplary configuration for components of a sample processing system, in accordance with various potential embodiments. [Figure 4] 1 depicts forward fluorescence data acquired using a single-detector sample processing system, according to various potential embodiments. [Diagram 5] 14 depicts histograms of forward and side fluorescence acquired using a dual detector sample processing system, according to various potential embodiments. [Figure 6] 1 depicts side fluorescence data acquired using a sample processing system, according to various potential embodiments. [Figure 7] 6 depicts the data of FIG. 5 showing regions corresponding to different orientation features of a cell, according to various potential embodiments. [Figure 8A] 1 includes cutaway views of an exemplary microfluidic chip with side ports and optical fibers, in accordance with various potential embodiments. [Figure 8B] 1 includes cutaway views of an exemplary microfluidic chip with side ports and optical fibers, in accordance with various potential embodiments. [Figure 8C] 1 includes cutaway views of an exemplary microfluidic chip with side ports and optical fibers, in accordance with various potential embodiments. [Figure 8D] 1 includes cutaway views of an exemplary microfluidic chip with side ports and optical fibers, in accordance with various potential embodiments. [Figure 9A] 1 depicts an exemplary configuration including three detectors as components of a sample processing system, according to various potential embodiments. [Figure 9B] 1 depicts an exemplary configuration including three detectors as components of a sample processing system, according to various potential embodiments. [Figure 10] 1 depicts an exemplary flowchart for processing a sample to obtain a modified particle population, in accordance with various potential embodiments. [Figure 11A] 1 illustrates a logically constructed gate separating male and female populations according to various potential embodiments. [Figure 11B] 1 illustrates a logically constructed gate separating male and female populations according to various potential embodiments. [Figure 12] 13 depicts an exemplary flowchart for logically imposing a saddle gate on forward and side fluorescence histograms, according to various potential embodiments. [Figure 13A] 14 illustrates the logical imposition of a kernel density estimation gate on a fluorescence histogram, according to various potential embodiments. [Figure 13B] 14 illustrates the logical imposition of a kernel density estimation gate on a fluorescence histogram, according to various potential embodiments. [Figure 13C] 14 illustrates the logical imposition of a kernel density estimation gate on a fluorescence histogram, according to various potential embodiments. [Figure 13D] 14 illustrates the logical imposition of a kernel density estimation gate on a fluorescence histogram, according to various potential embodiments. [Figure 14] 13 depicts an exemplary flowchart for logically imposing a kernel density estimation gate on a fluorescence histogram, according to various potential embodiments. [Figure 15] 1A-1D depict an exemplary photodiode detector assembly capable of detecting fluorescent light, in exploded and assembled views, respectively, in accordance with various embodiments. [Figure 16A] 11A-11C respectively depict a column histogram and a derivative of the column histogram, a row histogram with a number of points defined by the column histogram and a derivative of the column histogram, and various potential threshold points according to various potential embodiments. [Figure 16B] 11A-11C respectively depict a column histogram and a derivative of the column histogram, a row histogram with a number of points defined by the column histogram and a derivative of the column histogram, and various potential threshold points according to various potential embodiments. [Figure 16C] 11A-11C respectively depict a column histogram and a derivative of the column histogram, a row histogram with a number of points defined by the column histogram and a derivative of the column histogram, and various potential threshold points according to various potential embodiments. [Figure 17] 13C show row and column histograms of a pixel map region of interest (ROI) along with their derivatives and various threshold points overlaid on the image of FIG. 13C according to various potential embodiments. [Figure 18] 15 depicts a flow diagram of an example system configured to perform the methods disclosed by FIGS. 12 and 14 and otherwise perform portions of the present disclosure in accordance with various potential embodiments. [Figure 19] 15 depicts another flow diagram of an example system configured to perform the methods disclosed by FIGS. 12 and 14 and otherwise perform portions of the present disclosure in accordance with various potential embodiments.

[0077] The foregoing and other features of the present disclosure will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which the present disclosure is described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings depict only some embodiments in accordance with the present disclosure and are therefore not to be considered limiting of its scope. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments can be utilized and / or other changes can be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are expressly contemplated and made a part of this disclosure.

[0079] Various embodiments provide applications for detargeting particle populations in a sample to favor a first or second particle type. The particles can be cells or molecules. Biasing actions may be employed to select particular particles or cells, for example, by deactivating selected particles, activating selected particles, cauterizing, slicing, photodamaging, or otherwise defunctionalizing cells, and / or physically separating components of the sample. The effectiveness of the biasing action may depend on knowing the orientation or location of the particles. For example, the cells may be illuminated to excite fluorophore-tagged components of the cells, thereby producing detectable fluorescence. The intensity of the fluorescence may be considered to correspond to a property of the cell. In such cases, the area of ​​the detection surface (i.e., the surface on which the excitation light falls) may be positively correlated with the fluorescence intensity, such that the larger the surface area, the greater the excitation and therefore the greater the fluorescence. If a cell is irregularly shaped or otherwise asymmetric, the cell may effectively have multiple "surfaces" (i.e., the areas of the detection surfaces may vary), such as a primary surface having a relatively largest area, a tertiary surface having a relatively smallest area, and a secondary surface having an area between the area of ​​the primary surface and the area of ​​the tertiary surface.

[0080] In one example, a sample may include semen having both male and female sperm cells, and a sexual semen product having a predominantly Y chromosome ("Y asymmetric") may be desired. Standard flow cytometry techniques have been insufficient to effectively specifically differentiate sperm cells having a Y chromosome for the production of a sexual semen product having a Y asymmetric. In particular, the relative shapes and overlaps of sperm cells having an X chromosome and a Y chromosome as depicted on a histogram of fluorescence intensity (i.e., an observable approximately 4 percent difference in chromosomal DNA stained with a fluorescent DNA dye (e.g., Hoechst 33342 dye) between male and female sperm cells) under typical flow cytometer operation (i.e., a single detector oriented coaxially with the detection laser beam) do not allow for the effective differentiation of the Y chromosome peak from the X chromosome peak to a sufficient degree to improve the ability to remove sperm cells having an X chromosome at high throughput and asymmetric ratios associated with commercially viable products. Based on the combination of light detected in multiple directions, the position and / or orientation of cells or other particles can be identified and taken into account when distinguishing between different cell types. For example, in various embodiments, optical fibers can be incorporated into the microfluidic chip to collect emitted light, for example, in a direction orthogonal (or otherwise angled relative to) the illuminating laser axis (and also potentially orthogonal or otherwise angled relative to the direction of sample flow). Such optical fibers facilitate the collection of side fluorescence and side scatter data from cells. Identifying regions on the two-dimensional histogram helps to more effectively distinguish between different orientations and particle types.

[0081] With reference to FIG. 1, in various embodiments, a microfluidic system 100 can include a computing device 110 (or multiple computing devices that are co-located or remote from one another) and a sample processing system 150. In various embodiments, the computing device 110 (or components thereof) can be integrated with the sample processing system 150 (or components thereof). The sample processing system 150 can include a sample platform 154, which can accept a microfluidic chip 158 that receives the sample via a sample channel. Sample delivery and movement can be controlled by an actuator 162, which can include fluidics components 166 that control fluid flow or reposition the optical components. The optical components 170 can include optical fibers 172, beam splitters 174, or other components (e.g., optical filters, mirrors, and lenses) for receiving, separating, combining, delivering, transmitting, focusing, defocusing, collimating, or directing the emitted light. As used herein, "directing" can include focusing, defocusing, and / or collimating operations on various light beams. The light emission sources 178 can include various light sources of different wavelengths and power levels, such as illumination light 182 (e.g., excitation lasers) and inactivation and / or activation light 186 (e.g., "kill" lasers). The sample processing system 150 includes sensors 190, such as a set of light detectors 194 positioned at various locations. The sensors 190 can detect light scattering (e.g., light that hits the sample and is scattered in various directions) and fluorescent light (e.g., light emitted by fluorophores in cells or by other particles in the sample when excited by one of the light emission sources 178).

[0082] The computing device 110 (or multiple computing devices) can be used to control components of the sample processing system 150 (e.g., light detected using the light detector 194) and receive signals acquired via the components. The computing device 110 can include one or more processors and one or more volatile and non-volatile memories for storing captured, acquired, recorded, and / or generated computing code and data. The computing device 110 can include a control unit 114 configured to exchange control signals with the sample platform 154, the actuators 162, the optical components 170, the light source 178, and / or the sensors 190, allowing the computing device 110 to control, for example, the performance of sample delivery, illumination with light, detection, and deflection operations. Calculations and data analysis on data captured using the sample processing system 150 can be performed using an analysis module 118, which can include, for example, a histogram unit 122 that can generate and process histograms. The biasing module 126 may identify and perform actions based on the light detected using the sensor 190, such as by controlling the actuator 162 (e.g., to perform a gating action to reorient the light source to affect the sample flow or the aiming of the light emitted by the light source) and / or the light emission source 178 (e.g., to emit light to activate or deactivate particles).

[0083] The transceiver 130 allows the computing device 110 to exchange readings, control commands, and / or other data with the sample processing system 150 (or components thereof). One or more user interfaces 134 allow the computing device 110 to receive user input (e.g., via a keyboard, touch screen, microphone, camera, etc.) and provide output (e.g., via a display screen, audio speakers, etc.). The computing device 110 may additionally include one or more databases 138 for storing, for example, signals acquired via one or more sensors 190, generated histograms, etc. In some implementations, the database 138 (or a portion thereof) may alternatively or additionally be part of another computing device, either co-located or remote, and in communication with the computing device 110 and / or the sample processing system 150 (or components thereof).

[0084] Referring to FIG. 2, an exemplary configuration 200 is depicted. Laser light 204 may be emitted to a microfluidic chip 208. In a forward direction (e.g., in a first direction that may be along an illumination axis), laser scattering 220 and fluorescence 228 may be received from the chip 208. A forward scatter ("FS") detector 224 may be used to detect the laser scattering 220, and a forward fluorescence ("FF") detector 232 may be used to detect the fluorescence 228. In a side direction (e.g., in a second direction that may be substantially orthogonal to the first direction or at an angle to the first direction), laser scattering 250 and fluorescence 258 may be received from the chip 208. A side scatter ("SS") detector 254 may be used to detect the laser scattering 250, and a side fluorescence ("SF") detector 262 may be used to detect the fluorescence 258.

[0085] 3, an exemplary configuration 300 shows separation of side fluorescence and side scatter signals in a multi-side detector arrangement including a dichroic (e.g., single laser dichroic Z355rdc from Chroma Technology Corp.). Laser light 304 reaches a microfluidic chip 308, and in the forward direction, fluorescence 328 can be obtained using a dichroic 320 and a filter 324 (e.g., a long wavelength pass filter such as a color glass replacement CGA-395 filter from Newport Corp.) and received by a FF avalanche photodiode (APD) 332. In the side direction, fluorescence 362 and laser scatter 370 can be obtained using a dichroic 350. A filter 354 (e.g., a 360LP low-pass mask aligner filter from Omega Optical, LLC) can be used to filter the laser scatter 370 received by the SS photomultiplier tube (PMT) 374, and a filter 358 (e.g., a 360 / 23 bandpass filter) can be used to filter the fluorescent light 362 received by the SF PMT 366.

[0086] Figure 4 shows a single detector (one dimensional) histogram 400 obtained based on forward fluorescence (FF) alone (i.e., no light is detected from a second direction) illustrating an attempt to distinguish cell types in a cell population. Figure 4 shows the number of cells versus the forward fluorescence ("CH1 prominence") of the sample, and shows two peaks, with a low intensity peak on the left and a high intensity peak on the right. The peak on the left is expected to correspond to cells with relatively little DNA (e.g., male sperm cells) and the peak on the right is expected to correspond to cells with relatively more DNA (e.g., female sperm cells). However, the heads of sperm cells have a disk-like shape and can be oriented such that fluorescence can be received from a wider plane or "primary surface" (where the fluorescence has the relatively highest intensity detected from the sperm cell), a narrower edge or "tertiary surface" (where the fluorescence has the relatively lowest intensity detected from the sperm cell), or a transition orientation between the plane and the edge or "secondary surface" (where the fluorescence has an intensity between the higher plane intensity and the lower edge intensity). As a result, unless all cells are oriented to have the same orientation when the fluorescence is detected (i.e., the area of ​​the detection surface remains fairly constant), some female cells may be observed at a different intensity than expected for male cells (hence the left peak falls off) and some male cells may be observed at a different intensity than expected for female cells (hence the right peak falls off). In the one-detector setup of FIG. 4, cells that fall within region 410 may be considered to be male cells or other target cells (and cells that fall outside region 410 may be deactivated because they are not target cells, such as female cells), but some target (male) cells may have fallen outside region 410 and some non-target (female) cells may have fallen within region 410.

[0087] Referring to FIG. 5, an exemplary forward fluorescence / side fluorescence (FF / SF) two-dimensional (2D) histogram 500 is depicted, plotting forward fluorescence (FF) ("CH1 prominence") on the X-axis and side fluorescence (SF) ("CH2 prominence") on the Y-axis. The histogram 500 uses a two-detector system on a cell population 410 highlighted on the 2D histogram 400. Region 510 points to a discernible male population, and arrow 520 points to the "tail" of a female population that is contaminating the desired male population. The histogram 600 in FIG. 6 plots side fluorescence (SF) ("CH2 prominence") on the X-axis and number of cells ("count") on the Y-axis for a sample, with region 610 corresponding to the target cells. Here, the target cells refer to cells that remain alive and have not been inactivated, ablated, sliced, or destroyed. With reference to FIG. 7, it can therefore be determined that a particular region corresponds to a particular orientation and / or position (e.g., cells with a particular orientation feature). In histogram 700 (based on the same data as FIG. 5), a first exemplary region 710 can correspond to cells oriented flat side to the primary detector, a second exemplary region 720 can correspond to cells transitionally oriented (between flat and edge), and a third exemplary region 730 can correspond to cells oriented edge side to the primary detector. Based on the orientation feature and the set of light detected using the forward and side detectors, different cell types can be better distinguished.

[0088] In various embodiments, the bias cannot be achieved (or cannot be effectively or efficiently achieved) or is not otherwise performed with a single detection location alone. In various embodiments, the biasing action is not derivable from a single set of light (e.g., from only forward fluorescence or only side fluorescence), and thus the biasing action may require two sets of light traveling in multiple directions. In various embodiments, if the biasing can be achieved using a single set of light or from a single detection location, the biasing action cannot be performed as efficiently or effectively as with biasing using more than one set of light or light from more than one direction.

[0089] To facilitate collection of light in multiple directions, various embodiments may integrate optical fibers with the microfluidic chip to allow collection of emitted light (e.g., side fluorescence and side scattering from cells) in a direction substantially orthogonal (or otherwise angled) to the illumination laser axis. Referring to Figures 8A, 8B, 8C, and 8D, cutaway views of exemplary microfluidic chips 800a, 800b, 800c, and 800d, respectively, are shown having a sample channel 810 with a flow axis 815. Chips 800a, 800b, 800c, and 800d include access ports 820a, 820b, 820c, and 820d for optical fiber elements 830a, 830b, 830c, and 830d, respectively, which in various embodiments may be inserted orthogonal to or otherwise angled relative to one or both of the illumination optical axis and / or the flow axis. The access ports can have various configurations and can form various angles with respect to the flow axis and / or with respect to the "forward" illumination axis, as depicted in Figures 8A-8D. The fiber optic element (e.g., 830a, 830b, 830c, and 830d) can be selected for wavelength transmission, for example, capable of collecting light emitted orthogonally from cells being illuminated by an illumination laser (e.g., an excitation laser). In various embodiments, this fiber optic element ("fiber") can be relatively large. This enhances photon capture / detection (i.e., increases the number of photons captured and detected). A smaller fiber may not be able to collect a sufficient number of photons for discrimination, for example, a 4% difference in DNA content between X and Y chromosomes (e.g., a 4% difference in emission intensity). In various embodiments, the fiber optic element can be positioned such that emissions from or caused by excitation by additional light sources (e.g., a kill laser or a laser that changes the state of cells or other particles in the sample) are not captured. In certain embodiments, the fiber optic element may be partially masked or filtered to filter out such killing laser emissions (e.g., partial masks and / or filters 811 and 812 (FIG. 8D) which may include one or more suitable lenses or opaque components).Alternatively or additionally, the size of the fiber may be selected to be spatially selective with respect to the detection location for the set of lights. For example, a smaller fiber may be used to capture only the light associated with the first fluorescence or excitation event, and not from the second fluorescence or excitation event. However, a smaller fiber may reduce the amount of photons that can be collected. Alternatively or additionally, the fiber may be angled or curved to position the face of the fiber such that the laser emission / excitation emission is not captured by the fiber. These solutions may be more suitable for larger fibers. The diameter of the fiber optic element may be such that it may capture the emission associated with both the kill laser and the detection laser if not properly positioned, masked, or filtered.

[0090] In various embodiments, the fiber may be selected based on a desired diameter and / or numerical aperture. The size of the fiber may be limited by the thickness of the microfluidic chip or by the manner of connection of the fiber to the chip. In various embodiments, the fiber may be 10-600 microns in diameter. The diameter may be selected so that the fiber is large enough to collect enough light to provide sufficient data for the bias determination. The fiber may be selected based on, for example, the numerical aperture, or the amount of light the fiber can collect. This is based on the material of the fiber, and in various embodiments, a numerical aperture of 0.1-0.5 may be sufficient to provide enough light to be collected.

[0091] The light may be in at least one of two ranges: fluorescence (e.g., wavelengths of about 400 nanometers (nm) or greater), and scattered light, which will have the same wavelength as the illuminating laser (e.g., about 355 nm). Alternatively, a filter may be used that filters out light closer to the wavelength of the illuminating laser (e.g., greater than 360 nm). The optical fiber may terminate in a beam splitting cube that achieves wavelength-based separation using dichroic optics to separate the fluorescent wavelengths from the scattered wavelengths. The two wavelength ranges may be focused onto photodetectors (e.g., PMTs or APDs), and the signals from these detectors may be processed, for example, using embedded hardware (FPGAs). The corresponding peak intensity signals may be plotted as two-dimensional histograms, as shown in Figures 5 and 7. A second detector signal system (Figures 5 and 6) provides additional information over a single detector (Figure 4) that allows for the identification of contaminating female cells. With two detectors, the additional information allows for the creation of gates that can better separate the male population from the female population, in various embodiments. For example, the gates disclosed in Figures 11B and 13C. One skilled in the art will appreciate that additional gates may be used to separate various populations.

[0092] In various embodiments, rather than using two detectors, a single detector with many pixels may be used, and light from both detection points (e.g., along a first axis and a second axis corresponding to different directions, such as side and forward) may be routed to the same multi-pixel detector. In certain embodiments, a fiber bundle may be used in the side direction (e.g., the second axis), and the fibers may be split at the detector end of the bundle to different areas or zones on a detector, such as a multi-pixel APD or camera (e.g., a charge-coupled device (CCD) detector). Processing may be performed on the intensity of the emissions detected at different pixels or regions of the detector or camera, and this may be used to obtain additional information to functionally separate spatial locations or for comparative analysis of intensity at different pixels or areas.

[0093] Moreover, large fibers may be used (as described above) to capture more than one color or wavelength of laser or fluorescent emission at slightly different main channel locations. This information may be combined, added, or differentiated to obtain additional information such as particle size, shape, quantity, position, or orientation. Additionally or alternatively, a phase difference may be determined for the light captured by the fiber. These may be referred to as "physical or positional properties" of the particle.

[0094] In various embodiments, determining the positional characteristics of a particle using the light intensity measured at the first pixel detection area and the second pixel detection area can be very similar to the measured light from two separate detectors, except that the use of multiple pixel detection areas can be implemented using multiple pixels on a single detector. The light intensity and pixels can be from the same direction (fiber bundle) or from separate directions. Different measured light intensity levels can indicate a primary detection surface, a secondary detection surface, or a tertiary detection surface (e.g., flat-facing, edge-facing, or intermediate). Different measured light intensity levels can also indicate a location (X, Y, Z) within the core stream. The particular mathematical operations that will be performed to provide the positional characteristics depend on the location of the pixels and the expected cell orientation / location associated with those pixels. In various embodiments, feature extraction can result from mathematical manipulation, regression, machine learning, or other techniques.

[0095] In various embodiments, component 850 may be used to guide and / or detect light transmitted through optical fiber 830. Component 850 may be another optical fiber, such as an optical fiber that is part of sample platform 154 or another component of sample processing system 150. In certain embodiments, optical fiber 830 remains with removable microfluidic chip 158, while optical fiber 850 remains with the sample processing system. Optical fiber 850 may have various shapes (e.g., curved sections, etc.) and sizes to guide light toward a detector or otherwise reduce crosstalk between components. In some embodiments, component 850 may be or include a photodetector.

[0096] In various embodiments, the fiber or other waveguide may enter the microfluidic chip at the angle shown in Figure 8C. Such a configuration may prevent light, either direct laser emission or fluorescent emission from the kill site, from entering the fiber and causing signal interference or simply overpowering the detection signal. Alternatively or additionally, a partial filter or mask on either the microfluidic chip or the fiber may be used, as shown in Figure 8D.

[0097] In various embodiments, lenses may be used to filter, mask, or better image the light collected by the fiber. A filter at the tip end can provide masking or filtering at the detector end of the fiber. A lens may also provide better imaging for use with a camera (e.g., CCD) for analysis of a larger image at the detector end.

[0098] Referring to Figures 9A and 9B, an alternative arrangement of components of a microfluidic sample processing system is shown. In Figures 9A and 9B, a microfluidic chip 900 is shown from two different perspectives, a front view in Figure 9A, and an edge view in Figure 9B. The chip 900 includes a sample channel 910 through which the sample flows downward. In Figure 9A, a first light source L1 (e.g., an illumination laser) emits light from the back of the chip 900 in a first direction (e.g., a forward direction or illumination axis) traveling out of the page. The light coincides with the sample (and cells or other particles in the sample) in the sample channel 910 as the sample flows down. Light from the chip 900 traveling in the forward direction (e.g., fluorescence resulting from excitation resulting from light from L1 and / or scattering) can be detected using a first detector D1 positioned in front of the chip (opposite the side where L1 is positioned). The light from L1 may be at a first wavelength, while the light from the chip detected by D1 may be at a second wavelength (e.g., fluorescence). Light from the chip 900 traveling in a second direction (e.g., side fluorescence or side scattering) may be collected via a first fiber F1 and detected using a second detector D2. Note that F1 collects light traveling along an axis perpendicular to the direction of sample flow in the sample channel 910 as shown (see 920). Thus, the light detected at D2 may be traveling at a substantially 90 degree (or other) angle to both the illumination axis and the axis of sample flow. In various embodiments, the angle between the first and second directions may be 75-105 degrees, or 60-120 degrees, or 45-135 degrees, or 30-150 degrees. Similarly, the angle 920 between the first direction and the flow direction can be between 75 and 105 degrees, or between 60 and 120 degrees, or between 45 and 135 degrees, or between 30 and 150 degrees.

[0099] A second light source L2 may be positioned downstream (e.g., further down along the sample channel 910, as depicted in FIG. 9A). L2 may be a biased light (e.g., an ablation laser, an inactivation laser, an activation laser, etc.) that emits light from behind the tip 900 along an axis that is parallel to the first direction, such that the emitted light hits the sample in the sample channel 910 after the sample is no longer in the path of the light from L1. Light from the tip 900 traveling parallel to the second direction (e.g., side fluorescence or side scattering) may be collected via a second fiber F2 and detected using a third detector D3. The light from L2 may be used, for example, to inactivate (e.g., slice or otherwise "kill") cells in the path of the L2 light. Cells traveling through the sample channel 910 may travel through the sample channel 910 in a single line, one after the other, such that each cell in the sample is matched with both the L1 light and the L2 light. The light detected at D3 can, for example, serve as confirmation that each cell in the sample channel has been exposed to (and therefore inactivated by) light from L2. For example, the L2 light may also function to excite fluorophores in the cells and result in side fluorescence. In such a case, if a burst of side fluorescence is detected at D3 following emission of the L2 light, the side fluorescence can be used as an indication that the cells have been exposed to the inactivating light from L2.

[0100] In the perspective shown in FIG. 9B, light from L1 travels from the left of the chip 900 to the right in a first direction and strikes the sample in the sample channel of the chip 900. Light from the chip 900 traveling in a first direction along the same axis is detected at D1 on the right side of the chip 900. Light from the chip 900 traveling in a second direction (orthogonal to the first direction) travels out of the page to a second detector D2 (positioned in front of the chip from the perspective in FIG. 9B). Similarly, light from L2 travels from the left of the chip 900 to the right (parallel to the first direction) and strikes the sample downstream of the sample channel of the chip 900. Light from the chip 900 traveling along an axis that is parallel to the second direction is detected at a third detector D3 (positioned in front of the chip from the perspective in FIG. 9B). Data from D1 and D2 can be used to determine orientation characteristics and cell type, and data from D3 can be used to provide confirmation of a "kill shot."

[0101] Referring to FIG. 10, an exemplary process 1000 for enhancing differentiation of components of a sample is depicted, according to various potential embodiments. At 1005, a sample may be provided to a sample channel of a microfluidic chip. For example, the control unit 114 may signal the fluidics component 166 (e.g., based on user input received through the user interface 134) to effect delivery of the sample to the microfluidic chip 158 when the chip is on the sample platform 154. The sample may have an initial (first) ratio of a first particle type (e.g., a first cell type) to a second particle type (e.g., a second cell type). Cells or other particles in the sample may be stained with a fluorophore before being flowed through the sample channel.

[0102] At 1010, coherent light may be emitted to the sample as it flows through the sample in the sample channel. For example, the control unit 114 may send a control signal to the illumination light 182 (such as an excitation laser) to emit light (e.g., may have a wavelength of 355 nm). At 1015, a first set of light (having one or more wavelengths) traveling in a first direction (e.g., forward direction along the illumination axis) may be detected. The first set of light may be scattered light (e.g., the same wavelength as the illumination light, 355 nm) and / or fluorescent light (e.g., wavelength for about 400 nm). The control unit may control the first photodetector 194 to detect light along the illumination axis and receive a signal from the first photodetector 194 corresponding to the detected light. The signal may be provided by the control unit 114 to the analysis module 118 for processing. At 1020, a second set of light (having one or more wavelengths) traveling in a second direction (e.g., a lateral direction orthogonal or otherwise angled relative to the forward direction) may be detected at a second location using a second photodetector.

[0103] At 1025, a position and / or orientation of the cells in the sample may be determined based on the first and second sets of detected light (e.g., as described above with respect to FIGS. 7 and 9 ). For example, histogram unit 122 may identify an orientation feature of the cells based on which region in the histogram the light of the cells falls into. At 1030, a biasing operation may be performed based on the orientation feature and the detected light. For example, control unit 114 may control an inactivation light 186 (e.g., an ablation laser) that is directed at cells identified as being of an unwanted cell type. In this manner, a corrected proportion of cell types in the sample may be achieved.

[0104] 11A and 11B, multiple gates covering male and female populations are disclosed. The logical separation of each population can aid in counting, tracking, or observing the populations and can be used in conjunction with biasing methods and systems such as those described herein to result in biased populations (e.g., primarily male or primarily female). As one skilled in the art would understand, similar gates may be used to identify populations along other dimensions. For example, non-active or dead populations may be gated. Similarly, such populations may be biased, tracked, etc. using the gates in conjunction. The intensity map 1110 may be organized similarly to the histogram of FIG. 5 based on multiple peak forward and side fluorescence measurements, which may be determined by similar methods and systems. In some embodiments, additional or fewer sensors may be used to collect the intensity map. For example, additional sensors may be used to generate an n-dimensional intensity map 1110 or to improve the accuracy, detection rate, etc. of the two-dimensional intensity map 1110.

[0105] FIG. 11A depicts an intensity map without any overlaid gates. Intensity map 1110 depicts populations similar to FIG. 5 with a distinguishable male population 1120, a distinguishable female population 1130, and a tail 1140 that may contain mixed male and female specimens. In some cases, the distinguishable populations may be mixed as well. For example, a distinguishable male population may contain a small percentage of female specimens, or vice versa, and there is a dead zone between the populations that is sufficient to completely separate these populations. Each depicted population has a population center defined by the peak intensity within the distinguishable population. In the depicted embodiment, the male and female populations are approximately equal, and the peak intensity of each population is roughly equal. In some embodiments, the peak intensity of the two populations may not be equal. For example, populations resulting from an iterative biasing process to further increase population skew, naturally diffuse populations such as non-active specimens, bimodal populations, trimodal populations, etc. In some embodiments, the population center may be defined by alternative criteria, for example by the geographic average of the population rather than a point or area of ​​peak intensity.

[0106] In the depicted embodiment, two population centers are defined according to the two areas of highest intensity. The two population centers correspond to a male population and a female population, and a male population center 1122 and a female population center 1132 are depicted. In some embodiments, the two population centers may be located based on the intensity profile. For example, a first intensity profile 1162 may be disposed along one axis of the intensity map, showing the sum of the detected populations in the depicted row. The first intensity profile may include a first local maximum 1162a, which may correspond to the tail 1140 of the intensity map 1110, and a second local maximum 1162b, which may correspond to one or more of the population centers. In some embodiments, the first intensity profile may also include a third local maximum, which may correspond to an additional population center. For example, the male population center 1122 and the female population center 1132 may be separately identifiable from the first intensity profile. In the depicted embodiment, the two populations are not readily distinguishable as depicted, although further processing may enable distinction of these populations.

[0107] A second intensity profile 1164 is also depicted having two maxima, a first maximum 1164a associated with the male population center 1122 and a second maximum 1164b associated with the female population center 1132. A third intensity profile 1166 may then be calculated based on the intensities at a vertical line drawn along the second maximum 1164b of the second intensity profile to exclude the intensity contribution of the male population. A fourth intensity profile (not depicted) may then be calculated based on the intensities at a vertical line drawn along the first maximum 1164a of the second intensity profile 1164 to exclude the intensity contribution of the female population. Each of the third intensity profile 1166 and the fourth intensity profile may then be evaluated to determine the centers of the male and female population centers, respectively. In addition to the maximum intensity defined by the third intensity profile 1166, multiple additional intensities may be recorded along the vertical line passing through the female population center. For example, points at 75% of maximum intensity, 50% of maximum intensity, 25% of maximum intensity, and 10% of maximum intensity are selected. As one skilled in the art would understand, these values ​​are just one possible set, and other values ​​may be selected that may be better formed for the particular data. Furthermore, according to the coordinate system selected, any movements based on vertical or horizontal lines may be inverted or truncated, and such description is not intended to limit the present disclosure, but is only intended to illustrate the figures herein.

[0108] 11B, the same intensity map 1110 is drawn, with a first sloping line 1170 passing through the male population center 1122 and the female population center 1132. The distance between the two population centers may hereafter be referred to as W for use as a constant, and may refer to the vertical distance alone or the total distance, according to various potential embodiments. A fifth intensity profile is generated along the first sloping line 1170, and a saddle point (i.e., an intensity maximum disposed between two intensity maxima) is defined as a first point 1180. Additional sets of intensity profiles are calculated along additional corresponding lines that are parallel to the first sloping line 1170. For example, a second sloping line 1171 is drawn. Saddle points are determined along each subsequent line. Saddle points 1180, 1181, 1182, 1183, and 1184 are shown and may be determined based on the saddle points of the intensity profiles generated along various sloped lines (only a first 1170 and a second 1171 of which are shown). One or more gates may be bounded, in part, by lines formed along the saddle points. A first gate 1150 covering a primarily male population and a second gate 1155 covering a primarily female population are depicted.

[0109] In alternative embodiments, additional or fewer saddle points may be selected. In some embodiments, the saddle points may be skewed left or right (e.g., to minimize female specimens gated by the first gate, or male specimens gated by the second gate). Additionally, the final saddle point (depicted herein as 1184) may be adjusted upward (e.g., to maximize the number of gated specimens, which may maximize skew) or downward (e.g., to minimize the number of gated specimens, which may maximize eligibility). For example, the gate may extend through the entire tail, which may result in a substantial increase in gated specimens, but reduce skew between populations.

[0110] In some embodiments, the intensity map 1110 may be oriented based on two population centers. For example, the two population centers may be disposed along or approximately along the horizontal axis of the intensity map. For example, the intensity map 1110 may be plotted based on collected data or based on historical data to maintain a horizontal alignment of the population centers, which may minimize the calculated intensity. In such an embodiment, the slanted line may have a slope of zero. Alternatively, a slope of zero may be estimated to minimize or otherwise reduce computational requirements or to impose an offset on the plotted saddle point.

[0111] Also, by selection of appropriate boundary points, various gates can be bounded. The boundary points can be predefined or based on saddle points or other measurements. In the depicted intensity map 1110, the first gate 1150 is bounded by a first boundary point 1185, which is located at a horizontal point relative to the bottom saddle point 1184, at a distance equal to twice the distance between the center of the male population and the center of the female population. A second boundary point 1186 is located at a point along a vertical line extending upward from the first boundary point 1185 twice the distance between the top saddle point 1180 and the bottom saddle point 1184 (which may hereafter be referred to as "H", with the second boundary point 1186 being located 2H above the first boundary point 1185). A third boundary point 1187 is located at the point where the vertical line from 1180 intersects with the horizontal line from 1186. Other boundary point locations are possible. For example, the first boundary point 1185 may be located at 1H or 3.5H, with the other boundary points adjusted.

[0112] The second gate 1155 may be generally symmetrical or different from the first gate. In the depicted embodiment, at the top right boundary of the second gate, a fourth boundary point 1188 may be symmetrical to the second boundary point 1186 (i.e., located 2W from the third boundary point in the opposite direction). The lower boundary is defined at a fifth boundary point located H below the bottom saddle point 1184, and the boundary of the second gate 1155 is completed by referencing the point where a horizontal line drawn from the lower boundary 1189 intersects with a vertical line drawn from the right boundary 1188. Thus, the second gate 1155 may extend lower than the first gate 1150, which may advantageously increase the eligible specimens without the reduced distortion that a lower gate would bring to the first gate 1150 due to the mixed tail.

[0113] Although the additional populations may not be displayed on the intensity map 1110, in some iterations, there may be additional populations. For example, there may be non-active populations, XY samples, etc. Thus, the boundary points may require additional specificity in some embodiments. Additional or fewer boundary points may bound the gate. Furthermore, in some embodiments, multiple gates may be overlaid. For example, multiple gates may be provided and Boolean functions may be operated on these gates.

[0114] Referring to FIG. 12, a method 1200 for generating a saddle map is disclosed. In operation 1210, a fluorescence intensity peak is measured in a first direction. For example, according to various embodiments disclosed herein, the first direction may correspond to a fluorescence detector oriented to detect fluorescence in front of the sample ("forward" fluorescence). Further, in operation 1210, a fluorescence intensity peak is measured in a second direction. The second direction may or may not be disposed substantially orthogonal (e.g., to the side) to the first direction. For example, the second direction may be the same direction at a different time.

[0115] At operation 1220, an intensity map is generated. An intensity map is a two-dimensional representation of the peak intensities observed in each of two directions. Thus, similar to the histogram of FIG. 5, the intensity map shows a two-dimensional representation of a population. Creation of the map may reveal clustering of similar specimens of a population. For example, viable sperm may be highly clustered on such a map. Furthermore, male and female sperm may be distinguishable, but may require additional processing to delineate the population due to the proximity of these sperm.

[0116] In operation 1230, a population center is identified. The population center may be identified according to the peak intensity and / or expected location. For example, if two peak intensities are immediately adjacent and another slightly smaller peak intensity is disposed an expected distance or in an expected direction away, the smaller intensity may be selected as the population center. If a suitable population center cannot be located, additional data may be collected, an error may indicate a sensor problem, or the data may be archived for later review.

[0117] In operation 1240, saddle points are identified. Saddle points may be identified based on a local minimum bounded by two maxima. Saddle points may also be identified based on a sloping line parallel to a line between two population centers, a sloping line between two areas of equal density along a row, etc. Saddle points may be defined at regular intervals, or as a pattern selected to maximize distortion, or may be selected otherwise.

[0118] In operation 1250, the first population is bounded by forming a gate that includes the population between a saddle point and an outer boundary. The outer boundary may be defined statically, in relation to population characteristics, manually, etc. In some embodiments, the boundary (and / or saddle point) may be dynamically adjusted. For example, an offset from the saddle point may be used to bound the population. To maximize distortion, a gate may be formed that does not include the saddle point. For example, the gate may include a left or right shift point from the calculated saddle point to increase the percentage of the population bounded by the gate, or the overall number of samples bounded by the gate (e.g., by shrinking or increasing the mixed population captured).

[0119] In operation 1260, the second population is bounded in a manner similar to the first population. This operation may be performed in addition to or instead of operation 1260. For example, in many embodiments, only a single gate may be formed. In some embodiments, both gates may be formed (e.g., for data tracking / analysis), but only a single gate (or no gate) is used in conjunction with the biasing process. The disclosed steps disclose some embodiments of method 1200, but one of ordinary skill in the art will understand that certain steps may be added, omitted, modified, etc., according to other embodiments of this document. For example, related methods may be performed based on three measurements (e.g., fluorescence or other measurements) as a three-dimensional system.

[0120] With reference to FIG. 13A, a digital approximation of the Epanechnikov kernel 1301 is disclosed. Similar approximations may be considered with other kernels, such as Gaussian kernels. Multiple kernels may be arranged that may smooth the resulting data set. For example, the depicted kernel may be a digital approximation of the Epanechnikov kernel 1301 with n-bit depth for each pixel (i.e., each pixel is a 1-bit pixel with n-bit depth from 0 to 2). ’’1303) and a resolution of m pixels. For example, an 8-bit kernel may have 256 possible values ​​for each pixel of the kernel. Thus, the center value (i.e., root pixel 1302) may have a value of 200 and the outer pixels 1303 may have a value of 10. The drawn kernel has a radius of 7 pixels. Increasing the pixel resolution may better capture the kernel (e.g., parabolic Epanechnikov kernel) and thus smooth the display, but such smoothing may increase the computational load on the system. In some embodiments, the system must operate in real time (e.g., in certain systems that rely on classification of specimens to bias the specimens), and therefore the computational load may be managed according to available computing resources. For example, one embodiment may have a 4-bit depth and a radius of 5 pixels, while another embodiment may have an 8-bit depth and a radius of 32 pixels. Note that while the kernels are depicted figuratively, the pixels of FIG. 13A need not be presented on a display, presented to a user, etc., as with other data sets. The pixel nomenclature is intended to describe the various discrete locations used to sum the various kernels and does not necessarily relate to the display technology. Each kernel may represent a measurement of a cell (e.g., a sperm cell). Each measurement may include multiple detections. For example, the fluorescence of a cell may be measured in two directions and placed on a pixel map based on the dimensions (e.g., side fluorescence may determine location along the x-axis and frontal fluorescence may determine location along the y-axis). Additional or fewer measurements may result in a pixel map with additional or fewer dimensions.

[0121] With reference to FIG. 13B, multiple pixels may be summed onto a pixel map 1305. For example, if two partially offset pixels are overlaid such that a pixel value of 200 overlays a pixel value of 10, a value of 210 is generated. Additional kernels may continue to be added to form a pixel map 1305 that represents a density function of the kernel, such as in relation to the fluorescence of multiple cells measured from two directions. Thus, the fluorescence of various cells may be mapped to reveal the relative fluorescence of various identifiable cell populations. For example, a population of cells may be described as a population of non-activated cells 1340, male cells 1320, female cells 1330, etc. Some populations may describe instrumentation limitations or errors. For example, some cells may be detected with a very high level of fluorescence in one direction (e.g., the lateral direction) due to interference from other system components, and such cells may be referred to as saturated cells 1350. In some embodiments, such cells can be classified as male, female, non-viable, etc., such as by taking further measurements or by additional data processing steps.

[0122] Some populations or errors may be removed from the pixel map to better analyze one or more populations of interest. Populations of interest may be identified based on characteristics, location, or past experience. Referring again to FIG. 13B, two populations of interest are disclosed: male population 1320 and female population 1330. The populations exhibit the same characteristic shapes as in FIGS. 7 and 11. Furthermore, the populations are substantially denser than the other detected populations. For example, estimated inactive population 1340 and estimated saturated population 1350.

[0123] The populations of interest may be better analyzed by adjusting the pixel map 1305 to exclude irrelevant populations, no-population spaces, noise, etc. The columns of the pixel map may be summed to plot a histogram, and the derivative of the histogram may be used to locate the maxima and minima of the histogram based on the zero crossings of the derivative. In certain embodiments, a deadband is used to suppress noise-induced zero crossings. The deadband may be fixed or dynamic. For example, the deadband may be based on a percentage of the peak derivative value. For a defined population, various critical points may be generally predictable and may be defined based on the location of emergence, direction of exit / entrance from the deadband space, slope, etc. All columns outside the range of interest may be cleared to reduce noise and information about undesired populations.

[0124] Such filtering of column histograms may not adequately remove noise or irrelevant populations. For example, the saturated population 1350 may not be vertically aligned with the population of interest and, as a result, may not be removed by column filtering. Thus, a second row histogram is generated and the derivative of the histogram is generated. As with column filtering, known populations may be identified for inclusion or exclusion and noise may be removed by use of deadbands. The known or desired populations may be determined based on zero crossings of the derivative and / or row-by-row analysis (e.g., row-by-row analysis near local maxima or minima determined according to the derivative).

[0125] FIG. 13C illustrates an expanded version of FIG. 13B, focusing on the male / female populations. Specific regions of interest can be calculated according to the analysis of the histograms described above. Because the populations of interest are closely arranged and may be mixed, the division of the two populations is more computationally intensive. However, because much of the pixel map has been eliminated, the computationally intensive task may be performed in a localized portion of the pixel map, such as the depicted portion.

[0126] A second row of histograms is generated and used to determine a lower threshold. The lower threshold eliminates tails where male and female cells may be heavily mixed. The lower threshold may be defined according to a minimum density, or an inflection in the change in density. For example, the generally decreasing density of the pixel map along the negative y-axis of the pixel map may be slowed by the curvature of the tail, at which point an inflection point may establish the threshold for the tail.

[0127] Once the threshold is established, pixels above the threshold may be sampled row by row to establish a trough line 1390. The trough line 1390 is defined by a local minimum disposed horizontally between two local maxima. A critical point 1399 may be defined by a point on the trough line disposed vertically with the greatest density according to the row histogram, and may be the end of the trough line 1390. The slope of the trough line 1390 is zero (i.e., vertical, as depicted) at the critical point 1399. Also, additional points along the trough line are collected. A second trough line point 1398, and a third trough line point 1397 are depicted. Since the increased points generally improve the fitting of the trough line and therefore the distortion, several points may be defined. For example, a trough line point may be defined for every row.

[0128] In some embodiments, the plotted trough line 1390 may be used directly. In other embodiments, a function (e.g., a Lagrange polynomial) may be generated based on the trough line 1390. The generation of the trough line 1390 may include rotating the pixel map by 90 degrees to simplify the required calculations. Moreover, restricting the polynomial to a second order polynomial and forcing the polynomial to pass through critical points with a zero slope may further reduce the calculations and may fit some trough lines 1390 well. In other embodiments, a higher order polynomial may be selected and the best fitting line may be accepted. In some embodiments, either the generated trough line 1390 or the generated function may be approximated by a fixed number of points. For example, the polynomial may be reduced to four, six, or eight line segments. Advantageously, such approximation may reduce the calculations required to define gates based on the trough lines in a particular system.

[0129] Similar to the saddle of FIG. 11, the trough line 1390 may be bounded based on fixed points, relative distances, or other criteria. For example, the vertical distance between the critical point and the tail threshold, or the distance between two peaks in the row of critical points, may be used as a base measurement. The left or right population boundary points of the trough line 1390 may be some multiple (e.g., 0.1x, 0.25x, 0.5x, 1x, 2x, 5x, 10x, etc.) of the base measurement to define a gate. A base measurement of width may also be defined or calculated.

[0130] 13D, a close-up of the saturated population 1350 is shown, generally divided into two populations, a predominantly male saturated population 1352 and a predominantly female saturated population 1353. A vertical extension 1380 of a critical point 1399 is depicted that approximately bisects the saturated population into a male 1352 and a female 1353 component. By extending a gate vertically upward from the critical point, the saturated population 1350 may be included in a gate that includes populations to the left or right of the trough line 1390 (e.g., extending a polynomial trough line may not bisect the population or may impose additional computational burden relative to extending the critical point upward).

[0131] While that may be computationally acceptable (i.e., not overly computationally intensive and therefore not overly time consuming for a particular application), extending the critical point 1399 vertically upward may not be desirable since the concavity of the trough line 1390 above the critical point may affect the separation of the unsaturated male and female populations. Thus, the gate may be selectively extended based on the presence or absence of saturated cells. For example, the row histogram may indicate the presence or absence of a saturated population 1350. If there is a saturated population 1350, which may be determined according to the minimum density of the first row histogram, the gate may be extended upward to include the saturated population. Advantageously, this may maximize eligible cells. If there is no saturated population 1350, the gate may be constructed regardless of any potential saturated cells. Advantageously, such an embodiment may maintain the desired skewness in the main population by maintaining the concavity of the trough line above the critical point 1399 in the unsaturated population.

[0132] Alternatively, or in addition, it may be useful to bisect the population in another manner. For example, a polynomial may be generated regardless of the saturated population, and then a line segment may extend from above the critical point 1399 to the saturated bisection point 1392 on or above the saturated population 1350. Thus, the main population above the critical point may maintain distortion performance, and the population may be more accurately bisected.

[0133] The saturation bisection point 1392 may be calculated similarly to a point along the trough line 1390, for example, based on a row histogram showing a local peak kernel density near the center of the saturated cell 1350, and then offset (e.g., vertically up a fixed number of rows). Because the mixed "tail" cells of the saturated population may not be easily identifiable, potential gates may be more tightly bounded. A first boundary line 1321 is shown disposed near the center of the male saturated population 1352. The first boundary line 1321 may result in bordering many male specimens from the gate, which may also increase the specificity (i.e., skew) of the selection. The first boundary line 1321 may be moved to the left or right to balance skewness and eligibility. For example, the first boundary line 1321 may be placed to avoid excluding the entire saturated population, which may advantageously increase the number of cells gated. Similarly, another boundary line (not depicted) may be disposed along the right side of the saturated cell. However, because the saturated population 1350 exhibits similar characteristics to the unsaturated population, there may be limited mixing to the right, and therefore a wider boundary line may be selected, such as a boundary line extending from the right side of the population.

[0134] In some embodiments, multiple gates may be used in combination (e.g., separate gates may be used for saturated and non-saturated populations). Advantageously, additional gates for discrete populations may be generated in parallel to any other gates present, which may be preferable for at least some systems than a single, more complex gate. In such embodiments, the additional gates may be imposed in a similar manner to any gates associated with non-saturated populations, or may be imposed by a different method, such as the saddle method disclosed by FIG. 12, or another method.

[0135] With reference to FIG. 14, a method 1400 of generating a kernel density estimation gate is described. The method may be performed for many datasets according to various embodiments, such as the datasets illustrated in FIGS. 13B and 13C. In operation 1402, a kernel is generated. The kernel may be selected for computational performance, accuracy, etc. For example, the kernel may be selected according to the implied accuracy of various sensor devices detecting cells and according to the quality of the results. In some embodiments, multiple kernels may be generated for a dataset (e.g., real-time data, historical data, etc.), and the method 1400 may be run with each and the results compared. Once a kernel is selected, a digitally decomposing kernel is generated according to the system requirements (e.g., amount of computer memory or processing resources, detector resolution, etc.). In some embodiments, the minimum resolution, depth, etc. of the kernel may be determined, and the system may be designed to support such a system. In some embodiments, the digitally decomposing kernel may be configured to run on a particular available hardware.

[0136] In operation 1404, the system awaits a first data point. The data point relates to an attribute of a cell (i.e., an individual) of a population of cells. For example, the data point may be the fluorescence of DNA contained within various populations of sperm by multiple detectors. For example, one dimension of the data point may relate to the fluorescence detected in a first direction (e.g., a "head-on" direction) and a second data point may relate to the fluorescence detected in a second direction (e.g., a "side" direction). In some embodiments, absolute fluorescence may be graphed, and in other cases, the fluorescence may be normalized (e.g., to pre-process the data to account for detector differences, etc.). For example, the data may be range checked to minimize subsequent processing.

[0137] In operation 1406, kernels representing the data points are added to the pixel map. The data point addition may include a root pixel representing the probability center of the location data point, and remaining kernels representing the distant (typically decreasing) probabilities from the root pixel to the outer edge of the generated kernel. If the kernel addition overlays any existing kernel, each overlapping pixel is added to the pixel map such that the cumulative kernel may show a probability density on the pixel map beyond the probability density available at each pixel. For this reason, the depth of the pixel map may need to exceed the depth of the kernel (e.g., each pixel may require 32-bit depth to represent a pixel with 8-bit depth). Because solving the kernel for the desired resolution and depth may be computationally expensive, in many embodiments, the pre-generated kernels may be summed to each pixel based on an offset from the root pixel, which may or may not be at the center of the kernel.

[0138] In operation 1408, the number of data points is checked for reasonableness. In some embodiments, a simple numerical check (e.g., at least 100,000 data points, 120,000 data points, or 140,000 data points) may be performed. In such embodiments, this operation may further include incrementing a data point counter. In some embodiments, another check may be performed instead of or in addition to the counter. For example, the pixel map may be checked for a minimum density. For example, if any population exceeds a density of X, an adequate number of data points may be collected. Alternatively, or in addition, additional operations 1410-1434 may be tentatively performed (e.g., on a copy of the pixel map so that the data is not affected) to check whether the number of data points is adequate. Advantageously, such a method may allow for earlier identification of gating zones that may allow biasing or other operations on the population. If the number of data points is determined to be insufficient, the operation may proceed to operation 1438 to await future data points.

[0139] If the number of data points is determined to be adequate, a first histogram is generated in operation 1410. Although FIG. 14 describes a column histogram, the first histogram may be taken in any dimension (indeed, method 1400 may be suitable for implementation in an n-dimensional system, and thus the first histogram may not be a row or column histogram). The first histogram is taken to exclude certain populations and noise from the chart. For example, it may be desired to exclude dead cells, noise, and saturated cells from the pixel map. The histogram may be one-dimensional, or in some embodiments, n-1-dimensional, and may be a measure of the density function represented by the pixel map across at least one axis.

[0140] The generation of the first histogram may establish various points of interest in operation 1412 to identify populations of interest and eliminate other populations. For example, the density function of the pixel map may be plotted with additional processed functions (derivatives, keep out bands, minimums, etc.). Points of interest that exhibit local minima or maxima may be used to determine a center of population location and the population of interest may be selected according to a dynamic or static process. In some embodiments, a predetermined or set number of pixel columns (such as 500 pixel columns) may be selected to be centered on the center of the peak density or offset from this center. In other embodiments, a variable number of columns may be selected according to the sensed data (e.g., based on local minima or maxima). In some embodiments, a fixed number of columns, rows, pixels, etc. may be limited by available computing power. A region of interest (ROI) is determined based on the selected points of interest. Some embodiments include multiple regions of interest. For example, in one embodiment, the saturated population may be a region of interest in addition to the X / Y population discussed hereafter. In such an embodiment, additional processing may be performed within each region, which may be similar to or different from the method 1400.

[0141] In operation 1414, data for populations outside the region of interest are discarded, archived, cleared, etc., so that ongoing processing can be based on the selected region of interest. Alternatively, or in addition, elements of the pixel map may be filtered according to defined locations or attributes. For example, if the location of the population of interest is known prior to plotting, certain measurements may not need to be plotted, and pixels below a pixel threshold (e.g., due to noise, incorrect samples, etc.) may be suppressed regardless of region.

[0142] In operation 1416, another histogram, a first row histogram, is generated. In some embodiments, such as those depicted in Figures 13B and 13C, the first row histogram includes side fluorescence data taken of various sperm populations and is a density histogram of the rows of the pixel map. In alternative embodiments, the second histogram may be based on orthogonal axes that may or may not be related to physical dimensions (e.g., the first histogram is a first fluorescence measurement and the second measurement is based on a second orthogonal or non-orthogonal fluorescence measurement or another measurement such as velocity, position, etc.). In operation 1410, the second row histogram may be generated over a limited portion of the pixel map since a portion of the pixel map may have been cleared, zeroed, and archived.

[0143] In some embodiments, the second histogram may be omitted. For example, pre-processing or the use of an appropriate coordinate system may be generated to minimize populations not captured by the first histogram. In some embodiments, the second histogram, or the first histogram, may be generated only once (e.g., following determination of adequate sample size), which may advantageously minimize processing requirements that may reduce associated hardware requirements, or allow for improved resolution and / or depth.

[0144] In operation 1418, various points of interest are found on the first row histogram. Determining the points of interest may be based on maximum density or additional processing may be performed. For example, the derivative of the histogram may also be plotted to find zero points (e.g., local maxima and minima), and such maxima and minima or maximums and minima may be used to determine regions of interest for further inspection and processing.

[0145] In operation 1420, data relating to undesired populations may be discarded, archived, erased, etc. For example, all data outside of a region of interest on the pixel map may be so disposed of. In some embodiments, some data may be discarded regardless of what region the data is in. For example, pixels below a density threshold may be discarded (i.e., set to 0 density), or other pre-processing may be performed to simplify later analysis.

[0146] In operation 1422, a second row histogram is generated over the region of interest that may have been reduced by operations 1414 and 1420. With some data sets, the region of interest may be substantially smaller than the original pixel map, which may allow additional calculations.

[0147] In operation 1424, a density function of a row (or portion thereof) of the pixel map is generated, and first and second derivatives of the density function are generated, which may assist in determining inflection points. Inflection points may be used to identify points of interest (POIs) on the map. For example, a positive inflection point located below and away from the center of the population may indicate a lower threshold. Advantageously, such an approach may enable locating relatively complex pixel map features, such as those with tails, which may be difficult by considering only the density values ​​themselves, or the absolute rate of change of density values.

[0148] In operation 1426, a tail threshold is defined. The tail threshold may be defined based on an inflection point of the density function of the second row histogram of operation 1424. For example, the rate of decreasing density decreases as the reduced population density away from the population center reaches the tail and slows (i.e., due to the tail aligning along the row instead of perpendicular to the tail). In some embodiments, the point of inflection may be used as the tail threshold. In other embodiments, a fixed or variable offset may be used. For example, 25 pixels above or below the inflection point may be selected. In some embodiments, the threshold may be dynamically determined based on the current fitness or distortion of the sample (e.g., the threshold may be adjusted upward for increasing distortion or downward for increasing fitness).

[0149] In operation 1430, a trough line point is defined for each row. The row may be a single pixel in height or may be another height having more than one pixel, according to requirements and available computing power. A histogram is generated for each row to determine the location of the first population, the second population, and the trough line point disposed between the two populations. Of particular note is that there is a trough line point determined in the row having the maximum density according to the row histogram (defined as the critical point).

[0150] In operation 1432, a determination is made whether all rows have been completed. Rows may be completed according to any rows in the region of interest that exceed a tail threshold, or may be further restricted. For example, rows may be restricted by an upper boundary defined according to a density function (e.g., when a minimum density is reached), or by an inflection point or critical point, or by an offset from an inflection point or critical point (e.g., a row 10 pixels above a critical point may have an associated trough point calculated). In some embodiments, the trough line may be defined according to additional processing steps. For example, rows may be defined along a sloping line on a grid, similar to the process of method 1200.

[0151] In operation 1434, a regression is performed along the trough line to define a polynomial function that describes the trough line. The function may be constrained by multiple trough points, a slope at a critical point (e.g., the slope may be constrained to zero), etc. In some embodiments, the trough line may be constrained to a second order polynomial. For example, in embodiments where the trough line points are not sampled above the critical points, such an embodiment may enforce a desired concave shape. In some embodiments, the polynomial may be shifted left or right to balance a desired distortion or well-formedness metric. To calculate the polynomial, the pixel map may be rotated, since various references to "up," "down," etc. may be optionally defined. In some embodiments including multiple sensors, such references may be understood in terms of being n-1 dimensions, n-2 dimensions, etc. down to a single dimension. For example, in a five-dimensional system, the above may be performed in terms of one or four dimensions.

[0152] In operation 1436, a number of discrete line segments are generated to fit the polynomial trough line. The number of line segments may be determined according to the intended computing system or according to the desired performance of the gate. In one embodiment, an eight-segment line provides a reasonable gate selection and may be computationally manageable. Some embodiments may utilize additional or fewer segments. Some embodiments may constrain the gate to employ a polynomial function directly.

[0153] A line segment may bisect the region of interest / pixel map into two gates, or additional boundary points may define the gates. For example, the lower boundary of the gate may be defined by or relative to the tail threshold. The upper boundary of the gate may be fixed (e.g., defined by the top surface of the region of interest) or defined otherwise relative to a critical point (e.g., twice the vertical distance between the tail threshold and the critical point), based on a density function (e.g., minimum density, inflection point, etc.). Additional gates may be formed that may be non-contiguous. For example, one gate may include the left side of the image depicted in FIG. 13C and the left side of the saturated population depicted in FIG. 13B. Such non-contiguous gates may be referred to as single (non-contiguous) gates, or else as multiple (contiguous) gates.

[0154] Referring to FIG. 15A, an exemplary detector that can detect fluorescence from one or more directions is depicted. The detector comprises an avalanche photodiode 1510 having a light-sensitive surface 1510a. Light of various wavelengths can be focused onto the avalanche photodiode 1510 by a tube lens 1520 configured to direct the light onto the sensor surface 1510a. The light that enters the lens itself is aligned by a collimator 1530. The collimator 1530 receives light from an optical fiber (e.g., a low hydroxyl (low OH) 0.5 NA multimode fiber) that may be accompanied by additional interfaces. For example, the fiber may pass through voids in various bulkheads. Transmission through voids associated with the interfaces may not be desired, and a system may be designed with a reasonable margin such that some attenuation may be tolerable with little or no impact on system operation. As one skilled in the art would understand, various optical fibers may be selected according to the desired optical frequency, wavelength, etc. For example, a low OH fiber may not be selected for a particular optical wavelength. FIG. 15B depicts the assembled device of FIG. 15A.

[0155] Referring to FIG. 16A, a debug plot is depicted showing a histogram and a derivative of the histogram. The histogram may be associated with a pixel map such as the pixel map of FIG. 13B. At the first POI 1610, a first population peak is indicated by the maximum value of the histogram and zero of the derivative. The first POI 1610 is bounded by a first left boundary 1605, which may be determined based on an inflection point or based on a slope and / or deviation from zero (e.g., emergence from a dead zone). The first POI 1610 is also bounded on the right side. For example, a first right boundary inflection point 1620 and a first right boundary dead zone cutoff 1625 may define a region of interest. For example, the distance between the first POI 1610 and the right boundary dead zone cutoff 1625 may be used to determine a band that defines the first population, which may be known or may be inferred to be a population of inactive cells.

[0156] A second POI 1635 may be defined at the maximum, which may correlate to another population (e.g., a male population). A third POI 1640 may define a trough or saddle (e.g., representing the maximum of the female population) disposed between the second and fourth POIs. Those skilled in the art will appreciate that various maxima and minima may exist for various pixel maps. For example, a pixel map having two populations linearly disposed along an axis (e.g., along the X-axis pointing to the lateral fluorescence of a bimodal population of male and female sperm) may have a single maximum value of a histogram taken along an orthogonal axis. Another population 1650 (e.g., a saturated population) is recognized to exist, but no POI is recognized. This may be because the histogram and / or derivative does not leave the dead zone area due to small sample size, density, etc., or may be the result of a programmatic decision to discard this population.

[0157] 16B, various constraints are imposed on the plot of 16A. A first population center 1655 is disposed along the peak of density of the first POI 1610. This point may be selected according to the first histogram beyond a minimum value at a particular location, etc., or may be determined according to further additional constraints (e.g., disposed to the right of the first left boundary 1605 and to the right of one or more right boundaries). A first population threshold 1660 is shown disposed to the right of the first population center, which may be determined based on a location within a dead zone disposed between the first population center 1655 and an additional population, which may be useful for generating future pixel maps, truncating data from the pixel map being considered, etc. A second population threshold 1655 is shown as a right boundary of one or more populations. For example, the second population threshold may be generated based on a return to the dead zone, based on an additional POI, based on a predefined or manually entered point on the debug plot or pixel map.

[0158] 16C, another debug plot including additional POIs is disclosed along with an associated pixel map. The pixel map discloses the same second population threshold as in FIG. 16B, and a second population center 1675 that may specify a population of saturated cells. As discussed herein, such a histogram may be used to eliminate the second population from the pixel map, biasing in favor of or against the second population. In some embodiments, the second population is further processed, and further defining the second population (e.g., split into males and females) center 1675 may enable further processing steps.

[0159] Referring to Figure 17, the pixel map of Figure 13C is depicted with a first histogram 1702 and corresponding first derivative 1704 overlaid along the vertical axis. Also depicted is a vertical maximum density 1710 corresponding to the population center. Also overlaid along the horizontal axis is a second histogram 1706 and corresponding first derivative 1708. The second histogram includes a first maximum 1720, a trough point 1730, and a second maximum 1740, which may represent the density along any row of the pixel map. These maximum and minimum points may be plotted row by row to yield a first maximum density line 1725, a trough line 1735, and a second maximum density line 1745.

[0160] Referring to FIG. 18, information flow through the system is disclosed that may enable various methods (e.g., method 1200, method 1400, etc.). Sensor 1610 may be or may include one or more fluorescent sensors that measure the fluorescence of cells from multiple directions, or another sensor type that may measure other attributes of the sample or the environment associated with the sample. The sensor data may be passed in a first analog form 1815 that may minimize losses. Those skilled in the art will understand that the choice of digital or analog filtering may be selected according to the particular implementation. For example, in some embodiments, if transmission losses of the analog signal would degrade the signal, analog to digital conversion may be performed as soon as practical, whereas in systems with very low losses (or uniform losses) of the analog signal, various filtering and processing may be performed on the analog signal before digital conversion. In the depicted embodiment, analog filtering and measurement 1820 is performed on the transmitted analog signal resulting in digital signal data 1825 that is input to a digital processing system, which may include various field programmable gate arrays (FPGAs), application specific integrated circuit (ASIC) processors, and other components (e.g., embedded FPGA system 1830) for gating various populations, subpopulations, etc. Gating data 1860 may be provided to a biasing element 1865 (e.g., a laser or other deactivation device to deactivate selected cells) for biasing the population.

[0161] In addition to or instead of the gate execution data 1860, various data may be provided to the embedded software system 1840, which may perform real-time or non-real-time data analysis based on the digital event data, and may provide another component (e.g., the embedded FPGA system 1830) with various gate data, boundaries, configuration settings, and the like. In some embodiments, the embedded software system 1840 may also interface with a high-powered software computing system (e.g., to store data, offload certain processing, and the like) comprising a server 1850 and a client. The server 1850 may then provide summary reports, gate visualizations 1855, predicted or analyzed data, and the like, to the client for further processing, adjust various parameters, or provide a visual or other presentation to the user, such as via a graphic user interface that may present various data through graphical representations such as pixel maps, plots, and the like as disclosed herein.

[0162] Referring to FIG. 19, another flow diagram is presented. Cell data 1910 is transmitted through measurement medium A 1920. If the desired measurement is of forward fluorescence, measurement medium A 1920 may include various air gaps, optics, lenses, etc. The measurement medium may be intended to accurately measure cell data 1910 or to amplify certain features before passing the data to sensor A (e.g., the light-sensing surface of an APD). A second measurement (e.g., side fluorescence) may be passed to sensor B 1935 through measurement medium B 1925. Processing 1940 may be performed on the sensor measurements, which may include analog-to-digital conversion, data analysis as described herein, etc. Such processing may define gating 1950, the location of which may indicate that certain cells should be biased, which may include deactivating some cells (e.g., by a laser). In some embodiments, this deactivating laser may cause interference with a sensor, such as a fluorescent sensor, causing certain cells to exhibit a saturation level of fluorescence. This saturated population and other populations may be further processed by a second process 1960 to optimize gate locations, determine trend data, etc. In some embodiments, the second process 1960 determines all gate locations and the first process 1940 determines if a cell is within a given gate (or not within a gate). The second process 1960 may also interface with various input / output (I / O) devices 1970, such as to a server for offloading processing, or to a graphical user interface (GUI) for presenting various data. In some populations, gate criteria and other system parameters (e.g., parameters of the first process, gating, etc.) may be determined via the I / O device 1970.

[0163] As can be seen above, in various embodiments, the use of a dual detector setup or system provides the ability to distinguish a subset of the Y region in a histogram from an X slice of the histogram. For example, in a two-dimensional histogram of CH2 prominence on the X axis, 1.75 to 1.875 would cover all Y values ​​in the X range, but the use of a second detector provides for identifying and narrowing down the Y values ​​to a subset of all Y values, for example 2.00 to 2.375. Being able to define a subset of all values ​​can provide for automatic definition of a gate for sperm populations that have a Y chromosome.

[0164] In various embodiments, a semen sample, such as a bovine or porcine semen sample, containing a set or a plurality of sperm cells with an X chromosome and a set or a plurality of sperm cells with a Y chromosome, may be processed by a microfluidic system with a dual detector setup or system to generate a sexed or processed semen product. The sexed or processed semen product may have a "purity" or percentage of a desired first type of sperm cells (e.g., X-chromosomal sperm cells or Y-chromosomal sperm cells) relative to a population, set, or plurality of second type sperm cells (e.g., sperm cells containing DNA content other than that desired), with desired purities of the first population being 50-55 percent, 55-60 percent, 60-65 percent, 65-70 percent, 70-75 percent, 75-80 percent, 80-85 percent, 85-90 percent, and 90-95 percent. In various embodiments, the desired purity is at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent.

[0165] In various embodiments, two detection locations or two detector systems can be used to get a clearer understanding of what the X-gate should look like. The X-gate can be optimized, for example, to get a 1%-2% improvement in X-skewness. At the same skewness percentage, further improvements in collection efficiency of X-chromosome cells can be obtained. For example, two "comma" shaped tails in the histogram can be analyzed so that only the "ball" or head of the X-peak is not collected. Also, the tails can be collected at a high skewness percentage to increase the overall collection percentage of X-chromosome cells for higher qualification. This analysis technique can also be applied to other "physical or positional properties" of other particles to increase the collection percentage of a particular property.

[0166] In various embodiments, the detectors are substantially 90 degree orthogonal detectors, one oriented substantially perpendicular to the plane of the chip and one oriented substantially parallel to the plane of the chip (e.g., through or along the side of the chip). The use of two orthogonally oriented detectors can provide a better orientation measurement when compared to a single detector. One or both detectors can be, for example, a photomultiplier tube or an avalanche photodiode.

[0167] In various embodiments, the orthogonal positional relationship between the two detectors can provide the ability to identify both orientation and position information for cells in the flow stream. This is particularly valuable information for asymmetric particles such as sperm cells. The position and orientation information can be used to identify a subset of information from the histogram to automatically determine the bias for sperm cells with a Y chromosome and determine the optimal microfluidic conditions (e.g., flow rate, ramp used, etc.) for asymmetric cell alignment. For example, in one embodiment, the flow rate can be selected to be about 10,000 to about 30,000 cells per second, with any particular cell having a velocity of about 8 to about 15 meters per second. More specifically, a flow rate of about 13,000 to about 20,000 cells per second can be selected, with a velocity of about 10 to about 12 meters per second. Depending on the sensor sensitivity, emission rate, and computing power, additional or fewer cells can be passed per second and can have a higher or lower velocity.

[0168] In various embodiments, additional information that can be captured using two detectors that can be useful is the brightness of any signals captured by each detector, the duration of any signals captured by each detector, and the level or degree of synchronization for the detectors.

[0169] In various embodiments, the optical fibers used through the openings or access ports in the chip may be angled and / or have a curved profile to reduce crosstalk or interference from the kill laser beam. The orthogonal fibers may be fibers with a numerical aperture of a particular value, such as 0.4. A light collection system with an effective numerical aperture of 0.4 could provide sufficient collection of fluorescent information to use an avalanche photodiode in various embodiments.

[0170] In various embodiments, an important aspect in a dual detector system may be filtering of the signal from the kill laser. A curved or curvilinear profile for the orthogonal (side port) optical fiber may provide some or all of the required filtering of the kill laser signal. Software or electronics based solutions may also be used. The signal from the kill laser may be filtered from the detection laser and / or fluorescence signals because the pulse width of the kill laser is very short. The fluorescence intensity of the kill laser is sub-microsecond in duration, and much longer fluorescence pulses are detected from the cells hit by the excitation laser. Digital or analog electronic filters such as low pass filters may be used to filter out the signal from the kill laser to obtain only the fluorescence pulses from the cells.

[0171] In various embodiments, a second (orthogonal) detector may also be used for kill confirmation. Instead of or before filtering the kill signal (by physical or software means), the fluorescent pulse (short micropulse) caused by the kill laser may be processed to determine whether it successfully hit or inactivated the cell based on the intensity, duration, or timing of the signal. The kill laser signal may be filtered, removed, or split from the detection laser signal (fluorescent pulse from the cell) and processed separately to make a kill confirmation determination. Conventional single detector systems use masking or physical filters (e.g., diffraction gratings) to filter the kill laser signal and are not easily adaptable to process both the kill and detection signals as in a dual detector setup.

[0172] In various embodiments, the fiber used is an optical fiber tuned or configured for the wavelength of light to be collected. In various embodiments, the optical fiber in the microfluidic chip can be attached in such a way as to provide for easy, fast, and / or simple replacement or change of the microfluidic chip in the system. This can be an interface in the chip or a fixing means for the fiber that provides for changing the chip without the chip "snakes" or carefully feeding the optical fiber into place every time the chip is changed.

[0173] In various embodiments, an orthogonally positioned dual detector system provides effective discrimination of sperm cells with a Y chromosome from within a sample population. To separate sperm with a Y chromosome from the population, it may be necessary to also separate sperm with an X chromosome, which appear dim or have differences in orientation or position as they move through the cytometer. Using a second detector (e.g., an orthogonally positioned detector using optical fibers) can provide valuable orientation and position information. This can provide differentiation of fluorescent signals due to DNA content from similar signals caused by sequence, dose, lensing, and other factors.

[0174] In various embodiments, a flow cytometry system has two or fewer detectors. Such a configuration may advantageously provide the relatively smallest, most "lightweight," and least expensive system possible in various embodiments. This reduces complexity, operator training time, calibration complexity and time, and cost of goods for any products generated or processed by such a flow cytometry system.

[0175] As used herein, a gating operation may include determining a "gate" or "gating" based on an analysis of data collected by a detector. Gating may involve determining what is inside and what is outside the determined gate. A gating operation may involve determining how a particle is to be processed, or determining the size or parameters of the gate. In various embodiments, a gating operation may be followed by an "action step" or "action operation," which may be, for example, an enrichment step, a selection step, a sorting step, an inactivation step, an ablation step, a kill step, etc. In various embodiments, an action may be performed after 1) determining the size or parameters of the gate, and 2) determining whether a particle is inside or outside the gate.

[0176] Additionally, in various embodiments, a single-detector gate-based system may be optimized using information gathered from a dual-detector based system such that it may perform better relative to a non-optimized system, but may approach the performance levels of a dual-detector system.

[0177] In various embodiments, information from a multi-detector system (e.g., a two-detector system) may be used to perform biasing operations on a one-detector system. For example, in certain embodiments, a two-dimensional histogram is used to identify overlapping "tail" regions that would not be identifiable using a one-dimensional histogram. The two-detector system is used to perform gating, which is used by the one-detector system. Some feedback from the one-detector system may be provided to adjust based on the specific measured behavior of that system. Effectively, data from the two-detector system allows for better discrimination of male and female populations in the overlapping regions. With this knowledge, the two-detector data can be re-evaluated as if the data came from a single-detector system, and the gates can be mathematically optimized to increase distortion.

[0178] For example, in various embodiments, an exemplary process may involve measuring a property and performing gating using a two-detector system, calculating the best gate to use with a one-detector system based on the particular measured property and the operation of the two-detector system, and classifying the cells using the one-detector system with the gate provided by the two-detector system.

[0179] As used herein, the terms "approximately," "about," "substantially," and similar terms are intended to have a broad meaning consistent with common and accepted usage by those of ordinary skill in the art to which the subject matter of the present disclosure pertains. Those of ordinary skill in the art who review this disclosure will understand that these terms are intended to enable the description of certain features described and claimed without limiting the scope of these features to the precise numerical ranges provided. Thus, these terms should be interpreted to indicate that insubstantial or insignificant modifications or variations of the subject matter described and claimed are considered to be within the scope of the present disclosure as set forth in the appended claims.

[0180] It should be noted that the terms "exemplary," "example," and "potential," and variations thereof, used to describe various embodiments herein, are intended to indicate that such embodiments are possible examples, representations, or illustrations of possible embodiments (and such terms are not intended to imply that such embodiments are necessarily particular or best examples).

[0181] As used herein, the term "coupled" and variations thereof refer to the direct or indirect joining of two members to each other. Such joining may be static (e.g., permanent or fixed) or movable (e.g., removable or releasable). Such joining may be achieved with two members directly joined to each other, two members joined to each other using a separate intervening member and any additional intermediate member joined to each other, or two members joined to each other using an intervening member integrally formed as a unitary body with one of the two members. When "coupled" or variations thereof are modified by an additional term (e.g., directly coupled), the inclusive definition of "coupled" provided above is modified by the plain language meaning of the additional term (e.g., "directly coupled" means joining two members without any separate intervening member), resulting in a definition narrower than the inclusive definition of "coupled" provided above. Such joining may be mechanical, electrical, or fluid.

[0182] As used herein, the term "or," when used to connect a list of elements, is used in its inclusive (rather than its exclusive) sense, such that the term "or" refers to one, some, or all of the elements in the list. Unless otherwise noted, conjunctions such as the phrase "at least one of X, Y, and Z" are understood to convey that the elements can be any of X, Y, Z, X and Y, X and Z, Y and Z, or X and Y and Z (i.e., any combination of X, Y, and Z). Thus, such conjunctions are generally not intended to imply that a particular embodiment requires that at least one X, at least one Y, and at least one Z, respectively, be present, unless otherwise indicated.

[0183] References herein to the location of elements (e.g., "top", "bottom", "upper", "lower") are used merely to describe the orientation of the various elements in the figures. It should be noted that the orientation of the various elements may differ according to other exemplary embodiments, and that such variations are intended to be encompassed by the present disclosure.

[0184] The embodiments described herein have been described with reference to the drawings, which illustrate certain details of particular embodiments that implement the systems, methods, and programs described herein, however, describing the embodiments with the drawings should not be construed as imposing any limitations on the disclosure that may be present in the drawings.

[0185] It is important to note that the construction and arrangement of the devices, assemblies, and steps shown in the exemplary embodiment are merely illustrative. Additionally, any element disclosed in one embodiment may be combined or utilized with any other embodiment disclosed herein. Although only one example of an element from one embodiment that may be incorporated or utilized in another embodiment is described above, it should be understood that other elements of the various embodiments may be combined or utilized with any of the other embodiments disclosed herein.

[0186] The foregoing description of the embodiments has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from the present disclosure. The embodiments have been chosen and described to illustrate the principles of the disclosure and its practical application to enable those skilled in the art to utilize the various embodiments with various modifications as appropriate for the particular use envisioned. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of the embodiments without departing from the scope of the disclosure, as expressed in the appended claims.

[0187] In accordance with the entirety of this disclosure, several sample embodiments are disclosed below to represent non-tangible embodiments that one of ordinary skill in the art would understand. Embodiment AA: A method comprising: providing a sample including a particle population having a ratio of a first particle type to a second particle type to a microfluidic chip having a sample channel; emitting illumination light from a light source along a first axis as the sample passes through the channel such that the illumination light coincides with particles in the sample; detecting the first light emitted from the sample along the first axis using a first light detector; detecting the second light emitted from the sample along the second axis using the first light detector or a second light detector; and performing a biasing operation to modify the ratio of the first particle type to the second particle type based on the detected first light emitted along the first axis and the detected second light emitted along the second axis. Embodiment AB: Embodiment AA, wherein the first particle type and the second particle type are viable cells of two genders, and the different particle type is non-activated cells. Embodiment AC: Embodiment AA or AB, wherein the first light and the second light (i) result from the incidence of illumination light on particles in the sample, and (ii) are fluorescent light traveling in a plurality of different directions from fluorescent particles in the sample. Embodiment AD: Any of embodiments AA-AC comprising: generating an intensity map of peak fluorescence associated with each particle type based on light emitted from the sample along a first axis and a second axis; detecting a first population center associated with the first particle type and a second population center associated with the second particle type based on the intensity map; calculating a relative intensity along a first line passing through the first population center; calculating a slope of a second line connecting the first population center and the second population center; locating a first saddle point along a third line connecting the first population associated with the first particle type and the second population associated with the second particle type; locating a second saddle point along a fourth line connecting the first population and the second population; and generating a gate by bounding the first population or the second population, wherein the gate is bounded based on the first saddle point, the second saddle point, and the additional boundary point. Embodiment AE: Any of embodiments AA-AD including calculating a kernel in the pixel map, adding a first instance of the kernel to the pixel map based on light emitted from the sample along a first axis and a second axis, adding a second instance of the kernel to the pixel map based on light emitted from the sample along the first axis and a second axis, deriving a first histogram from the pixel map, removing data from the pixel map, deriving a second histogram from a first row of the pixel map, generating a first trough point from the second histogram, deriving a third histogram from a second row of the pixel map, generating a second trough point from the third histogram, generating a polynomial function based on the first trough point and the second trough point, and bounding a population based on the polynomial function and additional bounding points. Embodiment AF: Any of embodiments AA-AE, wherein the first axis is at an angle to the second axis, the angle being between 45 degrees and 135 degrees. Embodiment AG: Any of embodiments AA-AF, wherein the angle is between 75 degrees and 105 degrees. Embodiment AH: Any of embodiments AA-AG, wherein the angle is between 85 degrees and 95 degrees. Embodiment AI: Any of embodiments AA-AH, wherein the first axis is perpendicular to the second axis. Embodiment AJ: Any of embodiments AA to AI, in which the first axis and the second axis are along the same direction, and a beam splitter or dichroic mirror is used, and a first detector is used to observe the first light and the second light based on the difference in wavelength. Embodiment AK: Any of embodiments AA-AJ, wherein the first light is detected using a first detector positioned at a first position along a first axis, and the second light is detected using a second detector positioned at a second position along a second axis. Embodiment AL: Any of embodiments AA-AK, wherein no more than two detectors are used to detect emission from the sample. Embodiment AM: Any of embodiments AA-AL, in which (i) a first light is detected using a first detector positioned at a first position along a first axis, and (ii) a second light is detected using the first detector via a fiber optic element that transmits light from a second position along the second axis to a first position along the first axis. Embodiment AN: Any of embodiments AA-AM, wherein the first detector comprises a multi-pixel photodetector having a first pixel detection area and a second pixel detection area, and the method further includes determining a physical location characteristic of each particle based on the intensities of the first light and the second light in the first pixel detection area and the second pixel detection area, respectively. Embodiment AO: Any of embodiments AA-AN, wherein the biasing operation includes using a second light source to emit a third light into the sample to change the state of individual particles in the sample. Embodiment AP: Any of embodiments AA-AO, wherein a third light is emitted to activate particles in the sample. Embodiment AQ: Any of embodiments AA-AP, wherein the particles are a first particle type and activating the particles using a third light converts the particles to a second particle type. Embodiment AR: Any of embodiments AA-AQ, wherein a second light is emitted to inactivate particles in the sample. Embodiment AS: Any of embodiments AA-AR, in which an illumination light coincides with the sample at a first location in the path of the sample flowing through the sample channel, and a third light is emitted onto the sample so as to coincide with the sample at a second location in the path of the sample, the second location being downstream of the first location. Embodiment AT: Any of embodiments AA-AS including determining an orientation characteristic of each particle in a set of one or more particles in a sample. Embodiment AU: Any of embodiments AA-AT, wherein the orientation feature is determined based on the detected first light and the detected second light. Embodiment AV: Any of embodiments AA-AU, wherein a biasing operation is performed on each particle based on an orientation characteristic of each particle. Embodiment AW: Any of embodiments AA-AV, wherein the particle population is a cell population, the first particle type is a first cell type, and the second particle type is a second cell type. Embodiment AX: Any of embodiments AA-AW including determining an orientation characteristic of each cell in the sample based on the detected first light and the detected second light. Embodiment AY: Any of embodiments AA-AX, wherein the orientation feature includes a relative orientation of the primary surface of each cell with respect to a second axis. Embodiments AZ: Any of embodiments AA-AY, wherein the primary surface of each cell is the flat side of the cell or the edge side of the cell. Embodiment AAA: Any of embodiments AA-AZ, wherein the first cell type is male sperm cells that have a Y chromosome and the second cell type is female sperm cells that do not have a Y chromosome. Embodiment AAB: Any of embodiments AA-AAA, wherein the ratio corrected by the biasing action includes more male sperm cells relative to female sperm cells. Embodiment AAC: Any of embodiments AA-AAB, wherein performing the biasing operation includes inactivating female sperm cells in the sample. Embodiment AAD: Any of embodiments AA-AAC, wherein the light source is a laser having a first wavelength, and the first light and the second light are fluorescent light having a second wavelength different from the first wavelength. Embodiment AAE: Any of embodiments AA-AAD, wherein the light source is a first light source and performing the biasing operation includes emitting light to the sample using a second light source to change the state of particles of either the first particle type or the second particle type in the sample. Embodiment AAF: Any of embodiments AA-AAE, wherein the biasing action is performed based on fluorescence detected along a first axis in combination with fluorescence detected along a second axis. Embodiment AAG: Any of embodiments AA-AAF, including generating a histogram of intensities along a first axis and a second axis, and identifying regions in the histogram that correspond to a first type of particle or a second type of particle. Embodiment AAH: Any of embodiments AA-AAG, wherein the first light and the second light are fluorescence of particles in the sample and the histogram corresponds to fluorescence intensity along a first axis and a second axis. Embodiment AAI: Any of embodiments AA-AAH, wherein the first light and the second light are fluorescent light, and further comprising: (i) determining an orientation characteristic of each particle in the set particles in the sample based on the detected first light and the detected second light; (ii) generating a histogram of fluorescence intensity in a first axis and a second axis; and (iii) identifying regions in the histogram corresponding to particles having the first orientation characteristic and particles having the second orientation characteristic. Embodiment AAJ: Any of embodiments AA to AAI, wherein the method is implemented on a first microfluidic system in which light is detected in multiple directions, and the method further includes performing a second biasing operation based on the identified region to a second microfluidic system that does not have multi-directional light detection. Embodiment AAK: Any of embodiments AA-AAJ, wherein the microfluidic chip further comprises an access port through which the second light emitted along the second axis is collected. Embodiment AAL: Any of embodiments AA-AAK, wherein the access port of the microfluidic chip comprises a fiber optic element for transmitting the second set of light. Embodiment AAM: Any of embodiments AA-AAL in which the fiber optic element terminates in a beam splitting element configured to separate the fluorescent wavelengths from wavelengths corresponding to the illumination source. Embodiment AAN: Any of embodiments AA-AAM including performing a gating operation based on a detected first light emitted along a first axis and a detected second light emitted along a second axis. Embodiment AAO: Any of embodiments AA-AAN, wherein the gating action affects the hydrodynamic flow of the sample in the microfluidic chip. Embodiment AAP: Any of embodiments AA-AAO, wherein the biasing action requires both the detected first light and the detected second light to modify the ratio of the first particle type to the second particle type. Embodiment AAQ: Any of embodiments AA-AAP, including computing a kernel within the pixel map; adding a predefined number of instances of the kernel to the pixel map based on light emitted from a plurality of samples along a first axis and a second axis; deriving a first histogram from the pixel map along the first axis; removing data from the pixel map based on the first histogram; generating a second histogram from the pixel map along the second axis; determining a region of interest in the pixel map based on the second histogram; generating an additional plurality of histograms along the second axis in the region of interest of the pixel map; determining a plurality of trough points based on the additional plurality of histograms; generating a polynomial function based on the multiple trough points; and bounding a population based on the polynomial function and the additional boundary points. Embodiment BA: A product comprising a particle population having a modified ratio of a first particle type to a second particle type produced according to the method of embodiments AA-AAP. Embodiment CA: A method comprising: providing a sample in a microfluidic chip having a sample channel, the sample comprising a particle population having a first ratio of a first particle type to a second particle type; emitting coherent light using an illumination source along an illumination axis such that the coherent light coincides with the sample as it passes through the channel; detecting a first set of one or more light of one or more wavelengths traveling in a first direction from the sample using a first photodetector at a first location along a first direction from the microfluidic chip; detecting a second set of one or more light of one or more wavelengths traveling in a second direction from the sample using a second photodetector at the first location or at a second location along a second direction from the microfluidic chip; and performing a biasing operation to obtain a modified particle population having a second ratio of the first particle type to the second particle type based on the first set of light and the second set of light. Embodiment CB: Embodiment CA, wherein the first set of lights comprises fluorescence in a first direction from particles in the sample and the second set of lights comprises fluorescence in a second direction from particles in the sample. Embodiment CC: Embodiment CA or CB, wherein illumination light incident on particles in the sample causes fluorescence of the particles in the sample. Embodiment CD: Any of embodiments CA-CC, wherein the biasing action includes a gating action that affects sample flow in the microfluidic chip. Embodiment CE: Any of embodiments CA-CD, wherein the biasing action includes emitting a second coherent light to the sample. Embodiment CF: Any of embodiments CA-CE, wherein the second coherent light is emitted using a second light source. Embodiment CG: Any of embodiments CA-CF, wherein the second coherent light has a wavelength that is lower than the wavelength of the first coherent light. Embodiment CH: Any of embodiments CA-CG, wherein a first coherent light is coincident with the sample at a first location along the path of the sample flow, and a second coherent light is emitted to coincide with the sample at a second location along the path downstream from the first location. Embodiment CI: Any of embodiments CA-CH, wherein the second coherent light activates particles in the sample. Embodiment CJ: Any of embodiments CA-CI, wherein the second coherent light inactivates particles in the sample. Embodiment CK: Any of embodiments CA-CJ, wherein the first photodetector is a first pixel detection area of ​​the detector module and the second detector is a second pixel detection area of ​​the detector module. Embodiment CL: Any of embodiments CA-CK, including determining a physical location characteristic of each particle based on the intensity of the first light and the second light in the first pixel detection area and the second pixel detection area. Embodiment CM: Any of embodiments CA-CL, wherein the particle population is a cell population, the first particle type is a first cell type, and the second particle type is a second cell type. Embodiment CN: Any of embodiments CA-CM comprising determining an orientation characteristic of a plurality of cells in a sample. Embodiment CO: Any of embodiments CA-CN, wherein the orientation characteristic is determined based on the first set of detected light and the second set of detected light. Embodiment CP: Any of embodiments CA-CO, wherein the biasing action is performed (i) for each of the plurality of cells, and (ii) additionally based on the orientation characteristics of each of the plurality of cells. Embodiment CQ: Any of embodiments CA-CP, wherein the first cell type is a male sperm cell having a Y chromosome and the second cell type is a female sperm cell having no Y chromosome, or the first cell type is a female sperm cell having an X chromosome and the second cell type is a male sperm cell having no X chromosome. Embodiment CR: Any of embodiments CA-CQ, wherein the orientation features of the plurality of cells include (i) a first orientation corresponding to a primary surface of the cell facing the illumination access, and (ii) a second orientation corresponding to a secondary surface of the cell oriented facing the illumination axis. Embodiment CS: Any of embodiments CA-CR, wherein the orientation characteristics of the plurality of cells include (i) a first orientation corresponding to cells oriented flat side relative to at least one of the first detector or the second detector, and (ii) a second orientation corresponding to cells oriented edge side relative to at least one of the first detector or the second detector. Embodiment CT: Any of embodiments CA-CS, wherein the orientation characteristics of the plurality of cells further include a third orientation corresponding to cells transitionally oriented between the edge side and the flat side. Embodiment CU: Any of embodiments CA-CT, wherein the second proportion is greater than the first proportion, such that as a result of the biasing operation, the corrected cell population has more male sperm cells relative to female sperm cells. Embodiment CV: Any of embodiments CA-CU, wherein performing the biasing operation includes inactivating one or more cells of the first cell type or the second cell type in the sample. Embodiment CW: Any of embodiments CA-CV, wherein the first set of lights includes at least one of light scattering or fluorescence in a first direction and the second set of lights includes at least one of light scattering or fluorescence in a second direction. Embodiment CX: Any of embodiments CA-CW, wherein the illumination source is a laser having a first wavelength and the fluorescent light has a second wavelength different from the first wavelength. Embodiment CY: Any of embodiments CA-CX, wherein performing the biasing operation includes changing a state of one or more particles of the first particle type or the second particle type in the sample. Embodiment CZ: Any of embodiments CA-CY, wherein the biasing action is performed based on fluorescence detected in a first direction in combination with fluorescence detected in a second direction. Embodiment CAA: Any of embodiments CA-CZ, wherein the biasing action is performed based on (i) fluorescence in a first direction, and (ii) both light scattering and fluorescence in a second direction. Embodiment CAB: Any of embodiments CA-CAA, comprising generating a histogram of fluorescence intensity in a first direction and a second direction, and identifying regions in the histogram that correspond to particles of a first cell type or particles of a second cell type. Embodiment CAC: Any of embodiments CA to CAB, comprising generating a histogram of fluorescence intensity in a first direction and a second direction, and identifying regions in the histogram corresponding to particles having a first orientation feature and particles having a second orientation feature. Embodiment CAD: Any of embodiments CA-CAC, comprising generating a histogram of fluorescence intensity in a first direction and a second direction, and identifying regions in the histogram that correspond to at least one of: (i) particles of a first cell type, (ii) particles of a second cell type, (iii) particles having a first orientation characteristic, or (iv) particles having a second orientation characteristic. Embodiment CAE: Any of embodiments CA-CAD, wherein the method is implemented on a first microfluidic system in which light is detected in multiple directions, and the method further includes performing a second biasing operation based on the identified region on a second microfluidic system that does not have multi-directional light detection. Embodiment CAF: Any of embodiments CA-CAE, wherein the first direction is along the illumination axis and the second direction is along a second axis that is angled relative to the illumination axis. Embodiment CAG: Any of embodiments CA to CAF, wherein the second direction is perpendicular to the illumination axis. Embodiment CAH: Any of embodiments CA-CAG, wherein the second direction is at an angle to the illumination axis, the angle being between 45 degrees and 135 degrees. Embodiment CAI: Any of embodiments CA-CAH, wherein the microfluidic chip further comprises an access port through which the second set of light is collected. Embodiment CAJ: Any of embodiments CA-CAI, wherein the access port of the microfluidic chip comprises a fiber optic element for transmitting the second set of light. Embodiment CAK: Any of embodiments CA-CAJ, in which the fiber optic element terminates in a beam splitting element configured to separate the fluorescent wavelengths from wavelengths corresponding to the illumination source. Embodiment CAL: Any of embodiments CA-CAK, further comprising filtering the third set of light from the second illumination source. Embodiment CAM: Any of embodiments CA-CAL, wherein the filtering further comprises filtering with an optical fiber defined by a length having a curved shape. Embodiment CAN: Any of embodiments CA-CAM including determining inactivation of particles in the sample based on detecting a third set of characteristics of the light. Embodiment CAO: Any of embodiments CA-CAN, comprising modifying at least one of (i) the microfluidic chip, or (ii) the manner in which the sample is provided to the microfluidic chip, to obtain a third ratio of the first particle type to the second particle type based on the modified cell population. Embodiment CAP: Any of embodiments CA-CAO including collecting the sample via the channel following the biasing action. Embodiment DA: A product comprising a modified particle population having a second ratio of the first particle type to the second particle type produced according to embodiments AA-CAO. Embodiment EA: A flow cytometry system comprising: an illumination source configured to emit coherent light having a first wavelength and coinciding with a sample channel of a microfluidic chip; a first photodetector configured to detect light traveling in a first direction from the sample channel; and a second photodetector configured to detect light traveling in a second direction from the sample channel; and a control unit, wherein the control unit is configured to: (i) illuminate a sample passing through the sample channel of the microfluidic chip, the sample including a cell population having a first ratio of a first cell type to a second cell type; (ii) detect the first light using the first photodetector and detect the second light using the second photodetector; and (iii) control one or more biasing mechanisms to perform a biasing operation to modify the cell population to obtain a second ratio of the first cell type to the second cell type based on both the first light detected using the first photodetector and the second light detected using the second photodetector. Embodiment EB: Embodiment EA, in which the microfluidic chip includes a microfluidic chip with an access port having a fiber optic element through which the second light travels to reach the second detector. Embodiment EC: Embodiment EA or EB, wherein the first direction is an illumination axis of the illumination source and the second detector is positioned to detect light at an angle to the illumination axis. Embodiment ED: Any of embodiments EA-EC, wherein the angle is between 45 degrees and 135 degrees. Embodiment EE: Any of embodiments EA-ED, wherein the first direction is an illumination axis of the illumination source and the second detector is positioned to detect light traveling orthogonally to the illumination axis. Embodiment EF: Any of embodiments EA-EE, wherein the one or more biasing mechanisms include a second laser having a higher intensity or lower wavelength relative to the illuminating laser, and the control unit is further configured to use the second laser to inactivate one or more cells of the first cell type or the second cell type in the sample. Embodiment EG: Any of embodiments EA-EF, wherein the one or more biasing mechanisms include an actuator that affects sample flow in the microfluidic chip. Embodiment EH: Any of embodiments EA to EG, wherein the control unit is configured to perform a biasing operation based on forward fluorescence detected using a first detector and side fluorescence detected using a second detector. Embodiment EI: Any of embodiments EA-EH, wherein the control unit is further configured to generate histograms of fluorescence intensity in the first direction and the second direction. Embodiment EJ: Any of embodiments EA-EI, wherein the control unit is further configured to identify regions in the histogram corresponding to at least one of: (i) particles of a first cell type, (ii) particles of a second cell type, (iii) particles having a first orientation characteristic, or (iv) particles having a second orientation characteristic. Embodiment EK: Any of embodiments EA to EJ, wherein the control unit is further configured to control one or more biasing mechanisms to perform a second biasing operation on the second sample using only detected light traveling in the first direction and not in the second direction based on the identified region. Embodiment FA: A microfluidic chip comprising: a sample channel extending from a sample inlet to a sample outlet and configured to receive a sample comprising a cell population; an access port extending from an opening of the microfluidic chip to the sample channel; and a fiber optic element positioned within the access port and configured to transmit wavelengths of light having wavelengths corresponding to at least one of light scattering and fluorescence from a sample passing through the sample channel, wherein the microfluidic chip is configured to illuminate the sample using an illumination laser along an illumination axis and configured to collect light emitted coaxially to the illumination axis, and the fiber optic element is configured to collect light emitted orthogonally from cells illuminated using the illumination laser, and orientation characteristics of cells in the cell population of the sample are derivable from the light collected by the fiber optic element and the light collected coaxially to the illumination axis. Embodiment FB: Embodiment FA comprising a beam splitting element configured to separate fluorescent wavelengths of light from scattered wavelengths of light transmitted through the fiber optic element. Embodiment FC: Embodiment FA or FB, wherein the orientation characteristic of the cells in the cell population of the sample is the relative orientation of the primary surface of the particle with respect to the illumination axis. Embodiment GA: A product comprising: providing a sample in a microfluidic chip having a sample channel, the sample including a particle population having an initial ratio of a first particle type to a second particle type; emitting coherent light using an illumination source along an illumination axis such that the coherent light coincides with the sample as it passes through the channel; detecting a first set of one or more wavelengths of light using a first photodetector at the first location in a first direction from the microfluidic chip; detecting a first set of one or more wavelengths of light using a second photodetector at the second location in a second direction from the microfluidic chip; detecting a second set of one or more wavelengths of light, determining at least one of an orientation or position for each cell in the sample based on the first set of light and the second set of light, and performing a biasing operation to obtain a modified cell population having a modified ratio of the first cell type to the second cell type based on (i) the at least one of the determined orientations or positions and (ii) the first set of light detected and the second set of light detected. Embodiment GB: Embodiment GA, wherein the first cell type is male sperm cells that have a Y chromosome and the second cell type is female sperm cells that do not have a Y chromosome. Embodiment GC: Embodiment GA or GB, wherein as a result of the biasing operation, the corrected ratio is greater than the initial ratio, such that the corrected cell population has more male sperm cells relative to female sperm cells. Embodiment GD: Any of embodiments GA-GC, wherein the illumination source is a laser having a first wavelength and the fluorescent light has a second wavelength different from the first wavelength. Embodiment GE: Any of embodiments GA-GD, wherein performing the biasing operation includes ablating one or more cells of the first cell type or the second cell type in the sample. Embodiment GF: Any of embodiments GA-GE, in which the biasing action is performed based on fluorescence detected in a first direction in combination with fluorescence detected in a second direction. Embodiment GG: Any of embodiments GA-GF, wherein the biasing action is performed based on (i) fluorescence in a first direction, and (ii) both light scattering and fluorescence in a second direction. Embodiment GH: Any of embodiments GA to GG, wherein the method further comprises generating a histogram of fluorescence intensity in the first direction and the second direction, and identifying regions in the histogram that correspond to cells of the first cell type or cells of the second cell type. Embodiment GI: Any of embodiments GA-GH, wherein the first direction is along the illumination axis and the second direction is along a second axis that is angled relative to the illumination axis. Embodiment GJ: Any of embodiments GA to GI, wherein the second direction is perpendicular to the illumination axis. Embodiment GK: Any of embodiments GA-GJ, wherein the second direction is at an angle to the illumination axis, the angle being between 45 degrees and 135 degrees. Embodiment GL: Any of embodiments GA-GK, wherein the microfluidic chip further comprises an access port through which the second set of light is collected. Embodiment GM: Any of embodiments GA-GL, wherein the access port of the microfluidic chip comprises a fiber optic element for transmitting a second set of light. Embodiment GN: Any of embodiments GA-GM in which the fiber optic element terminates in a beam splitting element configured to separate the fluorescent wavelengths from the wavelengths corresponding to the illumination source. Embodiment GO: Any of embodiments GA-GN, wherein the method further includes filtering a third set of light from the second illumination source. Embodiment GP: Any of embodiments GA-GO, wherein the filtering further comprises filtering with an optical fiber defined by a length having a curved shape. Embodiment GQ: Any of embodiments GA-GP, wherein the method further comprises determining inactivation of cells in the sample based on a third set of light. Embodiment GR: Any of embodiments GA-GQ, wherein the method further includes filtering a third set of light from the second illumination source. Embodiment GS: Any of embodiments GA-GR, wherein the filtering further comprises filtering with an optical fiber defined by a length having a curved shape. Embodiment GT: Any of embodiments GA-GS, wherein the method further comprises determining inactivation of cells in the sample based on a third set of light. Embodiment HA: A method comprising: emitting coherent light using an illumination light source such that the coherent light coincides with the sample as the sample passes through a sample channel of a microfluidic chip, the sample including a cell population having a first ratio of a first cell type to a second cell type; detecting a first set of one or more lights of one or more wavelengths at a first location in a first direction from the microfluidic chip using a first light detector at the first location; detecting a second set of one or more lights of one or more wavelengths at a second location in a second direction from the microfluidic chip using a second light detector at the second location; determining an orientation characteristic of a plurality of cells in the sample based on the first set of lights and the second set of lights; and performing a biasing operation to obtain a modified cell population having a second ratio of the first cell type to the second cell type based on at least one of (i) the orientation characteristic of the plurality of cells and (ii) the first set of lights or the second set of lights. Embodiment HB: Embodiment HA, wherein the first cell type is male sperm cells that have a Y chromosome and the second cell type is female sperm cells that do not have a Y chromosome. Embodiment HC: Embodiment HA or HB, wherein the second ratio is greater than the first ratio, such that as a result of the biasing action, the corrected cell population has more male sperm cells relative to female sperm cells. Embodiment HD: Any of embodiments HA-HC, wherein the orientation characteristics of the plurality of cells include (i) a first orientation corresponding to cells oriented with their flat side relative to at least one of the first detector or the second detector, and (ii) a second orientation corresponding to cells oriented with their edge side relative to at least one of the first detector or the second detector. Embodiment HE: Any of embodiments HA-HD, wherein the orientation characteristics of the plurality of cells further include a third orientation corresponding to cells transitionally oriented between the edge side and the flat side. Embodiment HF: Any of embodiments HA-HE, wherein the first set of lights includes at least one of light scattering or fluorescence in a first direction and the second set of lights includes at least one of light scattering or fluorescence in a second direction. Embodiment HG: Any of embodiments HA-HF, wherein the illumination source is a laser having a first wavelength and the fluorescent light has a second wavelength different from the first wavelength. Embodiment HH: Any of embodiments HA-HG, wherein performing the biasing action includes ablating one or more cells of the first cell type or the second cell type in the sample. Embodiment HI: Any of the embodiments HA-HH, wherein the biasing action is performed based on the fluorescence detected in a first direction and the fluorescence detected in a second direction. Embodiment HJ: Any of embodiments HA-HI, further comprising generating a histogram of fluorescence intensity in a first direction and a second direction, and identifying at least one of (i) a first region in the histogram corresponding to cells of a first cell type or cells of a second cell type, or (ii) a second region in the histogram corresponding to cells having a first orientation characteristic and cells having a second orientation characteristic. and calculating a slope of a second line connecting the first population center and the second population center; locating a first saddle point along a third line connecting the first population associated with the first particle type and the second population associated with the second particle type; and locating a second saddle point along a fourth line connecting the first population and the second population; and generating a gate by bounding the first population or the second population, wherein the gate is bounded based on the first saddle point, the second saddle point, and the additional boundary point. Embodiment HI: Any of embodiments HA-HK including calculating a kernel in a pixel map, adding a first instance of the kernel to the pixel map based on light emitted from the sample along a first axis and a second axis, adding a second instance of the kernel to the pixel map based on light emitted from the sample along the first axis and a second axis, deriving a first histogram from the pixel map, removing data from the pixel map, deriving a second histogram from a first row of the pixel map, generating a first trough point from the second histogram, deriving a third histogram from a second row of the pixel map, generating a second trough point from the third histogram, generating a polynomial function based on the first trough point and the second trough point, and bounding a population based on the polynomial function and additional bounding points.

Claims

1. 1. A method comprising: providing a sample to a microfluidic chip having a sample channel, the sample including a particle population having a ratio of a first particle type to a second particle type; emitting illumination light from a light source along a first axis such that the illumination light coincides with particles in the sample as the sample passes through the channel; detecting a first light emitted from the sample along a first axis using a first light detector; detecting a second light emitted from the sample along a second axis using the first photodetector or a second photodetector; and performing a biasing operation to modify the ratio of the first particle type to the second particle type based on the detected first light emitted along the first axis and the detected second light emitted along the second axis.

2. 2. The method of claim 1 , wherein the first light and the second light (i) result from the illumination light being incident on particles in the sample, and (ii) are fluorescent light traveling in a plurality of different directions from fluorescent particles in the sample.

3. generating a peak fluorescence intensity map associated with each particle type based on the light emitted from the sample along the first axis and the second axis; detecting a first population center associated with the first particle type and a second population center associated with the second particle type based on the intensity map; calculating a relative intensity along a first line passing through the first population center; calculating a slope of a second line connecting the first population center and the second population center; locating a first saddle point along a third line connecting a first population associated with the first particle type and a second population associated with a second particle type; locating a second saddle point along a fourth line connecting the first population and the second population; 3. The method of claim 2, further comprising: generating a gate by bounding the first population or the second population, the gate being bounded based on the first saddle point, the second saddle point, and an additional boundary point.

4. Computing a kernel in a pixel map; adding a first instance of the kernel to the pixel map based on the light emitted from the sample along the first axis and the second axis; adding a second instance of the kernel to the pixel map based on the light emitted from the sample along the first axis and the second axis; deriving a first histogram from the pixel map; removing data from the pixel map; deriving a second histogram from a first row of the pixel map; generating a first trough point from the second histogram; and deriving a third histogram from a second row of the pixel map; and generating a second trough point from the third histogram; and generating a polynomial function based on the first trough point and the second trough point; The method of claim 2 , further comprising: bounding a population based on the polynomial function and additional boundary points.

5. Computing a kernel in a pixel map; adding a predefined number of instances of the kernel to the pixel map based on the light emitted from a plurality of samples along the first axis and the second axis; deriving a first histogram from the pixel map along the first axis; removing data from the pixel map based on the first histogram; generating a second histogram from the pixel map along the second axis; determining a region of interest within the pixel map based on the second histogram; and generating additional histograms along the second axis of the region of interest of the pixel map; and determining a plurality of trough points based on the additional plurality of histograms; and generating a polynomial function based on the plurality of trough points; The method of claim 2 , further comprising: bounding a population based on the polynomial function and additional boundary points.

6. The method of claim 1 , wherein the first axis is orthogonal to the second axis.

7. The method of claim 1 , wherein no more than two detectors are used to detect emission from the sample.

8. The method of claim 1 , wherein the biasing action includes emitting a third light using a second light source into the sample to change the state of individual particles in the sample.

9. The method of claim 8 , wherein the second light is emitted to inactivate particles in the sample.

10. 10. The method of claim 9, wherein the particles in the particle population are cells and inactivating the cells comprises ablating the cells.

11. 10. The method of claim 9, wherein the illumination light coincides with the sample at a first location in the path of the sample flowing through the sample channel, and the third light is emitted onto the sample to coincide with the sample at a second location in the path of the sample, the second location being downstream of the first location.

12. The method of claim 1 , further comprising determining an orientation characteristic of each particle in a set of one or more particles in the sample.

13. The method of claim 12 , wherein the orientation feature is determined based on the detected first light and the detected second light.

14. 2. The method of claim 1, wherein the particle population is a cell population, the first particle type is a first cell type, and the second particle type is a second cell type.

15. 15. The method of claim 14, wherein the first cell type is male sperm cells that have a Y chromosome and the second cell type is female sperm cells that do not have the Y chromosome.

16. 16. The method of claim 15, wherein the ratio modified by the biasing action includes more male sperm cells relative to female sperm cells.

17. The method of claim 1 , wherein the biasing action is performed based on fluorescence detected along the first axis in combination with fluorescence detected along the second axis.

18. 2. The method of claim 1, further comprising generating a histogram of intensities along the first axis and the second axis, and identifying regions in the histogram that correspond to the first type of particles or the second type of particles.

19. 20. The method of claim 18, wherein the first light and the second light are fluorescence of particles in the sample, and the histogram corresponds to fluorescence intensity along the first axis and the second axis.

20. 10. The method of claim 1, wherein the microfluidic chip further comprises an access port through which the second light emitted along the second axis is collected.

21. 21. The method of claim 20, wherein the access ports of the microfluidic chip comprise fiber optic elements for transmitting a second set of light.

22. 10. The method of claim 1, further comprising performing a gating operation based on the detected first light emitted along the first axis and the detected second light emitted along the second axis.

23. 1. A method comprising: providing a sample in a microfluidic chip having a sample channel, the sample comprising a particle population having a first ratio of a first particle type to a second particle type; using an illumination source to emit coherent light along an illumination axis such that the coherent light is coincident with the sample as it passes through the channel; detecting a first set of one or more wavelengths of light traveling in a first direction from the sample using a first photodetector at a first location along the first direction from the microfluidic chip; detecting a second set of one or more wavelengths of light traveling in the second direction from the sample using a second photodetector at the first location or at a second location along a second direction from the microfluidic chip; performing a biasing operation based on the first set of light and the second set of light to obtain a modified particle population having a second ratio of the first particle type to the second particle type.

24. generating a peak fluorescence intensity map associated with each particle type based on the light emitted from the sample along the first axis and the second axis; detecting a first population center associated with the first particle type and a second population center associated with the second particle type based on the intensity map; calculating a relative intensity along a first line passing through said first population center; calculating a slope of a second line connecting the first population center and the second population center; locating a first saddle point along a third line connecting a first population associated with the first particle type and a second population associated with a second particle type; locating a second saddle point along a fourth line connecting the first population and the second population; 24. The method of claim 23, further comprising generating a gate by bounding the first population or the second population, the gate being bounded based on the first saddle point, the second saddle point, and an additional boundary point.

25. Computing a kernel in a pixel map; adding a first instance of the kernel to the pixel map based on the light emitted from the sample along the first axis and the second axis; adding a second instance of the kernel to the pixel map based on the light emitted from the sample along the first axis and the second axis; deriving a first histogram from the pixel map; removing data from the pixel map; deriving a second histogram from a first row of the pixel map; generating a first trough point from the second histogram; and deriving a third histogram from a second row of the pixel map; and generating a second trough point from the third histogram; and generating a polynomial function based on the first trough point and the second trough point; 24. The method of claim 23, further comprising: bounding a population based on the polynomial function and additional boundary points.

26. 24. The method of claim 23, wherein the first cell type is a male sperm cell having a Y chromosome and the second cell type is a female sperm cell not having the Y chromosome, or the first cell type is a female sperm cell having an X chromosome and the second cell type is a male sperm cell not having the X chromosome.

27. 24. The method of claim 23, wherein the second proportion is greater than the first proportion such that, as a result of the biasing action, the modified cell population has more male sperm cells relative to female sperm cells.

28. 24. The method of claim 23, wherein the first set of lights comprises at least one of light scattering or fluorescence in the first direction and the second set of lights comprises at least one of light scattering or fluorescence in the second direction.

29. 24. The method of claim 23, further comprising generating a histogram of fluorescence intensity in the first direction and the second direction, and identifying regions in the histogram that correspond to at least one of: (i) particles of the first cell type, (ii) particles of the second cell type, (iii) particles having a first orientation characteristic, or (iv) particles having a second orientation characteristic.

30. 24. The method of claim 23, wherein the first direction is along the illumination axis and the second direction is along a second axis that is angled relative to the illumination axis.

31. 1. A flow cytometry system comprising: an illumination source configured to emit coherent light having a first wavelength and coincident with a sample channel of the microfluidic chip; a first photodetector configured to detect light traveling in a first direction from the sample channel; a second photodetector configured to detect light traveling in a second direction from the sample channel; and A control unit, the control unit comprising: (i) illuminating a sample passing through the sample channel of the microfluidic chip, the sample comprising a cell population having a first ratio of a first cell type to a second cell type; (ii) detecting a first light using the first photodetector and detecting a second light using the second photodetector; and (iii) controlling one or more biasing mechanisms to perform a biasing action to modify the cell population to obtain a second ratio of the first cell type to the second cell type based on both the first light detected using the first photodetector and the second light detected using the second photodetector.

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