Particle shape discrimination in flow cytometry

Abstract

A system for analyzing particles includes a flow chamber that streams the particles through an interrogation zone. A light emitting unit generates an excitation light beam directed toward the interrogation zone causing forward scattered light from the particles passing through the interrogation zone. A collection unit collects the forward scattered light, and includes a beam splitter conveying a first portion of the forward scattered light in a first direction and a second portion of the forward scattered light in a second direction, a first detector detecting the first portion of the forward scattered light, and a second detector detecting the second portion of the forward scattered light. The system generates waveforms from the second portion of the forward scattered light, determines one or more parameters from the waveforms, and discriminates the particles based on at least one of shape and morphology using the one or more parameters.

Description

PARTICLE SHAPE DISCRIMINATION IN FLOW CYTOMETRYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is being filed on July 26, 2024, as a PCT international patent application and claims the benefit of and priority to U.S. Provisional Application No. 63 / 579,071, filed August 28, 2023, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] In flow cytometry, particles are arranged in a sample stream, and typically pass one-by-one through one or more excitation light beams with which the particles interact. Light scattered or emitted by the particles upon interaction with the one or more excitation beams is collected and analyzed to characterize and differentiate the particles. In a sorting flow cytometer, particles may be extracted out of the sample stream after having been characterized by their interaction with the one or more excitation beams, and thereby sorted into different groups.

[0003] The light scattered by the particles is typically measured by two optical detectors: a forward scatter (FSC) detector that detects forw ard light scatter which is parallel with the excitation light beam, and a side scatter (SSC) detector which detects side light scatter which is orthogonal to the excitation light beam. The intensity' of the light scatter detected by the FSC detector is proportional to the diameters of the particles such that the forward light scatter can be used to discriminate particles by size. The side light scatter detected by the SSC detector can provide information about the shape, morphology, and internal complexities of the particles.

[0004] Relative to forward light scatter signals, side light scatter signals are weak. Also, conventional flow cytometers are ty pically not well-suited for detecting spatial features for discriminating the shape and morphology of particles due to collecting the side light scatter over a wide collection angle, which reduces the resolution of the side light scatter signals.SUMMARY

[0005] In general terms, the present disclosure relates to analyzing particles using flow cytometry’. In one possible configuration, forward scattered light from the particles is analyzed to discriminate the particles based on at least one of shape and morphology’.Various aspects are described in this disclosure, which include, but are not limited to, the following aspects.

[0006] One aspect relates to a system for analyzing particles, the detection system comprising: a flow chamber configured to stream the particles through an interrogation zone; a light emitting unit configured to generate an excitation light beam, the excitation light beam being directed toward the interrogation zone, the excitation light beam causing forward scattered light from the particles passing through the interrogation zone; a collection unit configured to collect the forward scattered light, the collection unit including: a beam splitter conveying a first portion of the forward scattered light in a first direction and a second portion of the forward scattered light in a second direction; a first detector detecting the first portion of the forward scattered light; and a second detector detecting the second portion of the forward scattered light; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: generate waveforms from the second portion of the forward scattered light; determine one or more parameters from the waveforms; and discriminate the particles based on at least one of shape and morphology using the one or more parameters determined from the waveforms.

[0007] Another aspect relates to a unit for analyzing scattered light from particles, the unit comprising: a beam splitter conveying first and second portions of forward scattered light; a first detector detecting the first portion of forward scattered light; a second detector detecting the second portion of the forward scattered light; and a processing circuitry' having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: generate waveforms from the second portion of the forward scattered light; determine one or more parameters from the waveforms; and discriminate the particles based on at least one of shape and morphology using the one or more parameters.

[0008] Another aspect relates to a method of analyzing particles, the method comprising: emitting an excitation light beam toward an interrogation zone; streaming the particles through the interrogation zone such that interactions of the particles with the excitation light beam cause forward scattered light; conveying a first portion of the forward scattered light to a first detector; conveying a second portion of the forward scattered light to a second detector; generating waveforms from the second portion ofthe forward scattered light; determining one or more parameters from the waveforms; and discriminating the particles based on at least one of shape and morphology using the one or more parameters determined from the waveforms.

[0009] A variety of additional aspects will be set forth in the description that follows. The aspects can relate to individual features and to combination of features. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.DESCRIPTION OF THE FIGURES

[0010] The following drawing figures, which form a part of this application, are illustrative of the described technology and are not meant to limit the scope of the disclosure in any manner.

[0011] FIG. 1 schematically illustrates an example of a detection system for detecting and analyzing particles.

[0012] FIG. 2 illustrates examples of scattering profiles of particles of different sizes being illuminated by an excitation light beam inside an interrogation zone of the detection system of FIG. 1.

[0013] FIG. 3 schematically illustrates an example of a forward collection unit of the detection system of FIG. 1.

[0014] FIG. 4 shows an example arrangement of the detection system of FIG. 1.

[0015] FIG. 5 schematically illustrates an example of a method that can be performed by the detection system of FIG. 1 for discriminating particles based on shape and morphology.

[0016] FIG. 6 schematically illustrates an example of an operation in the method of FIG. 5 that can be performed by the detection system of FIG. 1 .

[0017] FIG. 7 illustrates an example of a first waveform generated from side scattered light collected from a particle passing through an interrogation zone of the detection system of FIG. 1.

[0018] FIG. 8 illustrates an example of a second waveform generated from the side scattered light collected from another particle passing through the interrogation zone of the detection system of FIG. 1.

[0019] FIG. 9 illustrates a first plot of heights determined from waveforms of particles having a first shape and particles having a second shape passing through the interrogation zone of the detection system of FIG. 1.

[0020] FIG. 10 is a histogram plot showing an overlap of the waveform heights of the particles having the first and second shapes shown in FIG. 9.

[0021] FIG. 11 illustrates a second plot of areas determined from the waveforms of the particles having the first shape and the particles having the second shape passing through the interrogation zone of the detection system of FIG. 1.

[0022] FIG. 12 is a histogram plot showing an overlap of the waveform areas of the particles having the first and second shapes shown in FIG. 11.

[0023] FIG. 13 illustrates an example of a third waveform generated from forward scattered light collected over a reduced collection angle from a particle passing through the interrogation zone of the detection system of FIG. 1.

[0024] FIG. 14 illustrates an example of a fourth waveform generated from the forward scattered light collected over a reduced collection angle from another particle passing through the interrogation zone of the detection system of FIG. 1.

[0025] FIG. 15 illustrates a third plot of widths determined from w aveforms of particles having the first shape and particles having the second shape passing through the interrogation zone of the detection system of FIG. 1.

[0026] FIG. 16 is a histogram plot showing an overlap of the waveform widths of the particles having the first and second shapes shown in FIG. 15.

[0027] FIG. 17 illustrates a fourth plot of phases determined from the waveforms of the particles having the first shape and the particles having the second shape passing through the interrogation zone of the detection system of FIG. 1.

[0028] FIG. 18 is a histogram plot showing an overlap of the waveform phases of the particles having the first and second shapes shown in FIG. 17.

[0029] FIG. 19 schematically illustrates an example of a computing system for implementing aspects of the detection system of FIG. 1.

[0030] FIG. 20 schematically illustrates an example of a method of discriminating particle shape that can utilize features of the forward collection unit of the detection system of FIG. 1.

[0031] FIG. 21 illustrates examples of waveforms generated in an operation of the method of FIG. 20 based on forward scattered light collected over a reduced collection angle.

[0032] FIG. 22 illustrates an example of a fitting of raw data to the Morlet wavelet in an operation of the method of FIG. 20.

[0033] FIG. 23 illustrates an example of raw data and an envelope of the Morlet wavelet determined in an operation of the method of FIG. 20.

[0034] FIG. 24 illustrates a comparison of results for discriminating particles based on shape by traditional side scattering techniques versus the method of FIG. 20.

[0035] FIG. 25 illustrates another comparison of results for discriminating particles based on shape by traditional side scattering techniques versus the method of FIG. 20.DETAILED DESCRIPTION

[0036] Various embodiments will be described in detail with reference to the drawings, wherein like reference numerals represent like parts and assemblies throughout the several views. Reference to various embodiments does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments for the appended claims.

[0037] An example detection system is described herein for use in a flow cytometry analyzer. It should be understood that the present disclosure is not limited to the illustrated detection system, but may be applied to a flow cytometry analyzer with other structure or other types of detection systems. In particular, the present disclosure can be applied to various types of sample processing instruments for detecting, sorting, or otherwise processing particles.

[0038] FIG. 1 schematically illustrates an example of a detection system 100 for detecting and analyzing particles. In some examples, the detection system 100 can be incorporated into a flow cytometer such as a sorting flow cytometer.

[0039] As shown in FIG. 1, the detection system 100 includes a light emitting unit 110. The light emitting unit 110 emits one or more excitation light beams 12 for projection onto particles flowing inside a flow chamber 15 through an interrogation zone 18.

[0040] In the example shown in FIG. 1, the light emitting unit 110 includes multiple light sources, such as light sources I l la. 111b, 111c, and 11 Id. As anillustrative examples, the light sources 111 a- 111 d can include lasers. The light sources 11 la-11 Id are each configured to emit excitation light beams 12 with different wavelengths, for example, 405 nm, 488 nm, 561 nm, and 638 nm. In the example shown in FIG. 1, the light sources 11 la-11 Id are arranged in parallel. It should be understood that the number, the type, and the arrangement of the light sources are not limited to the example shown and described herein, and may be changed as needed. For example, the system may include three, five, six, or any other suitable number of light sources.

[0041] The light emitting unit 110 further includes a focal lens 119. The focal lens 119 is configured to focus the one or more excitation light beams 12 for high scatter intensity detection of particles. For example, the one or more excitation light beams 12 emitted by the light sources 11 la-11 Id pass through the focal lens 119, which focuses the one or more excitation light beams 12 to the interrogation zone 18 of the flow chamber 15. The interrogation zone 18 may also be referred to as a focus point where the focused excitation light beams meet the core sample stream of particles that stream through the flow chamber 15 in the detection system 100.

[0042] Dichroic mirrors 117a, 117b, 117c, and 117d are arranged between the focal lens 119 and the respective light sources 11 la-11 Id. Each of the dichroic mirrors 117a- 117d is configured to reflect a light beam of a corresponding one of the light sources 11 la- 11 Id and transmit the light beams of the other light sources. The dichroic mirrors 1 17a-l 17d are selected and configured according to the wavelengths of the light beams emitted by the respective light sources 11 la-11 Id. For example, the dichroic mirror 117a reflects light of the wavelength emitted by the light source 11 la toward the focal lens 119. the dichroic mirror 117b reflects light of the wavelength emitted by the light source 111b toward the focal lens 119 and transmits light of the wavelength emitted by the light source I l la, the dichroic mirror 117c reflects light of the wavelength emitted by the light source 111c toward the focal lens 119 and transmits light of the wavelengths emitted by the light sources I l la and 111b, and the dichroic mirror 117d reflects light of the wavelength emitted by the light source 11 Id toward the focal lens 119 and transmits light of the wavelengths emitted by the light sources I l la, 1 11b, and 111c.

[0043] The light beams emitted by the light sources 11 la-11 Id are reflected by or transmitted through the dichroic mirrors 117a-l 17d to form collinear beams. Thecollinear beams share an optical axis, and provide a confocal point of multiple light sources by focusing on the same interrogation point. The dichroic mirrors 117a-l 17d are adjustable in their positions or orientations, such that they can be used to adjust the position of the focus point of the light beams, especially, the position on a plane perpendicular to the optical axis.

[0044] Lenses 115a-l 15d are arranged between the respective light sources 111a-11 Id and the respective dichroic mirrors 117a- 117d. In some examples, the lenses 115a- 115d are long-focus lens. In some examples, the lenses 115a-l L5d are spherical lenses. In other examples, the lenses 115a-l 15d are aspheric lenses. Each of the lenses 115a-l 15d can convert light beams into parallel beams. In the example shown in FIG.1, each of the lenses 115a-115d is in the form of planoconvex lens with a flat surface and a convex surface opposite to each other.

[0045] The lenses 115a-l 15d are adjustable in their positions or orientations, so as to adjust the position of the focus point of the light beams, especially, the position on the plane perpendicular to the optical axis. Generally, the dichroic mirrors 117a-117d can be used to roughly adjust the position of the focus point of the light beams, whereas the lenses 115a-l 15d can be used to finely adjust the position of the focus point of the light beams.

[0046] It should be understood that the number, the type, and the arrangement of the dichroic mirrors 117a- 117d and the lenses 115a-115d may be changed as needed, and are not limited to the example illustrated herein. Also, the dichroic mirrors 1 17a- 117d and the lenses 115a-l 15d can be replaced with other optical elements or optical modules with similar functions.

[0047] Beam expanders 113a-l 13d are arranged between the respective light sources 11 la-1 l id and the respective lenses 115a- 115d. Each of the beam expanders 113a- 113d can change a sectional dimension and a divergence angle of a light beam. As such, each of the beam expanders 113a- 113d are configurable according to a desired size of a spot of a light beam.

[0048] The light beams irradiated on the particles by the focal lens 119 have a spot size that allows for more concentrated light beams with a higher power density. This can increase intensity of the light beams irradiated on the particles, and ultimately the intensity of the optical signals collected from the particles. This can improve theefficiency of collecting the optical signals, and thereby provide higher resolution and higher sensitivity for nanoparticle detection.

[0049] In the example shown in FIG. 1 , the light sources 111 a- 111 d are in the form of lasers that include respective laser diodes 112a-l 12d. As further show n in the example of FIG. 1, half-wave plates 116a-l 16d are provided betw een the dichroic mirrors 117a-117d and the lenses 115a- 115d, respectively. The spot of the light beam can be reduced by orientation of the light sources 11 la-11 Id and by use of the halfwave plates 116a- 116d.

[0050] As further shown in FIG. 1, cylindrical lenses 114a-l 14d are provided between the respective beam expanders 113a-l 13d and the respective lenses 115a- 115d. The horizontal size of the spot of the light beam focused in the flow chamber 15 can be adjusted by replacing the cylindrical lenses 114a-l 14d with replacement cylindrical lenses having different curvatures. The pow er of some or all of the light sources 11 la-11 Id can also be increased. The increased power of the light sources 11 la-11 Id can also improve detection sensitivity.

[0051] Each of the beam expanders 113a- 113d is formed of a first optical part and a second optical part. In the example shown in FIG. 1, each of the beam expanders 1 13 a- 113d includes a concave lens adjacent to the corresponding light source as the first optical part, and further includes a convex lens aw ay from the corresponding light source as the second optical part. It should be understood that each of the beam expanders 113a- 1 13d is not limited to the example shown in FIG. 1 . The beam expanders 113a- 113d may be formed of any suitable optical lens or lens group. For example, each of the first optical part and the second optical part can be selected from one of a convex lens, a convex lens group, a concave lens, and a concave lens group.

[0052] For each of the beam expanders 113a- 113d, the distance between the first optical part (e.g., the concave lens) and the second optical part (e.g., the convex lens) is adjustable. This allows for adjustment of a w aist position (the focus point) of the light beam on the optical axis.

[0053] As described above, by adjusting the dichroic mirrors 117a-l 17d. the lenses 115a-l 15d, and the beam expanders 1 13a-l 13d, the individual light beams can be focused at the desired interrogation point, and multiple light beams can be focused at the same interrogation point. It should be understood that the position of the focus point of the light beams may be adjusted by adopting any other optical element or in anyother adjustment manner. One or more adjustments to the dichroic mirrors 117a-l 17d, the lenses 115a-l 15d, and the beam expanders 113a-l 13d may be made manually, or may be made electronically using a computing device (e.g., a controller) that is associated with one or more actuators coupled to these components.

[0054] The detection system 100 further includes a light collection unit 120 that detects and / or analyzes particles that flow through the interrogation zone 18. The light collection unit 120 collects light scattered or emitted from the particles that flow through the interrogation zone 18 in the flow chamber 15 for analysis by a computing system 1900 (see FIG. 19).

[0055] The light collection unit 120 includes a side collection unit 130 and a forward collection unit 150. The side collection unit 130 collects side scattered light and fluorescent light from the particles as they are irradiated by the one or more excitation light beams 12 while streaming through the interrogation zone 18. The optical axis of light beams collected from the particles by the side collection unit 130 is approximately perpendicular to, or about 90 degrees, from the optical axis of the light beams emitted from the light sources 11 la-11 Id and directed by the dichroic mirrors 117a-l 17d toward the flow chamber 15. The detection system 100 analyzes the side scattered light to discriminate one or more shapes, morphologies, internal complexities, granularities, or compositions of the particles passing through the interrogation zone 18.

[0056] The forward collection unit 150 collects forward scattered light from the particles. The optical axis of light beams collected from the particles by the forward collection unit 150 is approximately parallel to, or about 0 degrees from, the optical axis of the light beams directed toward the flow chamber 15. The detection system 100 analyzes the forward scattered light to discriminate one or more sizes of the particles passing through the interrogation zone 18. As further shown in FIG. 1, the forward collection unit 150 includes a first detector 159 and a second detector 160, which are described in more detail below .

[0057] The side collection unit 130 includes an optical focusing lens group including a concave mirror 134 and an aspheric lens 135, a collection fiber 136, a beam splitter 133, a first wavelength division multiplexer 131, and a second wavelength division multiplexer 132. The concave mirror 134 reflects the scattered light and the fluorescent light that diverge in various directions at the interrogation point. Theconcave mirror 134 and the aspheric lens 135 focus the reflected light onto the collection fiber 136, for example, by focusing on the same point of the collection fiber 136 as shown in the dotted block 139 in FIG. 1. The concave mirror 134 can focus the reflected light on the fiber, while the aspheric lens 135 can make the focal point smaller (i.e., reduce the aberration). To prevent crosstalk, a beam splitter 133 is arranged to separate the scattered light with high intensity from the fluorescent light with low intensity. The separated scattered light and fluorescent light respectively enter the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 through first and second fibers 137, 138, respectively. Optical signals with different wavelengths are separated in the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 for analysis. In alternative examples, the optical focusing lens group may adopt other optical elements.

[0058] The beam splitter 133 includes a dichroic mirror 332 and a notch filter 334. Collected light is directed into the beam splitter toward the dichroic mirror 332 by the collection fiber 136, which may be oriented such that the light beam is directed toward the dichroic mirror 332 at an incident angle of, for example, 45 degrees. The dichroic mirror 332 reflects the side scattered light coming out of the collection fiber 136 such that the side scattered light enters the first wavelength division multiplexer 131 through the first fiber 137.

[0059] The fluorescent light coming out of the collection fiber 136 passes through dichroic mirror 332, and is incident to the notch filter 334 at an incident angle of about 90 degrees and then passes through the notch filter 334. The fluorescent light enters the second wavelength division multiplexer 132 through the second fiber 138. The dichroic mirror 332 and the notch filter 334 can each have multiple bands according to the confocal design of the light sources 11 la-11 Id. In this case, the dichroic mirror 332 and the notch filter 334 both have four bands that block four laser wavelengths. The number of bands of the dichroic mirror 332 and the notch filter 334 can correspond to the number of the light sources 11 la-11 Id.

[0060] The beam splitter 133 separates the side scattered light with high intensity from the fluorescent light with low intensity, reducing or preventing crosstalk of the side scattered light to the fluorescent light. In addition, by providing the beam splitter, it is possible to separate and transmit multiple light beams into two or more wavelength division multiplexers. The optical elements included in the beam splitter 133 and theirconfiguration may be changed, and are not limited to the example shown and described herein.

[0061] In some examples, the first wavelength division multiplexer 131 receives the side scattered light beams from the beam splitter 133 via the first fiber 137 and divides optical signals of the side scattered light with different wavelengths from each other. In the first wavelength division multiplexer 131, each optical signal is transmitted along an optical transmission path 310 corresponding to an optical channel of the optical signal.

[0062] The first wav elength division multiplexer 131 includes a first filter 311 and a second filter 312 for each optical channel. The first filter 311 and the second filter 312 are arranged at a certain distance from each other along the optical transmission path of the optical channel in a non-parallel manner. Crosstalk between side scattered lights can be reduced or prevented by providing the two filters. The first and second filters 311, 312 are not arranged in parallel so as to avoid multiple reflections of light between them and achieve a better optical density. Thereafter, the filtered light enters a light detection element 315 (e.g., a photodiode, an avalanche photodiode (APD), a photomultiplier tube) for further processing the light.

[0063] The second w avelength division multiplexer 132 receives a fluorescent beam from the beam splitter 133 via the second fiber 138, and divides the optical signals of the fluorescent beam having different wavelengths from each other. In the second wavelength division multiplexer 132, each optical signal is transmitted along an optical transmission path 320 corresponding to an optical channel of the optical signal. Since the fluorescent signal is weak, the second wav elength division multiplexer 132 includes a filter 321 for each optical channel. Thereafter, the filtered fluorescent light enters a light detection element 325 (e.g., a photodiode, an avalanche photodiode (APD), a photomultiplier tube) for further processing.

[0064] Alternative suitable configurations for the wavelength division multiplexers may be used. For example, the first and second wavelength division multiplexers 131, 132 can include notch filters corresponding to the respective fluorescence channels. The notch filters can reduce or eliminate the crosstalk of the side scattered light to the fluorescence light. In this case, the beam splitter 133 may only include the dichroic mirror 332 with no notch filter 334.

[0065] In the side collection unit 130, a diameter of the collection fiber 136 may be different from diameters of the first fiber 137 and the second fiber 138 according to the light transmission efficiency. Lenses in the beam splitter may cause aberration, and thus the output light spots may be larger than input of the beam splitter, and the fiber diameters may be selected accordingly.

[0066] The forward collection unit 150 includes an obscuration bar 155, a concave mirror 151, a filter 157, and a first detector 159. The obscuration bar 155 can be used to block a portion of the light transmitted through the flow chamber 15 to reduce background noise created by the one or more excitation light beams 12 transmitting directly through the flow chamber 15, and to allow collection of only forward scattered light from the particles.

[0067] The concave mirror 151 reflects a forward scattered beam emitted from the particles. The filter 157 allows forward scattered light with a high signal-to-noise ratio to pass, and block other light. The first detector 159 receives the filtered forw ard scattered light from the filter 157, and processes and analyzes the forward scattered light.

[0068] In the forw ard collection unit 150, the first detector 159 can be used to collect the forward scattered light for analysis by the computing system 1900 to determine sizes of the particles passing through the interrogation zone 18. Additionally, the second detector 160 can be used to collect the forward scattered light for analysis by the computing system 1900 to determine shapes and morphologies of the particles passing through the interrogation zone 18.

[0069] FIG. 2 illustrates examples of scattering profiles from particles 200 illuminated by an excitation light beam 12 inside the interrogation zone 18 of the detection system 100. The particles 200 include a first particle 202, which is an extracellular vesicle (EV) particle having a diameter of about 50 nanometers (nm), and a cross-sectional scattering profile that includes straight edges or sides resembling an octagon. The particles 200 further include a second particle 204, which is an EV particle having a diameter of about 100 nm. and a cross-sectional scattering profile that includes a rounded exterior resembling a circle or oval. The particles 200 further include a third particle 206, which is an EV particle having a diameter of about 250 nm, and a cross-sectional scattering profile that includes a rounded exterior portion 209, and a bulging portion 211 such that the scattering profile is asymmetrical. The particles 200further include a fourth particle 208. which is an EV particle having a diameter of about 1,000 nm, and a cross-sectional scattering profile that includes a plurality of gaps 213 between extensions 215 around the exterior of the scattering profile. The scattering profiles shown in FIG. 2 are provided by way of illustrative example, and it is contemplated that additional types of particles having additional diameter sizes and / or scattering profiles may be illuminated by the excitation light beam 12 inside the interrogation zone 18 for analysis by the detection system 100.

[0070] As shown in FIG. 2, the excitation light beam 12 causes forward scattered (FSC) light and side scattered (SSC) light from the particles 200 passing through the interrogation zone 18. The FSC light is scattered along a first direction such as in a direction parallel to the excitation light beam. The forward collection unit 150 collects the FSC light over a first collection angle al. As an illustrative example, the first collection angle al is about 20 degrees. The FSC light intensity is proportional to the diameters of the particles 200, and can be used by the detection system 100 for discrimination of the particles by size.

[0071] The SSC light is scattered in a second direction such as a direction orthogonal to the excitation light beam. The SSC light provides information about the internal and external complexity (e.g., shapes, morphologies, granularities, and compositions) of the particles 200. The side collection unit 130 collects the SSC light over a second collection angle a2. As shown in FIG. 2, the second collection angle a2 is larger than the first collection angle al . As an illustrative example, the second collection angle a2 is about 120 degrees.

[0072] The second collection angle a2 influences shape discrimination sensitivity' of the particles 200 due to the non-uniform nature of the SSC light. A large collection angle reduces the resolution of the shape discrimination due to lost information relevant to the peaks and troughs in the SSC light collected from the particles 200. For example, the second collection angle a2 reduces the resolution of the shape discrimination of the third particle 206 because information such as the bulging portion 211 is lost in the SSC light collected by the side collection unit 130. Additionally, the second collection angle a2 reduces the resolution of the shape discrimination of the fourth particle 208 because at least some of the information about the gaps 213 between extensions 215 is lost in the SSC light collected by the side collection unit 130.

[0073] FIG. 3 schematically illustrates an example of the forward collection unit 150 of the detection system 100. The forward collection unit 150 collects the FSC light for processing and analysis by the computing system 1900 (see FIG. 19). In this example, the FSC light collected by the forward collection unit 150 is processed and analyzed for shape and / or morphology discrimination of the particles 200 (see FIG. 2) passing through the interrogation zone 18 of the detection system 100. The forward collection unit 150 increases a resolution of the shape and / or morphologydiscrimination of the particles 200 passing through the interrogation zone 18 by decreasing the collection angle a used for collecting the scattered light from the particles.

[0074] As shown in FIG. 3, the forward collection unit 150 includes a beam splitter 170 that conveys a first portion FSC1 of the forward scattered light in a first direction DI and a second portion FSC2 of the forward scattered light in a second direction D2. For example, the first portion FSC1 of the forward scattered light is allowed to pass through the beam splitter 170 and reach the first detector 159, while the beam splitter 170 causes the second portion FSC2 of the forward scattered light to reflect toward the second detector 160. In the example illustrated in FIG. 3, the second direction D2 is orthogonal with respect to the first direction DI.

[0075] The detection system 100 analyzes the first portion FSC1 of the forward scattered light to determine sizes of the particles 200 passing through the interrogation zone 1 . Additionally, the detection system 100 analyzes the second portion FSC2 of the forward scattered light to determine at least one additional characteristic of the particles 200 passing through the interrogation zone 18. For example, the at least one additional characteristic determined by the detection system 100 from the second portion FSC2 of the forward scattered light can include one or more shapes, morphologies, internal complexities, granularities, or compositions of the particles 200 passing through the interrogation zone 18.

[0076] As further shown in FIG. 3, the forward collection unit 150 includes a lens 172 that focuses the second portion FSC2 of the forward scattered light. In some examples, the lens 172 is a collimating lens that collimates the second portion FSC2 of the forward scattered light.

[0077] In the example of FIG. 3, the second detector 160 is positioned within the collimated beam of the second portion FSC2 of the forward scattered light to achievedesirable signal -to-noise ratio. The second detector 160 includes one or more optical fibers 174 that collect the second portion FSC2 of the forward scattered light. In some examples, the second detector 160 includes a single optical fiber. In other alternative examples, such as the one shown in FIG. 3, the second detector 160 is a detector array that includes more than optical fiber, such as two, three, four, or more optical fibers.

[0078] The one or more optical fibers 174 collect the second portion FSC2 of the forward scattered light over a collection angle a having a reduced size. For example, the collection angle a of an optical fiber 174 in the second detector 160 is about 0.001 (i.e., 1 / 1000th) of the first collection angle al (see FIG. 2) of the first detector 159. As an illustrative example, the collection angle a of an optical fiber 174 in the second detector 160 is about 0.02 degrees.

[0079] Each of the one or more optical fibers 174 is connected to a photodetector 176 that receives the second portion FSC2 of the forward scattered light. The photodetectors 176 convert optical signals into electrical signals for processing by the computing system 1900 of the detection system 100. In some examples, the photodetectors 176 include avalanche photodiodes (APD) that amplify the light signal received from an optical fiber 174.

[0080] FIG. 4 shows an example arrangement of the detection system 100. As shown in FIG. 4. the forward collection unit 150 includes the beam splitter 170 that conveys the second portion FSC2 of the forward scattered light to the lens 172. In this example, lens 172 is a collimating lens that collimates the second portion FSC2 of the forward scattered light.

[0081] As further show n in the example of FIG. 4, the second detector 160 includes a single optical fiber 174 that collects a portion of the collimated beam of the second portion FSC2 of the forward scattered light for transfer to the photodetector 176, which can include an APD that amplifies the light signals received from the single optical fiber 174.

[0082] In this example shown in FIG. 4, the forward collection unit 150 is integrated on a CytoFLEX® flow cytometer from Beckman Coulter, Inc.®. Advantageously, the forward collection unit 150 can be integrated on an existing flow cytometer platform without replacing an existing FSC detector channel of the flow cytometer platform. Instead, components of the forward collection unit 150 such as the beam splitter 170, the lens 172, the optical fiber 174, and the photodetector 176 areadd-on components that do not require host system modifications. A further advantage of the forward collection unit 150 is that it can be implemented using a single optical fiber 174 instead of a plurality of optical fibers, such that the forward collection unit 150 is less bulky and easier to implement on existing flow cytometer platforms.

[0083] In some further examples, a tip of the optical fiber 174 that receives the second portion FSC2 of the forward scattered light can be moved about a plane orthogonal to the second direction D2 (see FIG. 3) of the second portion FSC2 of the forward scattered light. For example, the tip of the optical fiber 174 can be moved along at least two coplanar axes (e.g., X and Y) in the plane orthogonal to the second direction D2. This allows adjustment of the alignment of the optical fiber 174 with respect to the second portion FSC2 of the forward scattered light. For example, the alignment of the optical fiber 174 can be adjusted for optimal signal detection based on the size and shape of the particles passing through the interrogation zone 18.

[0084] As further shown in FIG. 4, a display device 180 is connected to the detection system 100. In some examples, the display device 180 is an oscilloscope. In FIG. 4, the display device 180 displays a first waveform 182 of a particle. The first waveform 182 is generated from side scattered light collected from the side collection unit 130. The display device 180 further displays a second waveform 184 of the particle. The second waveform 184 is generated from the second portion FSC2 of the forward scattered light detected by the second detector 160. The first and second waveforms 182, 184 have different shapes. For example, the first waveform 182 has a Gaussian shape, whereas the second waveform 184 does not have a Gaussian shape.

[0085] FIG. 5 schematically illustrates an example of a method 500 that can be performed by the detection system 100 for discriminating particles based on shape and morphology. As shown in FIG. 5, the method 500 includes an operation 502 of emitting the one or more excitation light beams 12 toward the interrogation zone 18. The detection system 100 can perform operation 502 by using the light emitting unit 110, which is described above in more detail.

[0086] The method 500 includes an operation 504 of streaming the particles through the interrogation zone 18 such that interactions of the particles with the excitation light beam 12 cause forward scattered light and side scattered light, such as is shown in FIG. 2.

[0087] The method 500 includes an operation 506 of causing a first portion FSC1 of the forward scattered light to reach the first detector 159. As shown in FIG. 4. the detection system 100 can perform operation 506 by using the beam splitter 170. For example, the beam splitter 170 allows the first portion FSC1 of the forward scattered light to pass through the beam splitter 170 and reach the first detector 159. In alternative examples, the beam splitter 170 can reflect the first portion FSC1 of the forward scattered light toward the first detector 159.

[0088] The method 500 includes an operation 508 of analyzing the first portion FSC1 of the forward scattered light. As described above, the intensity of the forward scattered light is proportional to the diameter of the particles passing through the interrogation zone 18. Thus, operation 508 can include analyzing the first portion FSC1 of the forward scattered light to determine the sizes of the particles. In some examples, operation 508 is optional.

[0089] The method 500 further includes an operation 510 of causing a second portion FSC2 of the forward scattered light to reach the second detector 160. As shown in FIG. 4, the detection system 100 can perform operation 510 by using the beam splitter 170 to reflect the second portion FSC2 of the forward scattered light toward the second detector 160. In alternative examples, the second portion FSC2 of the forward scattered light can pass through the beam splitter 170 to reach the second detector 160 of the forward collection unit 150.

[0090] The method 500 includes an operation 512 of analyzing the second portion FSC2 of the forward scattered light to discriminate shapes and / or morphologies of the particles. For example, operation 512 can include analyzing the second portion FSC2 of the forward scattered light to discriminate the different shapes of the particles. In some examples, operation 512 can include analyzing the second portion FSC2 of the forward scattered light to discriminate internal complexities, granularities, or compositions of the particles.

[0091] FIG. 6 schematically illustrates an example of the operation 512 in the method 500 that can be performed by the detection system 100 for analyzing the second portion FSC2 of the forward scattered light to discriminate one or more shapes or morphologies of the particles.

[0092] As show n in FIG. 6, the operation 512 includes a step 602 of generating a waveform from the second portion FSC2 of the forward scattered light collected by thesecond detector 160 in the forward collection unit 150. The waveform can resemble the second waveform 184 shown on the display device 180 connected to the detection system 100 in FIG. 4.

[0093] Operation 512 further includes a step 604 of determining one or more parameters from the waveform generated in step 602. For example, step 604 can include determining a height and an area of the waveform. In some further examples, step 604 can include determining additional parameters such as a phase and a width of the waveform.

[0094] Operation 512 includes a step 606 discriminating particle shape and / or morphology based on the one or more parameters determined in step 604. Discriminating particle shape and / or morphology based on the one or more parameters will now be described with reference to FIGS. 7-18. As shown in these figures, the method 500 when performed by the detection system 100 can significantly improve discrimination of particle shape and / or morphology.

[0095] FIG. 7 illustrates an example of a first waveform 700 generated from the side scattered (SSC) light collected from a particle passing through the interrogation zone 18. The side collection unit 130 collects the SSC light over the second collection angle a2 (see FIG. 2). In the example shown in FIG. 7, the first w aveform 700 is generated from the SSC light collected from a particle having a size of 4pm, and a first shape (e.g., spherical).

[0096] FIG. 8 illustrates an example of a second waveform 800 generated from the side scattered (SSC) light collected from another particle passing through the interrogation zone 18. The side collection unit 130 collects the SSC light over the second collection angle a2 (see FIG. 2). In the example shown in FIG. 8. the second waveform 800 is generated from the SSC light collected from a particle having a size of 4pm, and a second shape (e.g., peanut). Thus, the first and second waveforms 700, 800 respectively shown in FIGS. 7 and 8 are generated from the SSC light collected from particles having the same size, but different shapes.

[0097] One or more parameters can be determined from the first and second waveforms 700, 800. For example, a height (also called amplitude), an area, a phase, and a width can be derived from the first and second w aveforms 700, 800 generated from the SSC light.

[0098] FIG. 9 illustrates a first plot 900 of heights determined from waveforms of 1000 particles having the first shape (e.g.. spherical) and 1000 particles having the second shape (e.g., peanut) passing through the interrogation zone 18. The heights in the first plot 900 are determined from waveforms similar to the ones shown in FIGS. 7 and 8, which are generated from the SSC light collected over the second collection angle a2.

[0099] FIG. 10 is a histogram plot 1000 showing an overlap of waveform heights detected from the particles having the first and second shapes shown in FIG. 9. As shown in FIGS. 9 and 10, there is a large overlap of the waveform heights of the particles having the same size but different shapes. In view of FIGS. 9 and 10, shape discrimination of the particles based on the height of SSC light waveforms has low- accuracy or fidelity.

[0100] FIG. 11 illustrates a second plot 1100 of areas determined from the waveforms of 1000 particles having the first shape (e.g., spherical) and 1000 particles having the second shape (e.g., peanut) passing through the interrogation zone 18. The areas in the second plot 1100 are determined from waveforms similar to the ones shown in FIGS. 7 and 8, generated from the SSC light collected over the second collection angle a2.

[0101] FIG. 12 is a histogram plot 1200 showing an overlap of waveform areas detected from the particles having the first and second shapes shown in FIG. 11. As shown in FIGS. 1 1 and 12, there is a large overlap of the waveform areas of the particles having the same size but different shapes. In view of FIGS. 11 and 12, shape discrimination of the particles based on the area of SSC light waveforms has low accuracy or fidelity.

[0102] FIG. 13 illustrates an example of a third waveform 1300 generated from the second portion FSC2 of the forward scattered light collected from a particle passing through the interrogation zone 18, in accordance with the method 500 described above. In the example shown in FIG. 13, the third waveform 1300 is generated from the forward scattered light collected from a particle having a size of 4pm, and a first shape (e.g., spherical). In this example, the forw ard scattered light is collected by an optical fiber 174 over a collection angle a having a reduced size. For example, as described above, the collection angle a of the optical fiber 174 is about 0.001 (i.e., 1 / 1000th) ofthe first collection angle al of the first detector 159. In some examples, the collection angle a of the optical fiber 174 is about 0.02 degrees.

[0103] FIG. 14 illustrates an example of a fourth waveform 1400 generated from the second portion FSC2 of the forward scattered light collected from a particle passing through the interrogation zone 18, in accordance with the method 500 described above. In the example shown in FIG. 14, the fourth waveform 1400 is generated from the forward scattered light collected from a particle having a size of 4pm, and a second shape (e.g., peanut). In this example, the forward scattered light is collected by an optical fiber 174 over a collection angle a having a reduced size, as describe above with respect to the third waveform 1300 shown in FIG. 13.

[0104] The third and fourth waveforms 1300, 1400 shown in FIGS. 13 and 14. respectively, have a different shape than the first and second waveforms 700, 800 shown in FIGS. 7 and 9, respectively. For example, the first and second waveforms 700, 800 have a Gaussian shape. In contrast, the third and fourth waveforms 1300, 1400 have a sinusoidal shape that includes a peak 1302, 1402 and a trough 1304. 1404 of different magnitudes relative to a baseline 1306, 1406.

[0105] Like with the first and second waveforms 700, 800, one or more parameters can be determined from the third and fourth waveforms 1300, 1400. For example, a height (also called amplitude or magnitude), an area, a phase, and a width can be derived from the third and fourth waveforms 1300, 1400 that are generated from the forward scattered light.

[0106] FIG. 15 illustrates a third plot 1500 of w aveform widths determined from waveforms of 1000 particles having the first shape (e.g., spherical) and 1000 particles having the second shape (e.g., peanut) passing through the interrogation zone 18. The widths in the third plot 1500 are determined from waveforms similar to the ones shown in FIGS. 13 and 14, which are generated from the forward scattered light collected over a reduced collection angle.

[0107] FIG. 16 is a histogram plot 1600 showing an overlap of the waveform widths detected from the particles having the first and second shapes shown in FIG. 15. As shown in FIGS. 15 and 16, there is less overlap of the w aveform widths of the particles having the same size but different shapes, than the overlaps of the waveform heights and areas shown in FIGS. 9-12.

[0108] FIG. 17 illustrates a fourth plot 1700 of phases determined from the waveforms of 1000 particles having the first shape (e.g., spherical) and 1000 particles having the second shape (e.g., peanut) passing through the interrogation zone 18. The phases in the fourth plot 1700 are determined from waveforms similar to the ones shown in FIGS. 13 and 14, which are generated from the forward scattered light collected over a reduced collection angle.

[0109] FIG. 18 is a histogram plot 1800 showing an overlap of the waveform phases detected from the particles having the first and second shapes shown in FIG. 17. As show n in FIGS. 17 and 18, there is less overlap of the waveform phases of the particles having the same size but different shapes, than the overlaps of the w aveform heights and areas shown in FIGS. 9-12.

[0110] In view' of FIGS. 7-18, shape discrimination of the particles based on the waveforms generated from the forw ard scattered light collected over the reduced collection angle has a higher accuracy or fidelity than the shape discrimination based on the waveforms generated from the SSC light collected over the second collection angle a2. Accordingly, the forward collection unit 150 and the method 500 increase the accuracy and / or fidelity of shape discrimination of particles that pass through the interrogation zone 18 of the detection system 100.[OHl] FIG. 19 schematically illustrates an example of the computing system 1900 for implementing aspects of the detection system 100. For example, the computing system 1900 can process and analyze the portion of FSC light for particle shape and / or morphology discrimination. As show n in FIG. 19, the computing system 1900 of the detection system 100 includes one or more processing devices 1902, a memory storage device 1904, and a system bus 1906 that couples the memory storage device 1904 to the one or more processing devices 1902.

[0112] Examples of the one or more processing devices 1902 include central processing units (CPUs), digital signal processors, field-programmable gate arrays, and other types of electronic computing circuits. The one or more processing devices 1902 can be part of a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry' to perform the functionalities described herein.

[0113] The memory storage device 1904 can include a random-access memory (“RAM”) 1908 and a read-only memory (“ROM”) 1910. Basic input and output logichaving basic routines transferring information between elements in the detection system 100 can be stored in the ROM 1910. The detection system 100 can additionally include a mass storage device 1912 that can store an operating system 1914 and software instructions 1916. The mass storage device 1912 is connected to the processing device 1902 through the system bus 1906. The mass storage device 1912 and computer-readable data storage media provide non-volatile, non-transitory computer memory storage for the detection system 100.

[0114] Although the description of computer-readable data storage media contained herein refers to the mass storage device 1912, it should be appreciated by those skilled in the art that computer-readable data storage media can be any available non- transitory, physical device or article of manufacture from which the detection system 100 can read data and / or instructions. The computer-readable storage media can be comprised of entirely non-transitory media. The mass storage device 1912 is an example of a computer-readable storage device.

[0115] Computer-readable data storage media include volatile and non-volatile, removable, and non-removable media implemented in any method or technology for storage of information such as computer-readable software instructions, data structures, program modules or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, or any other medium which can be used to store information, and which can be accessed by the device.

[0116] The detection system 100 can operate in a networked environment using logical connections to the other devices through the communications network 1920. The detection system 100 connects to the communications network 1920 through a network interface unit 1918 connected to the system bus 1906. The network interface unit 1918 can also connect to other types of communications networks and devices, including through Bluetooth, Wi-Fi, and cellular telecommunications networks including 4G and 5G networks. The network interface unit 1918 can connect the detection system 100 to additional networks, systems, and devices. The detection system 100 also includes an input / output unit 1922 for receiving and processing inputs and outputs from one or more peripheral devices, and the user interface 1924.

[0117] The mass storage device 1912 and the RAM 1908 can store software instructions and data. The software instructions can include an operating system 1914suitable for controlling the operation of the detection system 100. The mass storage device 1912 and / or the RAM 1908 can also store the software instructions 1916. which when executed by the processing device 1902, provide the functionality of the detection system 100 discussed herein.

[0118] FIG. 20 schematically illustrates an example of a method 2000 of discriminating particle shape. The method 2000 can utilize the features of the forward collection unit 150 of the detection system 100. As described above, the forward collection unit 150 can be integrated into an existing flow cytometer platform without replacing an existing FSC detector channel of the flow cytometer platform. As described above, the forward collection unit 150 collects at least a portion of the forward scattered light over a reduced collection angle a. As an illustrative example, the reduced collection angle a is about 0.02 degrees.

[0119] As shown in FIG. 20, the method 2000 includes an operation 2002 of collecting forward scattered light over the reduced collection angle a. Operation 2002 can include using the second detector 160 of the forward collection unit 150 in the detection system 100 to collect the forward scattered light. For example, a second portion FSC2 of the forward scattered light can be directed by the beam splitter 170 toward the one or more optical fibers 174 of the second detector 160 for collecting the forward scattered light over the reduced collection angle a.

[0120] The method 2000 includes an operation 2004 of generating a digital waveform associated with a particle interrogation event based on the forward scattered light collected in operation 2002 over the reduced collection angle a.

[0121] FIG. 21 illustrates examples of waveforms 2102, 2104, 2106, 2108 generated in operation 2004 based on the forward scattered light collected over the reduced collection angle a. The waveforms 2102, 2104, 2106, 2108 are fittings that are based on data points after baseline correction. The data points are collected from polystyrene 4pm beads having a “snowman” type of shape (e.g., two or more spheres connected or linked together). The waveforms 2102, 2104, 2106, 2108 are referred to herein as small angle detection (SAD) waveforms because they are generated based on data points collected over the reduced collection angle a by the second detector 160 of the forward collection unit 150 in the detection system 100.

[0122] Referring back to FIG. 20, the method 2000 includes an operation 2006 of fitting the digital waveform generated in operation 2004 to the Morlet wavelet (orGabor wavelet) function. A Morlet wavelet can be defined as a sine wave tapered by a Gaussian. In mathematics, the Morlet wavelet is a wavelet composed of a complex exponential (earner) multiplied by a Gaussian envelope or window. As shown in FIG. 21, despite of the different shapes of the waveforms 2102, 2104, 2106, 2108, the data points of each of the waveforms can be fitted to the real-valued Morlet wavelet, which is defined by expression (1),where A is w aveform amplitude, / is frequency of carrier, q> is phase of carrier, p is envelope position, o is envelope width, d is waveform baseline, and t is time.

[0123] FIG. 22 illustrates an example of a fitting 2200 of raw data points (after baseline correction) to the real-valued Morlet wavelet defined by expression (1). In FIG. 22, the data points are collected by the second detector 160 over the reduced collection angle a.

[0124] Referring back to FIG. 20, the method 2000 includes an operation 2008 of determining an envelope or module of the Morlet wavelet. The envelope of the Morlet wavelet can be determined from expression (2),(2) Envelope = / C2+ S2

[0125] FIG. 23 illustrates an example of raw data 2302 and an envelope 2304 of the Morlet wavelet determined in operation 2008 of the method 2000. As shown in FIG.23. because the envelope 2304 is unique for the waveform generated from the raw data 2302, the envelope 2304 can be used for pulse analysis including discriminating particles by shape and / or morphology.

[0126] Referring back to FIG. 20, the method 2000 includes an operation 2010 of calculating one or more parameters from the envelope determined in operation 2008. As shown in FIG. 23, because the envelope 2304 has a Gaussian shape, the standard waveform analysis parameters - area, height, and width - can be determined from the envelope 2304.

[0127] Referring back to FIG. 20, the method 2000 includes an operation 2012 of discriminating or charactering particle shape and / or morphology based on the parameters calculated in operation 2010 from the envelope. For example, due to theGaussian shape of the envelope, the standard parameters area, height, and width can be used for particle discrimination.

[0128] The method 2000 includes several advantages over prior techniques for discriminating particles. For example, the digital waveforms can be processed by the method 2000 on the fly, which allows the particles to be sorted based on shape and / or morphology. Further, the method 2000 uses the standard parameters (e g., area, height, and width) derived from the envelope to discriminate the particles based on shape and / or morphology. Further, the method 2000 eliminates manual processing such that human errors are mitigated.

[0129] FIG. 24 illustrates a comparison of results for discriminating particles based on shape by traditional side scattering (SSC) techniques versus the method 2000 which uses data collected from the small angle detection (SAD) channels (i.e., the one or more optical fibers 174 of the second detector 160, as shown in FIG. 3). FIG. 24 includes a plurality of histograms that display results from discriminating two populations of polystyrene beads each having the same size (4 pm), but having different shapes (spherical shape versus peanut shape).

[0130] In FIG. 24, a first histogram 2402 shows results for discriminating the two populations of polysty rene beads using area determined from traditional side scattering (SSC) techniques. A second histogram 2404 shows results for discriminating the two populations of polystyrene beads using area determined from the method 2000 (which used data collected from the small angle detection (SAD) channels). As shown in FIG. 24, the second histogram 2404 does not have any overlap between the two populations of polysty rene beads, whereas the first histogram 2402 has overlap between the two populations of polystyrene beads. Thus, the area determined from the method 2000 is more successful in discriminating the two populations of polystyrene beads than the area determined from traditional SSC techniques.

[0131] FIG. 24 further includes a third histogram 2406 that shows results for discriminating the two populations of polystyrene beads using height determined from the traditional side scattering (SSC) techniques. A fourth histogram 2408 shows results for discriminating the two populations of polystyrene beads using height determined from the method 2000 (which used data collected from the small angle detection (SAD) channels). As shown in FIG. 24, the fourth histogram 2408 does not have any overlap between the two populations of polystyrene beads, whereas the third histogram 2406has overlap between the two populations of polystyrene beads. Thus, the height determined from the method 2000 more successfully discriminates the two populations of polystyrene beads than the height determined from SSC techniques.

[0132] FIG. 25 illustrates another comparison of results for discriminating particles based on shape by traditional side scattering (SSC) techniques versus the method 2000 which uses data collected from the small angle detection (SAD) channels (i.e., the one or more optical fibers 174 of the second detector 160. as shown in FIG. 3). FIG. 25 includes a plurality of histograms that display results from discriminating two populations of polystyrene beads each having the same size (4 pm), but having different shapes (snowman versus pear shape).

[0133] In FIG. 25. a first histogram 2502 shows results for discriminating the two populations of polystyrene beads using area determined from traditional side scattering (SSC) techniques. A second histogram 2504 shows results for discriminating the two populations of polystyrene beads using area determined from the method 2000 (which used data collected from the small angle detection (SAD) channels). As shown in FIG. 25, the second histogram 2504 exhibits less overlap between the two populations of polystyrene beads than the first histogram 2502. Thus, the area determined from the method 2000 more successfully discriminates the two populations of polystyrene beads than the area determined from traditional SSC techniques.

[0134] FIG. 25 further includes a third histogram 2506 that shows results for discriminating the two populations of polystyrene beads using height determined from the traditional side scattering (SSC) techniques. A fourth histogram 2508 shows results for discriminating the two populations of polystyrene beads using height determined from the method 2000 (which used data collected from the small angle detection (SAD) channels). As shown in FIG. 25, the fourth histogram 2408 has less overlap between the two populations of polystyrene beads than the third histogram 2506. Thus, the height determined from the method 2000 more successfully discriminates the two populations of polystyrene beads (e.g., snowman shape versus pear shape) than the height determined from SSC techniques.

[0135] The various embodiments described above are provided by way of illustration only and should not be construed to be limiting in any way. Various modifications can be made to the embodiments described above without departing from the true spirit and scope of the disclosure.

Claims

What is claimed is:

1. A system for analyzing particles, the detection system comprising: a flow chamber configured to stream the particles through an interrogation zone; a light emitting unit configured to generate an excitation light beam, the excitation light beam being directed toward the interrogation zone, the excitation light beam causing forward scattered light from the particles passing through the interrogation zone; a collection unit configured to collect the forward scattered light, the collection unit including: a beam splitter conveying a first portion of the forward scattered light in a first direction and a second portion of the forward scattered light in a second direction; a first detector detecting the first portion of the forward scattered light; and a second detector detecting the second portion of the forward scattered light; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: generate waveforms from the second portion of the forward scattered light; determine one or more parameters from the waveforms; and discriminate the particles based on at least one of shape and morphology using the one or more parameters determined from the waveforms.

2. The system of claim 1 , wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: determine widths of the waveforms; and discriminate the particles based on the at least one of shape and morphology using the widths determined from the waveforms.

3. The system of claim 1, wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: determine phases of the waveforms; and discriminate the particles based on the at least one of shape and morphology using the phases determined from the waveforms.

4. The system of claim 1, wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: analyze the first portion of the forward scattered light to determine sizes of the particles passing through the interrogation zone.

5. The system as in any of the preceding claims, further comprising: a collimating lens that collimates the second portion of the forward scattered light.

6. The system as in any of the preceding claims, wherein the second detector includes an optical fiber that collects the second portion of the forward scattered light over a collection angle having a reduced size.

7. The system of claim 6, wherein the collection angle of the optical fiber is about 0.001 of a first collection angle of the first detector, or wherein the collection angle of the optical fiber is about 0.02 degrees.

8. A unit for analyzing scattered light from particles, the unit comprising: a beam splitter conveying first and second portions of forward scattered light; a first detector detecting the first portion of forward scattered light; a second detector detecting the second portion of the forward scattered light; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: generate waveforms from the second portion of the forward scattered light; determine one or more parameters from the waveforms; anddiscriminate the particles based on at least one of shape and morphology using the one or more parameters determined from the waveforms.

9. The unit of claim 8, wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: determine widths of the waveforms; and discriminate the particles based on the at least one of shape and morphology using the widths determined from the waveforms.

10. The unit of claim 8, wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: determine phases of the waveforms; and discriminate the particles based on the at least one of shape and morphology using the phases determined from the waveforms.

11. The unit of claim 8, wherein the instructions, when executed by the processing circuitry, further cause the processing circuitry to: analyze the first portion of the forward scattered light to determine sizes of the particles.

12. The unit of claim 8, further comprising: a collimating lens that collimates the second portion of the forward scattered light.

13. The unit of any of claims 8-12, wherein the second detector includes an optical fiber that collects the second portion of the forward scattered light over a collection angle having a reduced size.

14. The unit of claim 13, wherein the collection angle of the optical fiber is about 0.001 of a first collection angle of the first detector, or wherein the collection angle of the optical fiber is about 0.02 degrees.

15. A method of analyzing particles, the method comprising:emitting an excitation light beam toward an interrogation zone; streaming the particles through the interrogation zone such that interactions of the particles with the excitation light beam cause forward scattered light; conveying a first portion of the forward scattered light to a first detector; conveying a second portion of the forward scattered light to a second detector; generating waveforms from the second portion of the forward scattered light; determining one or more parameters from the waveforms; and discriminate the particles based on at least one of shape and morphology using the one or more parameters determined from the waveforms.

Images