WO2023102675A1

WO2023102675A1 - Flow cytometry systems with image sensors

Info

Publication number: WO2023102675A1
Application number: PCT/CN2021/135594
Authority: WO
Inventors: Peiyan CAO; Yurun LIU
Original assignee: Shenzhen Genorivision Technology Co., Ltd.
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2023-06-15
Also published as: TW202338319A; CN118489055A

Abstract

A flow cytometry system(700) and a method. The system comprises: a light source system(710) configured to send light beams (i)(712), i=1, …, M respectively to interrogation points (i)(724), i=1, …, M in a tube(720), wherein the light beams (i)(712), i=1, …, M have different spectra, and wherein M is an integer greater than 1; detectors (i)(100), i=1, …, M; and a collimator(730) configured to, for each value of i, (A) provide a light path for light from the interrogation point (i)(724) to the detector (i)(100), and (B) prevent any light from the interrogation point (i)(724) from reaching any detector (j)(100), j=1, …, M and j≠ i.

Description

FLOW CYTOMETRY SYSTEMS WITH IMAGE SENSORS

Background

A radiation detector is a device that measures a property of a radiation. Examples of the property may include a spatial distribution of the intensity, phase, and polarization of the radiation. The radiation measured by the radiation detector may be a radiation that has transmitted through an object. The radiation measured by the radiation detector may be electromagnetic radiation such as infrared light, visible light, ultraviolet light, X-ray, or γ-ray. The radiation may be of other types such as α-rays and β-rays. An imaging system may include one or more image sensors each of which may have one or more radiation detectors.

Summary

Disclosed herein is a flow cytometry system, comprising: a light source system configured to send light beams (i) , i=1, …, M respectively to interrogation points (i) , i=1, …, M in a tube, wherein the light beams (i) , i=1, …, M have different spectra, and wherein M is an integer greater than 1; detectors (i) , i=1, …, M; and a collimator configured to, for each value of i, (A) provide a light path for light from the interrogation point (i) to the detector (i) , and (B) prevent any light from the interrogation point (i) from reaching any detector (j) , j=1, …, M and j ≠ i.

In an aspect, the light beams (i) , i=1, …, M have different maximum photon energies.

In an aspect, for each value of i, the light beam (i) does not incident on the interrogation point (j) , j=1, …, M and j ≠ i.

In an aspect, the collimator prevents any unscattered portion of the light beams (i) , i=1, …, M from reaching any point of the detectors (i) , i=1, …, M.

In an aspect, the flow cytometry system further comprises a sorter configured to sort particles flowing in single file along the tube based on light emitted by the particles under excitation by the light beams (i) , i=1, …, M or light scattered by the particles when the particles pass through the interrogation points (i) , i=1, …, M.

In an aspect, the flow cytometry system further comprises filters (i) , i=1, …, M, wherein for each value of i, the filter (i) is configured to prevent light from the interrogation point (i) and of pre-specified energies from reaching the detector (i) .

In an aspect, the light source system comprises light sources (i) , i=1, …, M configured to respectively generate the light beams (i) , i=1, …, M.

In an aspect, the light source system comprises a prism configured to receive as input a white light beam and thereby generate as output the light beams (i) , i=1, …, M.

In an aspect, the light beams (i) , i=1, …, M are pencil beams.

In an aspect, the light beams (i) , i=1, …, M are fan beams, and the light source system comprises an optical system configured to converge the light beams (i) , i=1, …, M respectively to the interrogation points (i) , i=1, …, M.

In an aspect, each detector of the detectors (i) , i=1, …, M comprises one or more avalanche photodiodes (APD) .

In an aspect, said one or more APDs are one or more single-photon avalanche diodes (SPAD) .

In an aspect, for each value of i, the detector (i) comprises Ni sensing elements, Ni being a positive integer, and for each value of i, the flow cytometry system is configured to add all picture elements of an image captured by the Ni sensing elements, resulting in a sum (i) .

Disclosed herein is a method, comprising: sending light beams (i) , i=1, …, M respectively to interrogation points (i) , i=1, …, M in a tube, wherein the light beams (i) , i=1, …, M have different spectra, and wherein M is an integer greater than 1; flowing particles in single file in the tube through the interrogation points (i) , i=1, …, M; and for each value of i, channeling with a collimator light from the interrogation point (i) to a detector (i) , wherein the collimator is configured to prevent any light from the interrogation point (i) from reaching any detector (j) , j=1, …, M and j ≠ i.

In an aspect, the method further comprises sorting the particles based on light emitted by the particles under excitation by the light beams (i) , i=1, …, M or light scattered by the particles when the particles pass through the interrogation points (i) , i=1, …, M.

Brief Description of Figures

Fig. 1 schematically shows a radiation detector, according to an embodiment.

Fig. 2 schematically shows a simplified cross-sectional view of the radiation detector, according to an embodiment.

Fig. 3 schematically shows a detailed cross-sectional view of the radiation detector, according to an embodiment.

Fig. 4 schematically shows a detailed cross-sectional view of the radiation detector, according to an alternative embodiment.

Fig. 5 schematically shows a top view of a radiation detector package including the radiation detector and a printed circuit board (PCB) , according to an embodiment.

Fig. 6 schematically shows a cross-sectional view of an image sensor including the packages of Fig. 5 mounted to a system PCB (printed circuit board) , according to an embodiment.

Fig. 7 schematically shows a flow cytometry system, according to an embodiment.

Fig. 8 shows a flowchart generalizing the operation of the flow cytometry system, according to an embodiment.

Detailed Description

RADIATION DETECTOR

Fig. 1 schematically shows a radiation detector 100, as an example. The radiation detector 100 may include an array of pixels 150 (also referred to as sensing elements 150) . The array may be a rectangular array (as shown in Fig. 1) , a honeycomb array, a hexagonal array, or any other suitable array. The array of pixels 150 in the example of Fig. 1 has 4 rows and 7 columns; however, in general, the array of pixels 150 may have any number of rows and any number of columns.

Each pixel 150 may be configured to detect radiation from a radiation source (not shown) incident thereon and may be configured to measure a characteristic (e.g., the energy of the particles, the wavelength, and the frequency) of the radiation. A radiation may include radiation particles such as photons (X-rays, gamma rays, etc. ) and subatomic particles (alpha particles, beta particles, etc. ) Each pixel 150 may be configured to count numbers of particles of radiation incident thereon whose energy falls in a plurality of bins of energy, within a period of time. All the pixels 150 may be configured to count the numbers of particles of radiation incident thereon within a plurality of bins of energy within the same period of time. When the incident particles of radiation have similar energy, the pixels 150 may be simply configured to count numbers of particles of radiation incident thereon within a period of time, without measuring the energy of the individual particles of radiation.

Each pixel 150 may have its own analog-to-digital converter (ADC) configured to digitize an analog signal representing the energy of an incident particle of radiation into a digital signal, or to digitize an analog signal representing the total energy of a plurality of incident particles of radiation into a digital signal. The pixels 150 may be configured to operate in parallel. For example, when one pixel 150 measures an incident particle of radiation, another pixel 150 may be waiting for a particle of radiation to arrive. The pixels 150 may not have to be individually addressable.

The radiation detector 100 described here may have applications such as in an X-ray telescope, X-ray mammography, industrial X-ray defect detection, X-ray microscopy or microradiography, X-ray casting inspection, X-ray non-destructive testing, X-ray weld inspection, X-ray digital subtraction angiography, etc. It may be suitable to use this radiation detector 100 in place of a photographic plate, a photographic film, a PSP plate, an X-ray image intensifier, a scintillator, or another semiconductor X-ray detector.

Fig. 2 schematically shows a simplified cross-sectional view of the radiation detector 100 of Fig. 1 along a line 2-2, according to an embodiment. Specifically, the radiation detector 100 may include a radiation absorption layer 110 and an electronics layer 120 (which may include one or more ASICs or application-specific integrated circuits) for processing or analyzing electrical signals which incident radiation generates in the radiation absorption layer 110. The radiation detector 100 may or may not include a scintillator (not shown) . The radiation absorption layer 110 may include a semiconductor material such as silicon, germanium, GaAs, CdTe, CdZnTe, or a combination thereof. The semiconductor material may have a high mass attenuation coefficient for the radiation of interest.

Fig. 3 schematically shows a detailed cross-sectional view of the radiation detector 100 of Fig. 1 along the line 2-2, as an example. Specifically, the radiation absorption layer 110 may include one or more diodes (e.g., p-i-n or p-n) formed by a first doped region 111, one or more discrete regions 114 of a second doped region 113. The second doped region 113 may be separated from the first doped region 111 by an optional intrinsic region 112. The discrete regions 114 may be separated from one another by the first doped region 111 or the intrinsic region 112. The first doped region 111 and the second doped region 113 may have opposite types of doping (e.g., region 111 is p-type and region 113 is n-type, or region 111 is n-type and region 113 is p-type) . In the example of Fig. 3, each of the discrete regions 114 of the second doped region 113 forms a diode with the first doped region 111 and the optional intrinsic region 112. Namely, in the example in Fig. 3, the radiation absorption layer 110 has a plurality of diodes (more specifically, 7 diodes corresponding to 7 pixels 150 of one row in the array of Fig. 1, of which only 2 pixels 150 are labeled in Fig. 3 for simplicity) . The plurality of diodes may have an electrical contact 119A as a shared (common) electrode. The first doped region 111 may also have discrete portions.

The electronics layer 120 may include an electronic system 121 suitable for processing or interpreting signals generated by the radiation incident on the radiation absorption layer 110. The electronic system 121 may include an analog circuitry such as a filter network, amplifiers, integrators, and comparators, or a digital circuitry such as a microprocessor, and memory. The electronic system 121 may include one or more ADCs (analog to digital converters) . The electronic system 121 may include components shared by the pixels 150 or components dedicated to a single pixel 150. For example, the electronic system 121 may include an amplifier dedicated to each pixel 150 and a microprocessor shared among all the pixels 150. The electronic system 121 may be electrically connected to the pixels 150 by vias 131. Space among the vias may be filled with a filler material 130, which may increase the mechanical stability of the connection of the electronics layer 120 to the radiation absorption layer 110. Other bonding techniques are possible to connect the electronic system 121 to the pixels 150 without using the vias 131.

When radiation from the radiation source (not shown) hits the radiation absorption layer 110 including diodes, particles of the radiation may be absorbed and generate one or more charge carriers (e.g., electrons, holes) by a number of mechanisms. The charge carriers may drift to the electrodes of one of the diodes under an electric field. The electric field may be an external electric field. The electrical contact 119B may include discrete portions each of which is in electrical contact with the discrete regions 114. The term “electrical contact” may be used interchangeably with the word “electrode. ” In an embodiment, the charge carriers may drift in directions such that the charge carriers generated by a single particle of the radiation are not substantially shared by two different discrete regions 114 ( “not substantially shared” here means less than 2%, less than 0.5%, less than 0.1%, or less than 0.01%of these charge carriers flow to a different one of the discrete regions 114 than the rest of the charge carriers) . Charge carriers generated by a particle of the radiation incident around the footprint of one of these discrete regions 114 are not substantially shared with another of these discrete regions 114. A pixel 150 associated with a discrete region 114 may be an area around the discrete region 114 in which substantially all (more than 98%, more than 99.5%, more than 99.9%, or more than 99.99%of) charge carriers generated by a particle of the radiation incident therein flow to the discrete region 114. Namely, less than 2%, less than 1%, less than 0.1%, or less than 0.01%of these charge carriers flow beyond the pixel 150.

Fig. 4 schematically shows a detailed cross-sectional view of the radiation detector 100 of Fig. 1 along the line 2-2, according to an alternative embodiment. More specifically, the radiation absorption layer 110 may include a resistor of a semiconductor material such as silicon, germanium, GaAs, CdTe, CdZnTe, or a combination thereof, but does not include a diode. The semiconductor material may have a high mass attenuation coefficient for the radiation of interest. In an embodiment, the electronics layer 120 of Fig. 4 is similar to the electronics layer 120 of Fig. 3 in terms of structure and function.

When the radiation hits the radiation absorption layer 110 including the resistor but not diodes, it may be absorbed and generate one or more charge carriers by a number of mechanisms. A particle of the radiation may generate 10 to 100,000 charge carriers. The charge carriers may drift to the

electrical contacts

119A and 119B under an electric field. The electric field may be an external electric field. The electrical contact 119B may include discrete portions. In an embodiment, the charge carriers may drift in directions such that the charge carriers generated by a single particle of the radiation are not substantially shared by two different discrete portions of the electrical contact 119B ( “not substantially shared” here means less than 2%, less than 0.5%, less than 0.1%, or less than 0.01%of these charge carriers flow to a different one of the discrete portions than the rest of the charge carriers) . Charge carriers generated by a particle of the radiation incident around the footprint of one of these discrete portions of the electrical contact 119B are not substantially shared with another of these discrete portions of the electrical contact 119B. A pixel 150 associated with a discrete portion of the electrical contact 119B may be an area around the discrete portion in which substantially all (more than 98%, more than 99.5%, more than 99.9%or more than 99.99%of) charge carriers generated by a particle of the radiation incident therein flow to the discrete portion of the electrical contact 119B. Namely, less than 2%, less than 0.5%, less than 0.1%, or less than 0.01%of these charge carriers flow beyond the pixel associated with the one discrete portion of the electrical contact 119B.

RADIATION DETECTOR PACKAGE

Fig. 5 schematically shows a top view of a radiation detector package 500 including the radiation detector 100 and a printed circuit board (PCB) 510. The term “PCB” as used herein is not limited to a particular material. For example, a PCB may include a semiconductor. The radiation detector 100 may be mounted to the PCB 510. The wiring between the radiation detector 100 and the PCB 510 is not shown for the sake of clarity. The package 500 may have one or more radiation detectors 100. The PCB 510 may include an input/output (I/O) area 512 not covered by the radiation detector 100 (e.g., for accommodating bonding wires 514) . The radiation detector 100 may have an active area 190 which is where the pixels 150 (Fig. 1) are located. The radiation detector 100 may have a perimeter zone 195 near the edges of the radiation detector 100. The perimeter zone 195 has no pixels 150, and the radiation detector 100 does not detect particles of radiation incident on the perimeter zone 195.

IMAGE SENSOR

Fig. 6 schematically shows a cross-sectional view of an image sensor 600, according to an embodiment. The image sensor 600 may include one or more radiation detector packages 500 of Fig. 5 mounted to a system PCB 650. The electrical connection between the PCBs 510 and the system PCB 650 may be made by bonding wires 514. In order to accommodate the bonding wires 514 on the PCB 510, the PCB 510 may have the I/O area 512 not covered by the radiation detectors 100. In order to accommodate the bonding wires 514 on the system PCB 650, the packages 500 may have gaps in between. The gaps may be approximately 1 mm or more. Particles of radiation incident on the perimeter zones 195, on the I/O area 512, or on the gaps cannot be detected by the packages 500 on the system PCB 650. A dead zone of a radiation detector (e.g., the radiation detector 100) is the area of the radiation-receiving surface of the radiation detector, on which incident particles of radiation cannot be detected by the radiation detector. A dead zone of a package (e.g., package 500) is the area of the radiation-receiving surface of the package, on which incident particles of radiation cannot be detected by the radiation detector or detectors in the package. In this example shown in Fig. 5 and Fig. 6, the dead zone of the package 500 includes the perimeter zones 195 and the I/O area 512. A dead zone (e.g., 688) of an image sensor (e.g., image sensor 600) with a group of packages (e.g., packages 500 mounted on the same PCB and arranged in the same layer or in different layers) includes the combination of the dead zones of the packages in the group and the gaps between the packages.

In an embodiment, the radiation detector 100 (Fig. 1) operating by itself may be considered an image sensor. In an embodiment, the package 500 (Fig. 5) operating by itself may be considered an image sensor.

The image sensor 600 including the radiation detectors 100 may have the dead zone 688 among the active areas 190 of the radiation detectors 100. However, the image sensor 600 may capture multiple partial images of an object or scene (not shown) one by one, and then these captured partial images may be stitched to form a stitched image of the entire object or scene.

The term “image” as used herein is not limited to spatial distribution of a property of a radiation (such as intensity) . For example, the term “image” may also include the spatial distribution of density of a substance or element or include a single value without spatial resolution.

FLOW CYTOMETRY SYSTEM

Fig. 7 schematically shows a flow cytometry system 700, according to an embodiment. In an embodiment, the flow cytometry system 700 may include a light source system 710, a tube 720, a collimator 730, and the radiation detector 100 of Fig. 1 –Fig. 4.

LIGHT SOURCE SYSTEM

In an embodiment, the light source system 710 may send light beams 712 (e.g., 712a, 712b, and 712c) respectively to interrogation points 724 (e.g., 724a, 724b, and 724c) in the tube 720. In an embodiment, each of the

light beams

712a, 712b, and 712c may include, among other things, visible light, infrared or ultraviolet or both. In an embodiment, the

light beams

712a, 712b, and 712c may have different spectra (e.g., with difference in terms of photon energy or photon wavelength) . Specifically, in an embodiment, the

light beams

712a, 712b, and 712c may have different maximum photon energies. For example, the

light beams

712a, 712b, and 712c may have maximum photon energies of 5 eV, 10 eV, and 20 eV, respectively.

In an embodiment, each light beam 712 does not incident on any interrogation point 724 other than the light beam’s corresponding interrogation point 724. In other words, the light beam 712a does not incident on any point of the

interrogation points

724b and 724c; the light beam 712b does not incident on any point of the

interrogation points

724c and 724a; and the light beam 712c does not incident on any point of the

interrogation points

724a and 724b.

TUBE AND PARTICLES

In an embodiment, the tube 720 may include any material so that particles (e.g., cells) 721.1, 721.2, and 721.3 can flow inside the tube 720. In an embodiment, a sheath fluid may be used to help carry the particles 721.1, 721.2, and 721.3 in single file inside the tube 720 in a direction 725 and through the

interrogation points

724a, 724b, and 724c.

In an embodiment, (A) the particle 721.1 may contain a first fluorophore, (B) the particle 721.2 may contain a second fluorophore, and (C) the particle 721.3 may contain a third fluorophore. For illustration, assume that (A) the first fluorophore is excited only by photons of at least 15 eV, (B) the second fluorophore is excited only by photons of at least 5 eV, and (C) the third fluorophore is excited only by photons of at least 3 eV. Assume further that the

light beams

712a, 712b, and 712c have maximum photon energies of 5 eV, 10 eV, and 20 eV, respectively.

The words “first” , “second” , and other ordinal numerals herein are used only for easy reference and do not imply any chronological order.

COLLIMATOR AND RADIATION DETECTOR

In an embodiment, the collimator 730 may include a material that blocks and absorbs (A) photons of the

light beams

712a, 712b, and 712c, and (B) photons scattered or generated at an interrogation point 724 when a particle 721 is illuminated by the corresponding light beam 712 at the interrogation point 724. In an embodiment, the collimator 730 may include tungsten.

In an embodiment, the collimator 730 may include

channels

736a, 736b, and 736c that respectively provide light paths for light from the

interrogation points

724a, 724b, and 724c to different active regions of the radiation detector 100.

Specifically, the channel 736a provides light path for light from the interrogation point 724a to the top 2 rows of sensing elements 150 (hereafter referred to as the top active region) of the radiation detector 100 as shown. The channel 736b provides light path for light from the interrogation point 724b to the middle 3 rows of sensing elements 150 (hereafter referred to as the middle active region) of the radiation detector 100 as shown. The channel 736c provides light path for light from the interrogation point 724c to the bottom 2 rows of sensing elements 150 (hereafter referred to as the bottom active region) of the radiation detector 100 as shown. In general, each active region of the top, middle, and bottom active regions may include one or more sensing elements 150.

In an embodiment, each of the

channels

736a, 736b, and 736c may include empty space (e.g., the air of the surrounding ambient) or a material transparent or not opaque to light from the

interrogation points

724a, 724b, and 724c.

In an embodiment, the collimator 730 may prevent any light from an interrogation point 724 from reaching any active region other than the interrogation point’s corresponding active region. In other words, the collimator 730 (A) prevents any light from the interrogation point 724a from reaching any point of the middle and bottom active regions of the radiation detector 100, (B) prevents any light from the interrogation point 724b from reaching any point of the top and bottom active regions of the radiation detector 100, and (C) prevents any light from the interrogation point 724c from reaching any point of the top and middle active regions of the radiation detector 100.

OPERATION OF THE FLOW CYTOMETRY SYSTEM

FIRST PARTICLE AT FIRST INTEROGATION POINT

In an embodiment, the flow cytometry system 700 may operate as follows. When the particle 721.1 is at the interrogation point 724a, the light beam 712a being aimed at the interrogation point 724a illuminates the particle 721.1 resulting in light propagating from the interrogation point 724a to the top active region of the radiation detector 100 via the channel 736a of the collimator 730.

The resulting light propagating from the interrogation point 724a to the top active region should include scattered light and no fluorescent light. Note that because (A) the light beam 712a has photons of up to 5 eV and (B) the first fluorophore in the particle 721.1 is excited only by photons of at least 15 eV, there is no fluorescent light generated by the first fluorophore in the particle 721.1 when the particle 721.1 is at the interrogation point 724a.

In an embodiment, when the particle 721.1 is at the interrogation point 724a, the top active region of the radiation detector 100 may capture an image using the light from the particle 721.1.

In an embodiment, the flow cytometry system 700 may add all picture elements of the captured image resulting a first sum. Note that the first sum is relatively low because there is no fluorescent light from the interrogation point 724a when the particle 721.1 is at the interrogation point 724a.

FIRST PARTICLE AT SECOND INTEROGATION POINT

Similarly, when the particle 721.1 is later at the interrogation point 724b, the flow cytometry system 700 may calculate a second sum for the particle 721.1. In an embodiment, the manner in which the flow cytometry system 700 calculates the second sum may be similar to the manner in which the flow cytometry system 700 calculates the first sum.

Note that because (A) the light beam 712b has photons of up to 10 eV and (B) the first fluorophore in the particle 721.1 is excited only by photons of at least 15 eV, there is no fluorescent light generated by the first fluorophore in the particle 721.1 when the particle 721.1 is at the interrogation point 724b. As a result, the second sum is relatively low.

FIRST PARTICLE AT THIRD INTEROGATION POINT

Similarly, when the particle 721.1 is later at the interrogation point 724c, the flow cytometry system 700 may calculate a third sum for the particle 721.1. In an embodiment, the manner in which the flow cytometry system 700 calculates the third sum may be similar to the manner in which the flow cytometry system 700 calculates the first sum.

Note that because (A) the light beam 712c has photons of up to 20 eV and (B) the first fluorophore in the particle 721.1 can be excited by photons of at least 15 eV, there is fluorescent light generated by the first fluorophore in the particle 721.1 when the particle 721.1 is at the interrogation point 724c. As a result, the third sum is relatively high.

THREE SUMS FOR SECOND PARTICLE

In an embodiment, the flow cytometry system 700 may calculate the first, second, and third sums for the particle 721.2. In an embodiment, the manner in which the flow cytometry system 700 calculates the first, second, and third sums for the particle 721.2 may be similar to the manner in which the flow cytometry system 700 calculates the first, second, and third sums for the particle 721.1 as described above.

Note that the first, second, and third sums for the particle 721.2 should be relatively low, relatively high, and relatively high respectively (or just low-high-high for short) .

THREE SUMS FOR THIRD PARTICLE

In an embodiment, the flow cytometry system 700 may calculate first, second, and third sums for the particle 721.3. In an embodiment, the manner in which the flow cytometry system 700 calculates the first, second, and third sums for the particle 721.3 may be similar to the manner in which the flow cytometry system 700 calculates the first, second, and third sums for the particle 721.1 as described above.

Note that the first, second, and third sums for the particle 721.3 should be relatively high, relatively high, and relatively high respectively (or just high-high-high for short) .

DETERMINATION AS TO WHICH FLUOROPHORE IS IN A PARTICLE

Assume that multiple particles each of which contains none or one of the first, second, and third fluorophores are flowed in the tube 720 in single file past the

interrogation points

724a, 724b, and 724c. In an embodiment, the flow cytometry system 700 may determine the first, second, and third sums for each particle of the multiple particles, and thereby determine which fluorophore is present in said each particle based on the determined first, second, and third sums for said each particle.

In an embodiment, if the first, second, and third sums for said each particle are low-low-low, the flow cytometry system 700 may determine that none of the first, second, and third fluorophores is present in said each particle.

If the first, second, and third sums for said each particle are low-low-high (like the case of the particle 721.1) , the flow cytometry system 700 may determine that the first fluorophore is present in said each particle.

If the first, second, and third sums for said each particle are low-high-high (like the case of the particle 721.2) , the flow cytometry system 700 may determine that the second fluorophore is present in said each particle.

If the first, second, and third sums for said each particle are high-high-high (like the case of the particle 721.3) , the flow cytometry system 700 may determine that the third fluorophore is present in said each particle.

FLOWCHART GENERALIZING OPERATION OF THE FLOW CYTOMETRY SYSTEM

Fig. 8 shows a flowchart 800 generalizing the operation of the flow cytometry system 700, according to an embodiment. Step 810 includes sending light beams (i) , i=1, …, M respectively to interrogation points (i) , i=1, …, M in a tube, and M is an integer greater than 1. For example, in the embodiments described above, with reference to Fig. 7, the

light beams

712a, 712b, and 712c are sent respectively to the

interrogation points

724a, 724b, and 724c in the tube 720 (here M = 3) .

In addition, in Step 810, the light beams (i) , i=1, …, M have different spectra. For example, in the embodiments described above, with reference to Fig. 7, the

light beams

712a, 712b, and 712c have different maximum photon energies (e.g., 5 eV, 10 eV, and 20 eV, respectively) .

Step 820 includes flowing particles in single file through the interrogation points (i) , i=1, …, M. For example, in the embodiments described above, with reference to Fig. 7, the particles 721.1, 721.2, and 721.3 are flowed in single file in the tube 720 through the

interrogation points

724a, 724b, and 724c.

Step 830 includes for each value of i, channeling with a collimator light from the interrogation point (i) to a detector (i) . For example, in the embodiments described above, with reference to Fig. 7, the collimator 730 channels light from the

interrogation points

724a, 724b, and 724c respectively to the top, middle, and bottom active regions of the radiation detector 100. Here, the top active region is considered the detector (1) ; the middle active region is considered the detector (2) ; and the bottom active region is considered the detector (3) .

In addition, in Step 830, the collimator is configured to prevent any light from the interrogation point (i) from reaching any detector (j) , j=1, …, M and j ≠ i. For example, in the embodiments described above, with reference to Fig. 7, the collimator 730 (A) prevents any light from the interrogation point 724a from reaching any point of the middle and bottom active regions of the radiation detector 100, (B) prevents any light from the interrogation point 724b from reaching any point of the top and bottom active regions of the radiation detector 100, and (C) prevents any light from the interrogation point 724c from reaching any point of the top and middle active regions of the radiation detector 100.

OTHER EMBODIMENTS

LIGHT BEAMS CANNOT GO STRAIGHT TO ACTIVE REGIONS

In an embodiment, with reference to Fig. 7, the collimator 730 may prevent any unscattered portion of the

light beams

712a, 712b, and 712c from reaching any point of the top, middle, and bottom active regions of the radiation detector 100.

PRISM EMITS LIGHT BEAMS

In an embodiment, with reference to Fig. 7, the light source system 710 may include a prism 713. In an embodiment, the prism 713 may receive as input a white light beam 715 and thereby generate as output the

light beams

712a, 712b, and 712c.

LIGHT BEAMS ARE PENCIL BEAMS OR FAN BEAMS

In an embodiment, with reference to Fig. 7, the

light beams

712a, 712b, and 712c may be pencil beams which are aimed at the

interrogation points

724a, 724b, and 724c respectively. Alternatively, the

light beams

712a, 712b, and 712c may be fan beams. As a result, the light source system 710 may include an optical system (not shown) that converge the

light beams

712a, 712b, and 712c respectively to the

interrogation points

724a, 724b, and 724c.

PARTICLE SORTER

In an embodiment, with reference to Fig. 7, the flow cytometry system 700 may further include a sorter 740 and reservoirs 750. In an embodiment, the sorter 740 may sort the particles 721.1, 721.2, and 721.3 into the reservoirs 750 based on (A) light emitted by the particles 721.1, 721.2, and 721.3 under excitation by the

light beams

712a, 712b, and 712c or (B) light scattered by the particles 721.1, 721.2, and 721.3, when the particles 721.1, 721.2, and 721.3 pass through the

interrogation points

724a, 724b, and 724c.

ALTERNATIVE EMBODIMENTS

LIGHT SOURCES GENERATE LIGHT BEAMS

In the embodiments described above, with reference to Fig. 7, the light source system 710 includes the prism 713 that is used to generate the

light beams

712a, 712b, and 712c. In an alternative embodiment, the light source system 710 may include a first light source, a second light source, and a third light source (not shown) that generate the

light beams

712a, 712b, and 712c respectively.

APD AND SPAD

In the embodiments described above, with reference to Fig. 7, the radiation detector 100 is used for detecting lights from the

interrogation points

724a, 724b, and 724c. Alternatively, a first detector, a second detector, and a third detector (not shown) may respectively substitute for the top, middle, and bottom active regions of the radiation detector 100. In an embodiment, each of the first, second, and third detectors may include one or more avalanche photodiodes (APD) . In an embodiment, each of the APDs that constitute the first, second, and third detectors mentioned above may be a single-photon avalanche diode (SPAD) .

FILTERING OUT SCATTERED LIGHTS FROM INTEROGATION POINTS

In the embodiments described above, with reference to Fig. 7, scattered lights from the

interrogation points

724a, 724b, and 724c are allowed to travel respectively to the top, middle, and bottom active regions of the radiation detector 100. In an alternative embodiment, the flow cytometry system 700 may further include a first filter, a second filter, and a third filter (not shown) that respectively prevent scattered lights from the

interrogation points

724a, 724b, and 724c from reaching the top, middle, and bottom active regions of the radiation detector 100.

In other words, the first filter prevents lights from the interrogation point 724a and of pre-specified energies (to be distinguished from the light emitted by the fluorophores) from reaching the top active region of the radiation detector 100.

Similarly, the second filter prevents lights from the interrogation point 724b and of the pre-specified energies (to be distinguished from the light emitted by the fluorophores) from reaching the middle active region of the radiation detector 100.

Similarly, the third filter prevents lights from the interrogation point 724c and of the pre-specified energies (to be distinguished from the light emitted by the fluorophores) from reaching the bottom active region of the radiation detector 100.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

A flow cytometry system, comprising:

a light source system configured to send light beams (i) , i=1, …, M respectively to interrogation points (i) , i=1, …, M in a tube, wherein the light beams (i) , i=1, …, M have different spectra, and wherein M is an integer greater than 1;

detectors (i) , i=1, …, M; and

a collimator configured to, for each value of i, (A) provide a light path for light from the interrogation point (i) to the detector (i) , and (B) prevent any light from the interrogation point (i) from reaching any detector (j) , j=1, …, M and j ≠ i.
The flow cytometry system of claim 1, wherein the light beams (i) , i=1, …, M have different maximum photon energies.
The flow cytometry system of claim 1, wherein for each value of i, the light beam (i) does not incident on the interrogation point (j) , j=1, …, M and j ≠ i.
The flow cytometry system of claim 1, wherein the collimator prevents any unscattered portion of the light beams (i) , i=1, …, M from reaching any point of the detectors (i) , i=1, …, M.
The flow cytometry system of claim 1, further comprising a sorter configured to sort particles flowing in single file along the tube based on light emitted by the particles under excitation by the light beams (i) , i=1, …, M or light scattered by the particles when the particles pass through the interrogation points (i) , i=1, …, M.
The flow cytometry system of claim 1, further comprising filters (i) , i=1, …, M, wherein for each value of i, the filter (i) is configured to prevent light from the interrogation point (i) and of pre-specified energies from reaching the detector (i) .
The flow cytometry system of claim 1, wherein the light source system comprises light sources (i) , i=1, …, M configured to respectively generate the light beams (i) , i=1, …, M.
The flow cytometry system of claim 1, wherein the light source system comprises a prism configured to receive as input a white light beam and thereby generate as output the light beams (i) , i=1, …, M.
The flow cytometry system of claim 1, wherein the light beams (i) , i=1, …, M are pencil beams.
The flow cytometry system of claim 1,

wherein the light beams (i) , i=1, …, M are fan beams, and

wherein the light source system comprises an optical system configured to converge the light beams (i) , i=1, …, M respectively to the interrogation points (i) , i=1, …, M.
The flow cytometry system of claim 1, wherein each detector of the detectors (i) , i=1, …, M comprises one or more avalanche photodiodes (APD) .
The flow cytometry system of claim 11, wherein said one or more APDs are one or more single-photon avalanche diodes (SPAD) .
The flow cytometry system of claim 1,

wherein for each value of i, the detector (i) comprises Ni sensing elements, Ni being a positive integer, and

wherein for each value of i, the flow cytometry system is configured to add all picture elements of an image captured by the Ni sensing elements, resulting in a sum (i) .
A method, comprising:

sending light beams (i) , i=1, …, M respectively to interrogation points (i) , i=1, …, M in a tube, wherein the light beams (i) , i=1, …, M have different spectra, and wherein M is an integer greater than 1;

flowing particles in single file in the tube through the interrogation points (i) , i=1, …, M; and

for each value of i, channeling with a collimator light from the interrogation point (i) to a detector (i) , wherein the collimator is configured to prevent any light from the interrogation point (i) from reaching any detector (j) , j=1, …, M and j ≠ i.
The method of claim 14, wherein the light beams (i) , i=1, …, M have different maximum photon energies.
The method of claim 14, wherein for each value of i, the light beam (i) does not incident on the interrogation point (j) , j=1, …, M and j ≠ i.
The method of claim 14, wherein the collimator prevents any unscattered portion of the light beams (i) , i=1, …, M from reaching any point of the detectors (i) , i=1, …, M.
The method of claim 14, further comprising sorting the particles based on light emitted by the particles under excitation by the light beams (i) , i=1, …, M or light scattered by the particles when the particles pass through the interrogation points (i) , i=1, …, M.
The method of claim 14, wherein each detector of the detectors (i) , i=1, …, M comprises one or more avalanche photodiodes (APD) .
The method of claim 19, wherein said one or more APDs are one or more single-photon avalanche diodes (SPAD) .