Extended dynamic range for flow cytometry

Abstract

A method of characterizing particles by flow cytometry includes determining a first median scatter intensity of a first particle population. The first median scatter intensity is based on light scatter collected by a first detector. The method includes determining a second median scatter intensity of the first particle population. The second median scatter intensity is based on light scatter collected by a second detector. The method includes determining a ratio of the second median scatter intensity relative to the first median scatter intensity. The method includes extending a dynamic range of the first detector by multiplying the ratio by a median scatter intensity of a second particle population. The median scatter intensity of the second particle population is based on the light scatter collected by the second detector.

Description

EXTENDED DYNAMIC RANGE FOR FLOW CYTOMETRYCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 608,615, filed December 11, 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 fluoresced 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 in two directions: forward scattered light which is parallel with the excitation light beams, and side scattered light which is orthogonal to the excitation light beams. Relative to optical signals from forward scattered light, optical signals from side scattered light are weak. Conventional flow cytometers typically include a single detector for detecting optical signals from side scattered light under a particular wavelength which limits the usage of side scattered light for characterizing the particles.SUMMARY

[0004] In general terms, the present disclosure relates to a system for characterizing particles by flow cytometry. In one possible configuration, the system includes first and second detectors that detect side scattered light, and a dynamic range of the first detector is extended to include at least six decades of side scattered light for display on a single display plot. Various aspects are described in this disclosure, which include, but are not limited to, the following aspects.

[0005] One aspect relates to a system for characterizing particles by flow cytometry, the system comprising: a light emitting unit configured to emit a light beam; a collection unit including: a first detector for collecting light scatter resulting from interrogation of the particles by the light beam; a second detector for collecting the light scatter resulting from the interrogation of the particles by the light beam; and a processing circuitry having a memory for storing instructions which, when executed bythe processing circuitry, cause the processing circuitry' to: determine a first median scatter intensity of a first particle population based on the light scatter collected by the first detector; determine a second median scatter intensity of the first particle population based on the light scatter collected by the second detector; determine a ratio of the second median scatter intensity relative to the first median scatter intensity; and extend a dynamic range of the first detector by multiplying the ratio by a median scatter intensity of a second particle population measured by the second detector.

[0006] Another aspect relates to a method of characterizing particles by floyv cytometry, the method comprising: determining a first median scatter intensity of a first particle population, the first median scatter intensity being based on light scatter collected by a first detector; determining a second median scatter intensity of the first particle population, the second median scatter intensity being based on light scatter collected by a second detector; determining a ratio of the second median scatter intensity relative to the first median scatter intensity ; and extending a dynamic range of the first detector by multiplying the ratio by a median scatter intensity of a second particle population, the median scatter intensity of the second particle population being based on the light scatter collected by the second detector.

[0007] Another aspect relates to a method of displaying data captured by floyv cytometry, the method comprising: receiving a first set of median scatter intensities from a first detector; receiving a second set of median scatter intensities from a second detector; extending the first set of median scatter intensities to include an extended set of median scatter intensifies, the extended set of median scatter intensities being calculated based on a ratio of the second set of median scatter intensities to the first set of median scatter intensities; and displaying the first set of median scatter intensities and the extended set of median scatter intensities on a single plot.

[0008] 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 yvhich the embodiments disclosed herein are based.DESCRIPTION OF THE FIGURES

[0009] The following drawing figures, yvhich 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.

[0010] FIG. 1 illustrates an example of a system for performing flow cytometry, the system including a flow cytometer and a workstation.

[0011] FIG. 2 schematically illustrates an example of the flow cytometer of FIG. 1.

[0012] FIG. 3 schematically illustrates an example of a method of characterizing particles by flow cy tometry7which can be performed by the flow cytometer of FIG. 2.

[0013] FIG. 4 illustrates an example of side scattered light data collected by first and second side scatter detectors of the flow cytometer of FIG. 2.

[0014] FIG. 5 illustrates another example of side scattered light data collected by the first and second side scatter detectors of the flow cytometer of FIG. 2.

[0015] FIG. 6 illustrates another example of side scattered light data collected by the first and second side scatter detectors of the flow cytometer of FIG. 2.

[0016] FIG. 7 illustrates an example of a display chart showing a dynamic range of the first side scatter detector extended in accordance with the method of FIG. 3.

[0017] FIG. 8 schematically illustrates an example of a method of displaying data captured by flow cytometry which can be performed by the flow cytometer of FIG. 2.

[0018] FIG. 9 schematically illustrates an example of a computing system for implementing aspects of the system of FIG. 1 .DETAILED DESCRIPTION

[0019] 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.

[0020] An example detection system is described herein for use in a flow cytometer. It should be understood that the present disclosure is not limited to the illustrated detection system, but may be applied to flow cytometers with other types of detection systems.

[0021] FIG. 1 illustrates an example of a system 10 that can be used to perform flow cytometry. The system 10 includes a llow cytometer 100 and a workstation 200. In general, the How cytometer 100 is an analytical instrument that detects physical and chemical properties of samples of cells or particles. In some examples, the flow' cytometer 100 is designed to capture robust and high quality data for characterizing biologically relevant nanoparticles. The flow cytometer 100 is a single instrument thatprovides simultaneous assessment of nanoparticle size, concentration, and cargo to understand biological mechanisms of action and nanoparticle origins. The flow cytometer 100 can collect data from millions of particles or cells in a matter of minutes for display in a variety of formats on a display monitor 204 of the workstation 200.

[0022] The flow cytometer 100 includes a housing 101 having a sample station 104 which receives a container 106 containing a sample of cells and / or particles. In some examples, the container 106 contains a sample of nanoparticles such as extracellular vesicles (EVs). A user of the system 10 can manually load the container 106 into the sample station 104. Once loaded in the sample station 104, the flow cytometer 100 can acquire the sample from the container 106 to perform the flow cytometry experiment. In some examples, the container 106 is a sample tube such as a 1.5-mL or 2-mL microtube, and / or has a diameter of 12 mm and a height of 75 mm.

[0023] The flow cytometer 100 can further include a sheath fluid container 107 for holding sheath fluid that is mixed with the sample during the flow cytometry experiment. The sheath fluid is pumped into the flow cytometer 100 causing a laminar flow. The sample is injected at a higher pressure into the center of the laminar flow of the sheath fluid. Hydrodynamic forces cause the particles to align, single file in the direction of the laminar flow. The sheath fluid container 107 is connected to the flow cytometer 100 via tubing 109a.

[0024] The flow cytometer 100 can further include a waste container 108 for collecting waste fluid. The waste container 108 is connected to the flow cytometer 100 via tubing 109b.

[0025] The workstation 200 connects to the flow cytometer 100 via a wired or wireless connection to receive data from the flow cytometer 100 for display on the display monitor 204. The workstation 200 includes one or more user input devices such as a mouse 206 and a keyboard 208 allowing a user of the system 10 to enter data and information, control the flow cytometer 100, and alter the display of the data on the display monitor 204.

[0026] The workstation 200 includes a computing device 900. The workstation 200 utilizes the computing device 900 to process raw data received from the flow cytometer 100. Alternatively, or additionally, the flow cytometer 100 includes a computing device 900 to process data collected from flow cytometry. In such examples, the flow cytometer 100 sends processed data to the workstation 200 for display on the displaymonitor 204.

[0027] FIG. 2 schematically illustrates an example of the flow cytometer 100. The flow cytometer 100 can detect and analyze particles on the nanoscopic scale such as particles having a diameter less than 100 nanometers (nm). Also, the flow cytometer can detect and analyze particles having a larger size such as particles having a diameter greater than 100 nm. The flow cytometer 100 includes a light emitting unit 110, and a light collection unit 120 that detects characteristics of particles passing through a flow chamber 15.

[0028] The light emitting unit 110 emits one or more excitation light beams for projection onto particles flowing through an interrogation zone 18 in the flow chamber 15. The light collection unit 120 collects light scattered or emitted from the particles that flow through the interrogation zone 18 for analysis by a computing device 900 (see FIG. 9).

[0029] The light emitting unit 110 includes a plurality' of light sources 11 la-11 Id such as a first light source I l la, a second light source 111b, a third light source 111c, and a fourth light source 11 Id. The plurality of light sources 11 la-11 Id can include more than four light sources, or fewer than four light sources. The plurality of light sources 11 la-1 l id can include lasers.

[0030] The plurality of light sources l l la-l l ld emit excitation light beams within a range of about 300 nm to about 825 nm. As a further example, the light sources l l la- l l ld emit excitation light beams within a range of about 325 nm to about 808 nm. Each of the plurality of light sources l l l a-l l l d emits an excitation light beam of a particular wavelength. As an illustrative example, the first light source I l la emits an excitation light beam in the red light spectrum (e.g., 625-825 nm), the second light source 111b emits an excitation light beam in the yellow light spectrum (e.g., 560-590 nm). the third light source 111c emits an excitation light beam in the blue light spectrum (e.g., 450- 490 nm), and the fourth light source 1 1 Id emits an excitation light beam in the violet light spectrum (e.g., 300-450 nm).

[0031] In the example shown in FIG. 2, 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 light emitting unit 110 may include any other suitable number of light sources.

[0032] The light emitting unit 110 further includes a focal lens 119. The focal lens119 is configured to focus the excitation light beams for high scatter intensity detectionof particles. For example, the excitation light beams emitted by the light sources 11 la- 11 Id pass through the focal lens 119, which focuses the excitation light beams 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 in the flow cytometer 100.

[0033] 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 ll la-ll ld and transmit the light beams of the other light sources. The dichroic mirrors 117a-l 17d are selected and configured according to the wavelengths of the light beams emitted by the respective light sources l l la-l l ld. 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 tow ard 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 1 11c 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 1 17d 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, 111b, and 111c.

[0034] The light beams emitted by the light sources 1 1 1 a-1 11 d are reflected by or transmitted through the dichroic mirrors 117a-l 17d to form collinear beams. The collinear 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.

[0035] Lenses 115a-l 15d are arranged between the respective light sources 111a- 11 Id and the respective dichroic mirrors 117a-l 17d. In some examples, the lenses 115a-l 15d 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. 2, each of the lenses 115a-l 15d is in the form of planoconvex lens with a flat surface and a convex surface opposite to each other.

[0036] The lenses 115a- 115d 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-l 17d 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.

[0037] It should be understood that the number, the type, and the arrangement of the dichroic mirrors 117a-l 17d and the lenses 115a-l 15d may be changed as needed, and are not limited to the example illustrated herein. Also, the dichroic mirrors 117a- 117d and the lenses 115a-l 15d can be replaced with other optical elements or optical modules with similar functions.

[0038] Beam expanders 113a- 113d are arranged between the respective light sources 11 la-1 l id and the respective lenses 115a-l 15d. 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.

[0039] The light beams irradiated on the particles by the focal lens 119 have a spot size that allow s for more concentrated light beams with a higher power density7. 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 the efficiency of collecting the optical signals, and thereby provide higher resolution and higher sensitivity' for nanoparticle detection.

[0040] In the example shown in FIG. 2, the light sources 11 la-11 Id are in the form of lasers that include respective laser diodes 112a-l 12d. As further shown in the example of FIG. 2, half-wave plates 1 16a-l 16d are provided between the dichroic mirrors 117a-l 17d and the lenses 115a-l 15d, 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.

[0041] As further shown in FIG. 2, 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 power of some or all of the lightsources 11 la-11 Id can also be increased. The increased power of the light sources 11 la-11 Id can also improve detection sensitivity.

[0042] 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. 2, each of the beam expanders 113a- 113d includes a concave lens adjacent to the corresponding light source as the first optical part, and further includes a convex lens away from the corresponding light source as the second optical part. It should be understood that each of the beam expanders 113a-l 13d is not limited to the example shown in FIG. 2. The beam expanders 113a-l 13d 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.

[0043] 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 waist position (the focus point) of the light beam on the optical axis.

[0044] As described above, by adjusting the dichroic mirrors 117a- 117d, 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 any other adjustment manner. One or more adjustments to the dichroic mirrors 1 17a- 117d, the lenses 115a- 115d, and the beam expanders 113a- 113d 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.

[0045] 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 emitted from the particles in the sample as they are irradiated by the excitation light beams while passing through the flow chamber 15. The optical axis of light beams collected by the side collection unit 130 is approximately perpendicular to, or about 90 degrees, from the optical axis of the light beams directed toward the interrogation zone 18 in the flow chamber 15.

[0046] The forward collection unit 150 collects forward scattered light from the particles. The optical axis of light beams collected by the forward collection unit 150 is approximately parallel to, or about 0 degrees from, the optical axis of the light beamsdirected toward the interrogation zone 18 in the flow chamber 15. The side collection unit 130 and the forward collection unit 150 are each described now in further detail below.

[0047] 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 side scattered light and the fluorescent light that diverge in various directions from the particles at the interrogation zone 18.

[0048] The concave mirror 134 and the aspheric lens 135 focus the side scattered light and the fluorescent light onto the collection fiber 136 by converging the side scattered light and the fluorescent light on the same point of the collection fiber 136 as shown in the dotted block 139 in FIG. 2. The concave mirror 134 focuses the side scattered light and the fluorescent light on the fiber, while the aspheric lens 135 reduces the focal point to mitigate aberration.

[0049] To prevent crosstalk, the beam splitter 133 is arranged to separate the side scattered light that has high intensity from the fluorescent light that has low intensity. The side scattered light is directed toward the first wavelength division multiplexer 131 via a first fiber 137, and the fluorescent light is directed toward the second wavelength division multiplexer 132 via a second fiber 138. Optical signals of different wavelengths are separated in the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 for analysis.

[0050] The beam splitter 133 includes a dichroic mirror 160 and a notch filter 162. Collected light is directed into the beam splitter 133 toward the dichroic minor 160 by the collection fiber 136, which may be oriented such that the light beam is directed toward the dichroic mirror 160 at an incident angle of, for example, 45 degrees. The dichroic mirror 160 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.

[0051] The fluorescent light coming out of the collection fiber 136 passes through dichroic mirror 160, and is incident to the notch filter 162 at an incident angle of about 90 degrees and then passes through the notch filter 162. The fluorescent light enters the second wavelength division multiplexer 132 through the second fiber 138.

[0052] The dichroic mirror 160 and the notch filter 162 can each have multiple bands according to the arrangement of the light sources 11 la- 11 Id. In the example shown in FIG. 2, the dichroic mirror 160 and the notch filter 162 both have four bands that filter four laser wavelengths. The number of bands on the dichroic mirror 160 and the notch filter 162 can correspond to the number of the light sources 111 a- 111 d.

[0053] The beam splitter 133 separates the side scattered light having high intensity from the fluorescent light having low intensity, which reduces or prevents crosstalk of the side scattered light to the fluorescent light. In addition, by providing the beam splitter 133, 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 their configuration may be changed, and are not limited to the example shown and described herein.

[0054] The first wavelength division multiplexer 131 separates optical signals within the side scattered light based on wavelength. For example, the first wavelength division multiplexer 131 separates optical signals including red side scattered light caused by the light beam emitted by the first light source I l la, yellow side scattered light caused by the light beam emitted by the second light source 111b, blue side scattered light caused by the light beam emitted by the third light source 111c, and violet side scattered light caused by the light beam emitted by the fourth light source 11 Id. As shown in FIG. 2. the separated optical signals are transmitted along an optical transmission path 164 toward a side scatter (SSC) detector 172.

[0055] The first wavelength division multiplexer 131 includes a plurality of SSC detectors 172a-172f for collecting side scatter light across multiple optical signals including violet side scattered light, blue side scattered light, yellow side scattered light, and red side scattered light. Examples of the SSC detectors 172a-172f include photodiodes, avalanche photodiodes (APDs), or photomultiplier tubes for collecting side scattered light corresponding to a given optical signal (e.g., violet, blue, yellow7, red). The first wavelength division multiplexer 131 includes two SSC detectors 172a, 172b for collecting side scattered light across the violet light spectrum.

[0056] Each optical transmission path 164 includes filters 166, 168 and a collimating lens 170 for each optical signal of the side scattered light. The filters 166, 168 are arranged at a predetermined distance from each other along the optical transmission path 164. Crosstalk between the side scattered light can be reduced orprevented by providing the filters 166, 168 in a non-parallel arrangement to avoid reflections of light and to achieve a better optical density.

[0057] The second wavelength division multiplexer 132 receives fluorescent light from the beam splitter 133 via the second fiber 138, and separates optical signals having different wavelengths within the fluorescent light. In the second wavelength division multiplexer 132, each optical signal of the fluorescent light is transmitted along an optical transmission path 180. Since the optical signals of the fluorescent light is weak, each of the optical transmission paths 180 includes a single filter 186. The filtered fluorescent light is then received by a light detection element 182 (e.g., a photodiode, an avalanche photodiode (APD), a photomultiplier tube).

[0058] 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 optical signals. 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 160 with no notch filter 162.

[0059] 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.

[0060] The forward collection unit 150 includes an obscuration bar 155, a concave mirror 151, a filter 157, and a forward detector 159. The obscuration bar 155 blocks a large portion of the light transmitted through the flow chamber 15 to reduce background noise created by the excitation light beams transmitting directly through the flow chamber 15, and to allow collection of only forward scattered light from the particles. In some examples, the majority of the transmitted light is blocked so as not to saturate the forward detector 159.

[0061] The concave mirror 151 reflects a forward scattered beam toward the forward detector 159. The filter 157 allows forward scattered light with a high signal - to-noise ratio to pass, and blocks other light. The forward detector 159 receives the filtered forward scattered light from the filter 157, for capturing and processing the forward scattered light.

[0062] Referring back to the side collection unit 130 that collects the side scattered light of the particles passing through the interrogation zone 18, the first wavelength division multiplexer 131 includes a plurality of SSC detectors 172a-172f. In the illustrative example shown in FIG. 2, the first wavelength division multiplexer 131 includes a first SSC detector 172a, a second SSC detector 172b, a third SSC detector 172c, a fourth SSC detector 172d, a fifth SSC detector 172e, and a sixth SSC detector 172f. In alternative examples, the first wavelength division multiplexer 131 can include more than six SSC detectors 172, or fewer than six SSC detectors 172.

[0063] The first wavelength division multiplexer 131 receives the side scattered light from the beam splitter 133 via the first fiber 137, and the first wavelength division multiplexer 131 separates optical signals of the side scattered light based on wavelength. A first range of optical signals in the violet light spectrum emitted by the fourth light source 11 Id pass through a first filter 166a while the remaining optical signals in the side scattered light are reflected by the first filter 166a, allowing capture of the first range of optical signals in the violet light spectrum by the first SSC detector 172a. The remaining optical signals reflect off a mirror 174 toward a second filter 166b. A second range of optical signals in the violet light spectrum emitted by the fourth light source 11 Id pass through the second filter 166b while the remaining optical signals in the side scattered light are reflected by the second filter 166b, allowing capture of the second range of optical signals in the violet light spectrum by the second SSC detector 172b.

[0064] In view of the foregoing, the flow cytometer 100 includes tw o SSC detectors 172a. 172b for collecting side scattered light across the violet light spectrum. The first and second SSC detectors 172a, 172b allow the flow cytometer 100 to cover the entire spectrum of side scattered light supported by the fourth light source 11 Id which emits light in the violet light spectrum (e.g., 380-450 nanometers). The first SSC detector 172a captures a first range within the violet light spectrum that is optimal for particles ranging in size from 40 nanometers to 150 nanometers while the second SSC detector 172b captures a second range within the violet light spectrum that is optimal for particles ranging in size from 80 nanometers to 1,000 nanometers. The first SSC detector 172a has a first sensitivity for detecting the violet side scattered light, and the second SSC detector 172b has a second sensitivity for detecting the violet side scattered light, where the first sensitivity of the first SSC detector 172a is higher than the second sensitivity of the second SSC detector 172b. This arrangement allows the second SSCdetector 172b to capture violet side scattered light for particles saturated by the first sensitivity of the first SSC detector 172a.

[0065] As described in more detail, the light collected by the second SSC detector 172b is used to extend the dynamic range of the first SSC detector 172a to include at least six decades of violet side scattered light. For example, the dynamic range of the first SSC detector 172a is extended to include violet side scattered light from particles ranging in size from 40 nanometers to 1000 nanometers, which can be displayed on a single plot on the display monitor 204.

[0066] Sill referring to FIG. 2, the remaining optical signals reflected by the second filter 166b reflect off the mirror 174 toward a third filter 166c. Optical signals in the blue light spectrum emitted by the third light source 111c pass through the third filter 166c while the remaining optical signals in the side scattered light are reflected by the third filter 166c, allowing capture of the optical signals in the blue light spectrum by the third SSC detector 172c.

[0067] As further shown in FIG. 2, the remaining optical signals reflect off the mirror 174 toward a fourth filter 166d. Optical signals in the yellow light spectrum emitted by the second light source 111b pass through the fourth filter 166d while the remaining optical signals in the side scattered light are reflected by the fourth filter 166d, allowing capture of the optical signals in the yellow light spectrum by the fourth SSC detector 172d.

[0068] As further shown in the first wavelength division multiplexer 131 of FIG. 2, the remaining optical signals reflect off the mirror 174 toward a fifth filter 166e.Optical signals in the red light spectrum emitted by the first light source I l la pass through the fifth filter 166e while the remaining optical signals in the side scattered light are reflected by the fifth filter 166e, allowing capture of the optical signals in the red light spectrum by the fifth SSC detector 172e. Thereafter, the remaining optical signals in the side scattered light are reflected by the mirror 174 toward a sixth filter 166f for collection by a sixth SSC detector 172f.

[0069] FIG. 3 schematically illustrates an example of a method 300 of characterizing particles by flow cytometry . In some instances, the method 300 is performed by the flow cytometer 100. Alternatively, the method 300 can be performed by the workstation 200. Under either example embodiment, the method 300 extends the dynamic range of the of the first SSC detector 172a based on the light captured by the second SSC detector 172b. As describe above, the first and second SSC detectors 172a,172b both capture side scattered light in the violet light spectrum, and the first and second SSC detectors 172a, 172b can include photodiodes, avalanche photodiodes (APD), or photomultiplier tubes for collecting the violet side scattered light.

[0070] The method 300 includes an operation 302 of determining a first median scatter intensity for one or more particle populations based on the light scatter collected by the first SSC detector 172a. The one or more particle populations can range in size from particles having a diameter of 40 nanometers to particles having a diameter of 150 nanometers.

[0071] FIG. 4 illustrates an example of side scattered light data 400 collected by the first and second SSC detectors 172a, 172b of the flow cytometer 100. In this illustrative example, the side scattered light data 400 includes a first set of violet side scattered light data 402 collected by the first SSC detector 172a and a second set of violet side scattered light data 404 collected by the second SSC detector 172b. The first and second sets of violet side scattered light data 402, 404 are each collected using a gain of 50 for both the first and second SSC detectors 172a, 172b.

[0072] The side scattered light data 400 further includes a table 406 having a first column 408 of median side scattered light intensities determined based on the optical signals collected by the first SSC detector 172a and a second column 410 of median side scattered light intensities determined based on the optical signals collected by the second SSC detector 172b for particle populations having diameters of 44 nanometers. 80 nanometers, 100 nanometers, 144 nanometers, 300 nanometers, 600 nanometers, and 1000 nanometers.

[0073] FIG. 5 illustrates another example of side scattered light data 500 collected by the first and second SSC detectors 172a, 172b of the flow cytometer 100. In this illustrative example, the side scattered light data 500 includes a first set of violet side scattered light data 502 collected by the first SSC detector 172a under a gain of 100 and a second set of violet side scattered light data 504 collected by the second SSC detector 172b under a gain of 100.

[0074] The side scattered light data 500 further includes a table 506 having a first column 508 of median side scattered light intensities determined based on the optical signals collected by the first SSC detector 172a and a second column 510 of median side scattered light intensities determined based on the optical signals collected by the second SSC detector 172b for particle populations having diameters of 44 nanometers.80 nanometers, 100 nanometers, 144 nanometers, 300 nanometers, 600 nanometers, and 1000 nanometers.

[0075] FIG. 6 illustrates another example of side scattered light data 600 collected by the first and second SSC detectors 172a, 172b of the flow cytometer 100. In this illustrative example, the side scattered light data 600 includes a first set of violet side scattered light data 602 collected by the first SSC detector 172a under a gain of 150 and a second set of violet side scattered light data 604 collected by the second SSC detector 172b under a gain of 100.

[0076] The side scattered light data 600 further includes a table 606 having a first column 608 of median side scattered light intensities determined based on the optical signals collected by the first SSC detector 172a and a second column 610 of median side scattered light intensities determined based on the optical signals collected by the second SSC detector 172b for particle populations having diameters of 44 nanometers, 80 nanometers, 100 nanometers, 144 nanometers, 300 nanometers, 600 nanometers, and 1000 nanometers.

[0077] Referring now to FIGS. 3-6, operation 302 of the method 300 can include determining first median violet side scattered light intensities for particle populations of 44 nanometers, 80 nanometers, 100 nanometers, and 144 nanometers based on the first range of optical signals in the violet light spectrum captured by the first SSC detector 172a. The first median violet side scattered light intensities determined in operation 302 are highlighted in the first columns 408, 508, and 608 respectively in tables 406, 506, and 606 of FIGS. 4-6.

[0078] The method 300 includes an operation 304 of determining a second median scatter intensity for one or more particle populations based on the light scatter collected by the second SSC detector 172b. The one or more particle populations can range in size from particles having a diameter of 80 nanometers to particles having a diameter of 1,000 nanometers.

[0079] Operation 304 can include determining second median violet side scattered light intensities for particle populations of 80 nanometers, 100 nanometers. 144 nanometers, 300 nanometers, 600 nanometers, and 1,000 nanometers based on the second range of optical signals in the violet light spectrum captured by the second SSC detector 172b. The second median violet side scattered light intensities determined in operation 304 are highlighted in the second columns 410. 510, and 610 respectively in the tables 406, 506, and 606 of FIGS. 4-6.

[0080] The method 300 includes an operation 306 of determining a ratio of the second median scatter intensity relative to the first median scatter intensity for one or more overlapping particle populations. Examples of the ratios determined for the second median scatter intensity relative to the first median scatter intensity for overlapping particle populations are shown in columns 412, 512, and 612 respectively in the tables 406, 506, and 606 of FIGS. 4-6.

[0081] Operation 306 can include determining a ratio of the second median scatter intensity relative to the first median scatter intensity for an overlapping particle population having a diameter of 80 nanometers. In table 406, where the gain for the first and second SSC detectors 172a, 172b is set at 50, the ratio is about 125. In table 506, where the gain for the first and second SSC detectors 172a, 172b is set at 100, the ratio is about 128. In table 606, where the gain for the first and second SSC detectors 172a, 172b is set at 150, the ratio is about 127.

[0082] Alternatively, or additionally , operation 306 can include determining a ratio of the second median scatter intensity relative to the first median scatter intensity for an overlapping particle population having a diameter of 100 nanometers. In table 406, the ratio is about 127. In table 506, the ratio is about 126. In table 606, the ratio is about 127.

[0083] Alternatively, or additionally, operation 306 can include determining a ratio of the second median scatter intensity relative to the first median scatter intensity for an overlapping particle population having a diameter of 144 nanometers. In table 406, the ratio is about 125. In table 506, the ratio is about 126. In table 606, the ratio is about 126.

[0084] In examples where operation 306 includes determining ratios of the second median scatter intensity relative to the first median scatter intensity for multiple overlapping particle populations (e.g., 80 nanometers, and / or 100 nanometers, and / or 144 nanometers), operation 306 can include calculating an average ratio. As shown in the columns 412, 512, and 612, the average ratio of the second median scatter intensity to the first median scatter intensity’ is about 125-127.

[0085] The method 300 includes an operation 308 of extending a dynamic range of the first SSC detector 172a by multiplying the ratio determined in operation 306 by a median scatter intensity of one or more second particle populations measured by the second SSC detector 172b.

[0086] In the illustrative example shown in FIG. 4, a dynamic range of the first SSC detector 172a shown in column 414 includes the first set of median scatter intensities for particles having a diameter of 44 nanometers, 80 nanometers, 100 nanometers, and 144 nanometers, and is extended to include an extended set of median scatter intensities 416 for particles having a diameter of 300 nanometers, 600 nanometers, and 1.000 nanometers.

[0087] The extended set of median scatter intensities 416 would be saturated by the sensitivity of the first SSC detector 172a. The side scattered light data collected by the second SSC detector 172b can extend the dynamic range of the first SSC detector 172a to include at least six decades of violet side scattered light. As an illustrative example, the dynamic range of the first detector is extended to include violet side scattered light from particles ranging in size from 40 nanometers to 1000 nanometers

[0088] As shown in column 414, the median scatter intensity for particles having a size of 300 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (125.4854827) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 300 nanometers (76,240.9) resulting in a stitched value of 9,567,126. 137 in the extended set of median scatter intensities 416.

[0089] The median scatter intensity for particles having a size of 600 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (125.4854827) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 600 nanometers (301,302.9) resulting in a stitched value of 37,809,139.84 in the extended set of median scatter intensities 416.

[0090] The median scatter intensity for particles having a size of 1,000 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (125.4854827) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 1,000 nanometers (1,033,122.3) resulting in a stitched value of 129,641,850.5 in the extended set of median scatter intensities 416.

[0091] In the illustrative example show n in FIG. 5, the dynamic range of the firstSSC detector 172a includes the first set of median scatter intensities for particles having a diameter of 44 nanometers, 80 nanometers, 100 nanometers, and 144 nanometers, andis extended to include an extended set of median scatter intensities 516 for particles having a diameter of 300 nanometers, 600 nanometers, and 1,000 nanometers.

[0092] As shown in column 514, the median scatter intensity for particles having a size of 300 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (125.5925671) and the median scatter intensity7of particles captured by the second SSC detector 172b for particles having a size of 300 nanometers ( 151,760.7) resulting in a stitched value of 19,060,015.9 in the extended set of median scatter intensities 516.

[0093] The median scatter intensity for particles having a size of 600 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (125.5925671) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 600 nanometers (601,426) resulting in a stitched value of 75,534,635.27 in the extended set of median scatter intensities 516.

[0094] The median scatter intensity for particles having a size of 1.000 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (125.5925671) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 1,000 nanometers (2,042,295.8) resulting in a stitched value of 256,497,172 in the extended set of median scatter intensities 516.

[0095] In the illustrative example shown in FIG. 6, the dynamic range of the first SSC detector 172a includes the first set of median scatter intensities for particles having a diameter of 44 nanometers, 80 nanometers, 100 nanometers, and 144 nanometers, and is extended to include an extended set of median scatter intensities 616 for particles having a diameter of 300 nanometers, 600 nanometers, and 1,000 nanometers.

[0096] As shown in the column 614, the median scatter intensity for particles having a size of 300 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (126. 1021014) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 300 nanometers (189,916.6) resulting in a stitched value of 23,948,882.35 in the extended set of median scatter intensities 616.

[0097] The median scatter intensity for particles having a size of 600 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (126.1021014) and the median scatter intensity of particles captured by thesecond SSC detector 172b for particles having a size of 600 nanometers (749,973.9) resulting in a stitched value of 94,573,284.78 in the extended set of median scatter intensities 616.

[0098] The median scatter intensity for particles having a size of 1,000 nanometers is calculated by multiplying the ratio determined for particles having a size of 144 nanometers (126.1021014) and the median scatter intensity of particles captured by the second SSC detector 172b for particles having a size of 1,000 nanometers (2.514,912.5) resulting in a stitched value of 317,135,751.1 in the extended set of median scatter intensities 616.

[0099] FIG. 7 illustrates an example of a display chart 700 showing the dynamic range of the first SSC detector 172a that has been extended in accordance with the method 300. The dynamic range of the first SSC detector 172a displayed in the display chart 700 is extended to include median scatter intensities of particles ranging in size from a diameter of 40 nanometers to 1,000 nanometers. The dynamic range of the first SSC detector 172a can be extended to include at least six decades of violet side scattered light for display on the display chart 700.

[0100] FIG. 8 schematically illustrates an example of a method 800 of displaying data captured by flow cytometry'. The method 800 can be performed by the workstation 200.

[0101] The method 800 includes an operation 802 of receiving a first set of median scatter intensities from the first SSC detector 172a. Illustrative examples of the first set of median scatter intensities from the first SSC detector 172a are show n in the first columns 408, 508, and 608 respectively shown in the tables 406, 506, and 606 in FIGS. 4-6. In such examples, the first set of median scatter intensities received from the first SSC detector 172a includes particles ranging in size from 40 nanometers to 150 nanometers. In operation 802, the w orkstation 200 can receive the first set of median scatter intensities from the first SSC detector 172a via a wired or wireless connection to the flow cytometer 100.

[0102] The method 800 includes an operation 804 of receiving a second set of median scatter intensities from the second SSC detector 172b. Illustrative examples of the second set of median scatter intensities from the second SSC detector 172b are shown in the second columns 410, 510, and 610 respectively shown in the tables 406, 506, and 606 in FIGS. 4-6. In such examples, the second set of median scatter intensities received from the second detector 172b includes particles ranging in sizefrom 80 nanometers to 1,000 nanometers. In operation 804, the workstation 200 can receive the second set of median scatter intensities from the second SSC detector 172b via a wired or wireless connection to the flow cytometer 100.

[0103] The method 800 includes an operation 806 of extending the first set of median scatter intensities to include an extended set of median scatter intensities. Illustrative examples of the extended set of median scatter intensities are shown by reference numerals 416, 516, and 616 in FIGS. 4-6. The extended set of median scatter intensities is calculated based on a ratio (see columns 412, 512, and 612 in FIGS. 4-6) of the second set of median scatter intensities received in operation 804 to the first set of median scatter intensities received in operation 802.

[0104] The method 800 includes an operation 808 of displaying on a single plot a dynamic range that includes the first set of median scatter intensities received in operation 802 and the extended set of median scatter intensities determined in operation 806. An illustrative example of the single plot displayed in operation 808 is show n in FIG. 7. In this manner, the dynamic range displayed on a single plot for violet side scattered light can include at least six decades of violet side scattered light for particles ranging in size from 40 nanometers to 1000 nanometers.

[0105] FIG. 9 schematically illustrates an example of a computing device 900 for implementing aspects of the system 10, including functions of the flow cytometer 100 and the workstation 200. Examples of the computing device 900 include a server computer, a desktop computer, a laptop computer, a tablet computer, a mobile computing device (such as a smartphone), or other devices configured to process digital instructions.

[0106] The computing device 900 includes one or more processing devices 902. Examples of the one or more processing devices 902 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 902 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.

[0107] The computing device 900 further includes a system memory 904, and a system bus 906 that couples various system components including the system memory 904 to the one or more processing devices 902. The system bus 906 is one of anynumber of types of bus structures including a memory bus, or a memory controller; a peripheral bus; and a local bus using any of a variety of bus architectures.

[0108] The system memory 904 can include a read only memory (ROM) 908 and a random access memory (RAM) 910. A basic input / output system (BIOS) 912 containing the basic routines that act to transfer information within computing device 900, such as during start up, can be stored in the system memory 904. The RAM 910 can be used for loading and subsequently analyzing the waveform data (e.g.. stored in a raw waveform data file, which can include digitalized raw waveform data).

[0109] The computing device 900 can also include one or more secondary storage devices 914 such as a hard disk drive for storing digital data. The one or more secondary storage devices 914 are connected to the system bus 906 by a secondary storage interface 916. The one or more secondary storage devices 914 and associated computer readable media provide nonvolatile storage of computer readable instructions (including application programs and program modules), data structures, and other data for the computing device 900. Although the example described herein employs a hard disk drive as a secondary storage device, other types of computer readable storage media are used in other embodiments. Examples of these other types of computer readable storage media include the ROM 908 and / or the RAM 910. Some examples include non-transitory media. Additionally, such computer readable storage media can include local storage or cloud-based storage.

[0110] The computing device 900 typically includes at least some form of computer readable media. Computer readable media includes any available media that can be accessed by the computing device 900. By way of example, computer readable media include computer readable storage media and computer readable communication media.

[0111] Computer readable storage media includes volatile and nonvolatile, removable and non-removable media implemented in any device configured to store information such as computer readable instructions, data structures, program modules or other data. Computer readable storage media includes, but is not limited to, random access memory, read only memory, electrically erasable programmable read only memory, flash memory or other memory technology7, or any other medium that can be used to store the desired information and that can be accessed by the computing device 900.

[0112] Computer readable communication media can embody computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term "modulated data signal” refers to a signal that has one or more characteristics set in a manner as to encode information in the signal. For example, computer readable communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency, infrared, and other wireless media. Combinations of any of the above are also included within the scope of computer readable media.

[0113] A number of program modules can be stored in secondary storage device 914 or the system memory 904, including an operating system 918. application programs 920, program modules 922 (such as the software engines), and program data 924. The computing device 900 can utilize any suitable operating system, such as Microsoft Windows™, Google Chrome™, Apple OS, and any other operating system suitable for a computing device.

[0114] A user provides inputs to the computing device 900 through one or more input devices 926. Examples of input devices 926 include the mouse 206, the keyboard 208, a microphone 932, and a touch sensor 934 (such as a touchpad or touch sensitive display). Additional types of the input devices 926 are contemplated. The input devices 926 are often connected to the one or more processing devices 902 through an input / output interface 936 that is coupled to the system bus 906. These input devices 926 can be connected by any number of input / output interfaces, such as a parallel port, serial port, game port, or a universal serial bus. Wireless communication between input devices and the input / output interface 936 is possible as well, and includes infrared, BLUETOOTH® wireless technology, 802.11a / b / g / n, cellular, or other radio frequency communication systems in some possible embodiments.

[0115] The display monitor 204 can include a liquid crystal display device, a touch sensitive display device, and the like. The display monitor 204 connects to the system bus 906 via an interface, such as a video adapter 940. In addition to the display monitor 204, the computing device 900 can include various other peripheral devices, such as speakers or a printer.

[0116] When used in a local area networking environment or a wide area networking environment (such as the Internet), the computing device 900 is typically connected to a network 944 through a network interface 942, such as an Ethernetinterface. Other possible embodiments use other communication devices. For example, some embodiments of the computing device 900 include a modem for communicating across the network 944.

[0117] The computing device 900 is an example of programmable electronics, which may include one or more such computing devices. When multiple computing devices are included, such computing devices can be coupled together with a suitable data communication network so as to collectively perform the various functions, methods, or operations disclosed herein.

[0118] 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 characterizing particles by flow cytometry, the system comprising: a light emitting unit configured to emit a light beam; a collection unit including: a first detector for collecting light scatter resulting from interrogation of the particles by the light beam; a second detector for collecting the light scatter resulting from the interrogation of the particles by the light beam; and a processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: determine a first median scatter intensity of a first particle population based on the light scatter collected by the first detector; determine a second median scatter intensity7of the first particle population based on the light scatter collected by the second detector: determine a ratio of the second median scatter intensity relative to the first median scatter intensity; and extend a dynamic range of the first detector by multiplying the ratio by a median scatter intensity of a second particle population measured by the second detector.

2. The system of claim 1, wherein the light beam is in the violet light spectrum, and the first and second detectors are configured to detect violet side scattered light.

3. The system of claim 2, wherein the first detector has a first sensitivity7for detecting the violet side scattered light, and the second detector has a second sensitivity for detecting the violet side scattered light, and wherein the first sensitivity is higher than the second sensitivity.

4. The system of claim 3, wherein the second particle population is saturated by the first sensitivity7of the first detector.

5. The system of any of claims 1-4, wherein the dynamic range of the first detector is extended to include at least six decades of violet side scattered light.

6. The system of any of claims 1-5, wherein the dynamic range of the first detector is extended to include violet side scattered light from particles ranging in size from 40 nanometers to 1000 nanometers.

7. The system of any of claims 1-6, wherein the first particle population ranges in size from 40 nanometers to 150 nanometers.

8. The system of any of claims 1-7, wherein the second particle population ranges in size from 80 nanometers to 1,000 nanometers.

9. A method of characterizing particles by flow cytometry, the method comprising: determining a first median scatter intensity of a first particle population, the first median scatter intensity being based on light scatter collected by a first detector; determining a second median scatter intensity of the first particle population, the second median scatter intensity being based on light scatter collected by a second detector; determining a ratio of the second median scatter intensity relative to the first median scatter intensity; and extending a dynamic range of the first detector by multiplying the ratio by a median scatter intensity of a second particle population, the median scatter intensity of the second particle population being based on the light scatter collected by the second detector.

10. The method of claim 9, wherein the first and second detectors are configured to detect violet side scattered light.

11. The method of claim 10. wherein the first detector has a first sensitivity for detecting the violet side scattered light, and the second detector has a second sensitivity for detecting the violet side scattered light, and wherein the first sensitivity is higher than the second sensitivity.12 The method of claim 11, wherein the second particle population is saturated by the first sensitivity of the first detector.

13. The method of any of claims 9-12, wherein the dynamic range of the first detector is extended to include at least six decades of violet side scattered light.

14. The method of any of claims 9-13. wherein the dynamic range of the first detector is extended to include violet side scattered light from particles ranging in size from 40 nanometers to 1000 nanometers.

15. The method of any of claims 9-14. wherein the first particle population ranges in size from 40 nanometers to 150 nanometers, and wherein the second particle population ranges in size from 80 nanometers to 1,000 nanometers.

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