Suppression element for flow cytometer

Abstract

A detection system for a flow cytometer includes a light source configured to generate an excitation beam. The suppression element has: a first portion having a first feature; and a second portion surrounding the first portion. One or more differences between the first feature and the second feature cause a phase shift between a light stripe of the excitation beam and the high intensity center portion, such that the light stripe is suppressed.

Description

[0001] This application was filed as a PCT international patent application on May 12, 2023 and claims the benefit and priority of U.S. Provisional Patent Application Serial No. 63 / 341,805 filed on May 13, 2022 and U.S. Provisional Patent Application Serial No. 63 / 349,274 filed on June 6, 2022. The entire disclosure of the above-mentioned U.S. Provisional Patent Applications is incorporated herein by reference in its entirety. Background Art

[0002] In flow cytometry, particles are arranged in a sample stream and typically pass one by one through one or more excitation beams with which the particles interact. Light scattered or emitted by the particles when interacting with the one or more excitation beams is collected and analyzed to characterize and differentiate the particles. In sorting flow cytometry, particles can be extracted from the sample stream after being characterized by their interaction with the one or more excitation beams and thereby sorted into different groups.

[0003] Conventional flow cytometers are generally suitable for detecting samples with particles or cells that are generally larger than 1000 nm in size. However, conventional flow cytometers are less suitable for detecting very small particles, such as biological nanoparticles (e.g., extracellular vesicles) or non-biological nanoparticles (e.g., nanobeads). For example, many conventional flow cytometers are simply not sensitive enough to detect or distinguish optical signals from these very small particles, resulting in inaccurate detection results.

[0004] Designing a flow cytometer for nanoparticle detection is challenging because the light scattering of nanoparticles is several orders of magnitude lower than that of microparticles, which makes light scattering difficult to detect. A method for analyzing nanoparticles using flow cytometry is to focus the excitation laser beam more compactly to increase the intensity of light scattering. However, due to the diffraction limitations of the focusing element for focusing the excitation laser beam, diffraction fringes appear. Diffraction fringes produce additional noise that is difficult to filter out without reducing resolution, making it difficult to accurately detect nanoparticles. In addition, the quality of the excitation beam and the aberrations from the optical components can produce additional light fringes in the excitation beam distribution at the interrogation point, which may produce additional noise and difficulty in detecting nanoparticles. Summary of the invention

[0005] The present disclosure relates to detection systems for sample processing instruments, such as flow cytometric sorters and / or analyzers, and in particular to inhibitory elements for sample processing instruments. Various aspects are described in the present disclosure, including but not limited to the following aspects.

[0006] One aspect relates to a detection system, which includes: a light source configured to generate an excitation light beam having a first portion and a second portion; an inhibition element having: a first portion having a first characteristic; and a second portion surrounding the first portion, the second portion of the inhibition element having a second characteristic, the second characteristic causing a phase shift between the first portion and the second portion of the excitation light beam; a focusing lens configured to focus the excitation light beam for high scattering intensity detection of particles; and a light collection unit configured to detect particles.

[0007] On the other hand, a suppression element is provided for a flow cytometer configured to detect nanoparticles, the suppression element comprising: a first portion having a first characteristic; and a second portion surrounding the first portion, the second portion having a second characteristic, wherein the second characteristic causes a phase shift between low-intensity light stripes and a high-intensity core of an excitation light beam.

[0008] On the other hand, a method for detecting nanoparticles in a flow cytometer is provided, the method comprising: emitting an excitation light beam having a first portion including a high-intensity central portion and a second portion including low-intensity light stripes; passing the excitation light beam through an inhibition element, wherein the first portion of the excitation light beam passes through a first portion of the inhibition element and the second portion of the excitation light beam passes through a second portion of the inhibition element; focusing the excitation light beam for high scattered intensity detection of nanoparticles; and detecting nanoparticles having a size of 80 nanometers or less. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following drawings, which form a part of this application, are illustrative of the described technology and are not intended to limit the scope of the present disclosure in any way.

[0010] Figure 1 An example of a detection system for detecting and analyzing nanoparticles is schematically shown.

[0011] Figure 2 Shows Figure 1 Examples of the distribution and cross-sectional diagrams of the excitation beam at the interrogation point in the detection system.

[0012] Figure 3 Shows Figure 1 Another example of a cross-sectional view of an excitation beam at an interrogation point in a detection system of FIG.

[0013] Figure 4 Shown for Figure 1 An example of a suppressor element in a detection system to reduce and / or eliminate light streaks present in an excitation beam.

[0014] Figure 5 Schematic diagram showing the inhibition Figure 1 An example of a method of detecting light fringes in a system.

[0015] Figure 6 Schematically shows Figure 4 Examples of first and second embodiments of the suppression element. DETAILED DESCRIPTION

[0016] Various embodiments will be described in detail with reference to the accompanying drawings, wherein the same reference numerals represent the same parts and components in several views. Reference to various embodiments does not limit the scope of the claims attached hereto. In addition, any examples set forth in this specification are not intended to be limiting, but only set forth some of the many possible embodiments of the attached claims.

[0017] This article describes an example detection system for use in a flow cytometer. It should be understood that the disclosure is not limited to the detection system shown, but can be applied to flow cytometers with other structures or other types of detection systems. In particular, the disclosure can be applied to various types of sample processing instruments for detecting, sorting or otherwise processing nanoparticles.

[0018] In addition, the detection system described herein is used to detect nanoparticles, which refer to nanoscale particles. For example, particles can have a size (e.g., diameter, maximum size or average size) less than or equal to 1000nm (nanometer), especially, a size range of from 40nm to 200nm. Nanoparticles can be biological nanoparticles (e.g., extracellular vesicles) or non-biological nanoparticles (e.g., nanobeads).

[0019] Figure 1 An example of a detection system 100 for detecting and analyzing nanoparticles is schematically shown. According to the examples described herein, the detection system 100 can be incorporated into a flow cytometer and / or a sorting flow cytometer. Figure 1 As shown, the detection system 100 includes a light emitting unit 110 and a light collecting unit 120 for detecting and / or analyzing nanoparticles flowing through a cuvette 15 .

[0020] The light emitting unit 110 is configured to emit a light beam and project the light beam onto the nanoparticles flowing through the detection channel 18 of the cuvette 15. The light collecting unit 120 is configured to collect light scattered or emitted from the nanoparticles so as to analyze the nanoparticles based on the collected light.

[0021] The light emitting unit 110 includes a plurality of light sources, for example Figure 11 and 2. As an illustrative example, the light sources 111a to 111d may include lasers. The light sources 111a to 111d are each configured to emit an excitation light beam having a different wavelength, for example, 405 nm, 488 nm, 561 nm, and 638 nm. Figure 1 In the example shown, light sources 111a to 111d are arranged in parallel. It should be understood that the number, type and arrangement of light sources are not limited to the examples shown and described herein, and can be changed as needed. For example, the system can include three, five, six or any other suitable number of light sources.

[0022] The light emitting unit 110 also includes a focusing lens 119. The excitation light beams emitted by the light sources 111a to 111d pass through the focusing lens 119, which focuses the excitation light beams to have the same interrogation point in the detection channel 18 of the cuvette 15. The interrogation point may also be referred to as a focal point, where the focused excitation light beam meets the central portion of the sample flow in the detection system 100. As will be described in more detail, the focusing lens 119 is configured to focus the excitation light beam for high scattering intensity detection of nanoparticles.

[0023] The dichroic mirrors 117a, 117b, 117c, and 117d are arranged between the focusing lens 119 and the corresponding light sources 111a to 111d. Each of the dichroic mirrors 117a to 117d is configured to reflect a light beam of a corresponding one of the light sources 111a to 111d and transmit light beams of the other light sources. The dichroic mirrors 117a to 117d are selected and configured according to the wavelength of the light beam emitted by the corresponding light source 111a to 111d. For example, the dichroic mirror 117b may be configured to reflect light of a wavelength emitted by the light source 111b, and configured to transmit light of a wavelength emitted by the light source 111a; the dichroic mirror 117c may be configured to reflect light of a wavelength emitted by the light source 111c, and configured to transmit light of a wavelength emitted by the light sources 111a and 111b; and the dichroic mirror 117d may be configured to reflect light of a wavelength emitted by the light source 111d, and configured to transmit light of a wavelength emitted by the light sources 111a, 111b, and 111c.

[0024] The light beam emitted by light source 111a to 111d is reflected or transmitted by dichroic mirror 117a to 117d to form collinear light beam. Collinear light beam shares optical axis, and provides the common focus of multiple light sources by focusing on the same inquiry point. The position or orientation of dichroic mirror 117a to 117d is adjustable so that it can be used to adjust the position of the focus of the light beam, especially the position on the plane perpendicular to the optical axis. In some examples, the light beam can be configured so that it is not collinear, but still focuses on the converging light beam of the same point. That is, the light beam may not all have the same optical axis, but it is all configured to focus on a single point in the sample channel of cuvette 15.

[0025] The lenses 115a to 115d are arranged between the corresponding light sources 111a to 111d and the corresponding dichroic mirrors 117a to 117d. In some examples, the lenses 115a to 115d are telephoto lenses. In some examples, the lenses 115a to 115d are spherical lenses. In other examples, the lenses 115a to 115d are aspherical lenses. Each of the lenses 115a to 115d can convert a light beam into a parallel light beam. Figure 1 In the example shown, each of the lenses 115a to 115d is in the form of a plano-convex lens having a flat surface and a convex surface opposite to each other. For example, the convex surface of the plano-convex lens may have a focal length of 2400 mm.

[0026] The position or orientation of lenses 115a to 115d is adjustable to adjust the position of the beam focus, especially the position on the plane perpendicular to the optical axis. Generally, dichroic mirrors 117a to 117d can be used to roughly adjust the position of the beam focus, while lenses 115a to 115d can be used to finely adjust the position of the beam focus.

[0027] It should be understood that the number, type and arrangement of the dichroic mirrors 117a to 117d and the lenses 115a to 115d can be changed as needed and are not limited to the examples shown herein. In addition, the dichroic mirrors 117a to 117d and the lenses 115a to 115d can be replaced with other optical elements or optical modules having similar functions.

[0028] The beam expanders 113a to 113d may be arranged between the corresponding light sources 111a to 111d and the corresponding lenses 115a to 115d. Each of the beam expanders 113a to 113d may change the cross-sectional size and divergence angle of the light beam. In this way, each of the beam expanders 113a to 113d may be configured according to the desired size of the light beam spot.

[0029] The light beam irradiated on the nanoparticles by the focusing lens 119 has a spot size smaller than the spot size provided by the detection system in a conventional flow cytometer. A smaller spot size allows a more concentrated light beam with a higher power density. This can increase the intensity of the light beam irradiated on the nanoparticles, and ultimately increase the intensity of the optical signal collected from the nanoparticles. This can improve the efficiency of collecting optical signals and thereby provide higher resolution and higher sensitivity for nanoparticle detection. For example, the spot size can be about 15×3 μm.

[0030] exist Figure 1 In the example shown, the light sources 111a to 111d are in the form of lasers including respective laser diodes 112a to 112d. Figure 1 As further shown in the example of , half-wave plates 116a to 116d are respectively disposed between dichroic mirrors 117a to 117d and lenses 115a to 115d. The light spot of the light beam can be reduced by the orientation of the light sources 111a to 111d and by using the half-wave plates 116a to 116d.

[0031] like Figure 1 As further shown in FIG. 1 , cylindrical lenses 114a to 114d are disposed between respective beam expanders 113a to 113d and respective lenses 115a to 115d. The horizontal size of the spot of the light beam focused in the cell 15 can be adjusted by replacing the cylindrical lenses 114a to 114d with replacement cylindrical lenses having different curvatures.

[0032] Additionally or alternatively, the power of some or all of the light sources 111a to 111d may be increased compared to conventional detection systems. For example, a particular light source of a conventional detection system may have a power of 30 mW, while the light sources 111a to 111d of the detection system 100 may have an increased power of 50 mW. The increased power of the light sources 111a to 111d may also improve detection sensitivity.

[0033] Each of the beam expanders 113a to 113d is formed by a first optical portion and a second optical portion. Figure 1 In the example shown, each of the beam expanders 113a to 113d includes a concave lens adjacent to the corresponding light source as a first optical portion, and also includes a convex lens away from the corresponding light source as a second optical portion. It should be understood that each of the beam expanders 113a to 113d is not limited to Figure 1 The example shown. The beam expanders 113a to 113d may be formed by any suitable optical lens or lens group. For example, each of the first optical portion and the second optical portion may be selected from one of a convex lens, a convex lens group, a concave lens, and a concave lens group.

[0034] For each of the beam expanders 113a to 113d, the distance between the first optical portion (eg, concave lens) and the second optical portion (eg, convex lens) is adjustable. This allows the waist position (focus) of the light beam on the optical axis to be adjusted.

[0035] As described above, by adjusting the dichroic mirrors 117a to 117d, the lenses 115a to 115d, and the beam expanders 113a to 113d, each beam can be focused at a desired interrogation point, and multiple beams can be focused at the same interrogation point. It should be understood that the position of the beam focus can be adjusted by using any other optical element or in any other adjustment manner. One or more adjustments to the dichroic mirrors 117a to 117d, the lenses 115a to 115d, and the beam expanders 113a to 113d can be made manually, or can be made electronically using a computing device (e.g., a controller) associated with one or more actuators coupled to these components.

[0036] The light collecting unit 120 includes a lateral collecting portion 130 and a forward collecting portion 150. The lateral collecting portion 130 serves as a lateral scattering unit that collects lateral scattered light and fluorescence scattered or emitted from the nanoparticles in the sample when the nanoparticles are irradiated by the light beam while passing through the cuvette 15. The optical axis of the light beam collected from the nanoparticles by the lateral collecting portion 130 is substantially perpendicular or at about 90 degrees to the optical axis of the light beam emitted from the light sources 111a to 111d and directed toward the cuvette 15 by the dichroic mirrors 117a to 117d.

[0037] The forward collecting section 150 is used as a forward scattering unit that collects forward scattered light from the nanoparticles. The optical axis of the light beam collected from the nanoparticles by the forward collecting section 150 can be approximately parallel or about 0 degrees to the optical axis of the light beam directed toward the cuvette 15. The lateral collecting section 130 and the forward collecting section 150 are described in further detail below.

[0038] The lateral collection unit 130 includes: an optical focusing lens group, which includes a concave mirror 134 and an aspheric lens 135; a collection optical fiber 136; a beam splitter 133; a first wavelength division multiplexer 131; and a second wavelength division multiplexer 132. The concave mirror 134 reflects the scattered light and the fluorescence that are diverged in different directions at the interrogation point. The concave mirror 134 and the aspheric lens 135 focus the reflected light on the same point of the collection optical fiber 136 (such as Figure 1The reflected light is focused on the collection optical fiber 136 by focusing the reflected light on the collection optical fiber 136 (as shown in the dotted block 139 in FIG. 1 ). The concave mirror 134 can focus the reflected light on the optical fiber, and the aspherical lens 135 can make the focus smaller (i.e., reduce aberrations). In order to prevent crosstalk, the beam splitter 133 is arranged to separate the scattered light with high intensity from the fluorescence with low intensity. The separated scattered light and fluorescence enter the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 respectively through the first optical fiber 137 and the second optical fiber 138. Optical signals with different wavelengths are separated in the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 for analysis. It should be noted that the optical focusing lens group can adopt other optical elements.

[0039] The beam splitter 133 includes a dichroic mirror 1332 and a notch filter 1334. The collected light is directed into the beam splitter toward the dichroic mirror 1132 through the collection fiber 136, which can be oriented so that the light beam is directed toward the dichroic mirror 1332 at an incident angle of, for example, 45 degrees. The dichroic mirror 1332 reflects the side scattered light coming out of the collection fiber 136 so that the side scattered light enters the first wavelength division multiplexer 131 through the first optical fiber 137.

[0040] The fluorescence coming out of the collection optical fiber 136 passes through the dichroic mirror 1332, and is incident on the notch filter 1334 at an incident angle of about 90 degrees and then passes through the notch filter 1334. The fluorescence enters the second wavelength division multiplexer 132 through the second optical fiber 138. According to the confocal design of the light sources 111a to 111d, the dichroic mirror 1332 and the notch filter 1334 can each have a plurality of frequency bands. In this case, both the dichroic mirror 1332 and the notch filter 1334 have four frequency bands that block four laser wavelengths. The number of frequency bands of the dichroic mirror 1332 and the notch filter 1334 can correspond to the number of light sources 111a to 111d.

[0041] The beam splitter 133 separates the side scattered light with high intensity from the fluorescence with low intensity, thereby reducing or preventing the crosstalk of the side scattered light to the fluorescence. In addition, by setting the beam splitter, multiple light beams can be separated and transmitted to two or more wavelength division multiplexers. Most existing wavelength division multiplexers have limited signal channels, such as six signal channels. In the case of more than six optical signals, a single wavelength division multiplexer with six signal channels is not enough. Using existing wavelength division multiplexers can significantly reduce costs. The optical elements and their configurations included in the beam splitter 133 can be changed and are not limited to the examples shown.

[0042] In some examples, the first wavelength division multiplexer 131 can be configured to receive the side scattered light beam from the beam splitter 133 via the first optical fiber 137, and separate the optical signals of the side scattered light with different wavelengths from each other. In the first wavelength division multiplexer 131, each optical signal is transmitted along the optical transmission path 1310 corresponding to the optical channel of the optical signal. The first wavelength division multiplexer 131 may include a first filter 1311 and a second filter 1312 for each optical channel. The first filter 1311 and the second filter 1312 can be arranged at a certain distance from each other in a non-parallel manner along the optical transmission path of the optical channel. Crosstalk between side scattered light can be reduced or prevented by setting two filters. The first filter 1311 and the second filter 1312 are not arranged in parallel to avoid multiple reflections of light between them and obtain better optical density. Thereafter, the filtered light enters a light detection element 1315 (e.g., a photodiode, an avalanche photodiode (APD), a photomultiplier tube) for further processing of the light.

[0043] As in Figure 1 As further shown in the illustrated example, the second wavelength division multiplexer 132 can be configured to receive the fluorescent light beam from the beam splitter 133 via the second optical fiber 138, and separate the optical signals of the fluorescent light beams having different wavelengths from each other. In the second wavelength division multiplexer 132, each optical signal is transmitted along an optical transmission path 1320 corresponding to the optical channel of the optical signal. Since the fluorescent signal is weak, the second wavelength division multiplexer 132 may include a single filter 1321 for each optical channel. Thereafter, the filtered fluorescent light enters a light detection element 1325 (e.g., a photodiode, an avalanche photodiode (APD), a photomultiplier tube) for further processing of the light.

[0044] An alternative suitable configuration for the wavelength division multiplexer may be used. For example, the first wavelength division multiplexer 131 and the second wavelength division multiplexer 132 may include notch filters corresponding to the respective fluorescence channels. The notch filters may reduce or eliminate the crosstalk of the side scattered light to the fluorescence. In this case, the beam splitter 133 may include only the dichroic mirror 1332 without the notch filter 1334.

[0045] In the lateral collection section 130, the diameter of the collection fiber 136 may be different from the diameters of the first fiber 137 and the second fiber 138 according to the light transmission efficiency. The lens in the beam splitter may cause aberrations, and thus the output spot may be larger than the input of the beam splitter, and the fiber diameter may be selected accordingly.

[0046] 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 is configured to block most of the light transmitted through the small pool 15 to reduce the background noise generated by the light beam directly passing through the small pool 15, and allows only the forward scattered light from the nanoparticles to be collected. In some examples, most of the transmitted light can be blocked so that the forward detector 159 is not saturated. The concave mirror 151 is configured to reflect the forward scattered light beam emitted from the nanoparticles. The filter 157 is configured to allow the forward scattered light with a high signal-to-noise ratio to pass through and block other light. The forward detector 159 receives the filtered forward scattered light from the filter 157 and processes and analyzes the forward scattered light.

[0047] Figure 2 An example of a distribution and cross-sectional view of an excitation beam 200 at an interrogation point in a detection system 100 is shown. The focusing lens 119 can focus the excitation beam emitted from the light sources 111a to 111d into a spot size smaller than the spot size provided by a conventional detection system. In this example, the excitation beam has a spot size of approximately 13.4 μm×3.5 μm. A smaller spot size can provide higher resolution and higher sensitivity for nanoparticle detection. Figure 2 As shown, the excitation beam 200 has a symmetric Gaussian distribution along the horizontal beam cross section (x-axis), but exhibits diffraction fringes 202 along the vertical beam cross section (y-axis).

[0048] Figure 3 Another example of a cross-sectional view of the excitation beam 300 taken along the y-axis at an interrogation point in the detection system 100 is shown. Figure 3 In FIG. 3 , the excitation light beam 300 similarly shows diffraction fringes 302 along the y-axis. Figure 2 and Figure 3 The diffraction fringes 202, 302 shown in FIG are low intensity light ripples caused by bending around the edge of the focusing lens 119 instead of passing directly through the focusing lens 119. Due to the small spot size (about 15 μm×3 μm) used by the detection system 100 to detect nanoparticles, the diffraction fringes can increase to more than a manageable noise level. The diffraction fringes are an optical effect that occurs naturally due to the diffraction limitation of the focusing lens 119.

[0049] The diffraction fringes may cause the light collection unit 120 to detect multiple excitations of a single nanoparticle by the light beam emitted from the light emitting unit 110. Multiple excitations may cause the light collection unit 120 to erroneously detect multiple nanoparticles, although only a single nanoparticle is excited by the light beam. In addition, the diffraction fringes have a lower intensity than the central portion of the excitation light beam 200, 300, which may cause the light collection unit 120 to detect multiple nanoparticles with different sizes, although only a single nanoparticle is excited by the light beam. Therefore, the diffraction fringes may cause errors in the detection of nanoparticles by the detection system 100.

[0050] Except in Figure 2 and Figure 3 In addition to the example shown in FIG. 1 , which illustrates the presence of diffraction fringes due to the diffraction limitation of focusing lens 119, irregularities in the shape of the excitation beam relative to a Gaussian distribution can cause additional light fringes to appear at the interrogation point. Furthermore, aberrations in one or more optical components in detection system 100 can cause additional light fringes at the interrogation point.

[0051] Figure 4 An example of a suppressor element 400 is shown, which is a general solution in the detection system 100 for suppressing light streaks that may be caused by diffraction limitations of a focusing lens, imperfections in the shape of the excitation beam (e.g., non-Gaussian), aberrations from one or more optical components in the detection system 100, and other possible causes of light streaks at the interrogation point of the detection system 100. In one specific example, the suppressor element 400 is used in the detection system 100 to reduce and / or eliminate Figure 2 and Figure 3 Diffraction fringes shown in .

[0052] The suppression element 400 includes a first portion 402 and a second portion 404 surrounding the first portion. The first portion 402 has a first characteristic, and the second portion 404 has a second characteristic, which causes a phase shift between the two portions of the excitation beam when the two portions of the excitation beam pass through the suppression element 400. As an illustrative example, a first portion of the excitation beam having high intensity light passes through the first portion 402, and a second portion of the excitation beam having low intensity light (e.g., a stripe) passes through the second portion 404. At an interrogation point in the detection system 100, both the first portion and the second portion of the excitation beam interfere with each other to produce destructive interference that suppresses the low intensity light (e.g., a stripe) of the second portion of the excitation beam, and produces a maximum contrast between the high intensity light of the first portion of the excitation beam and the low intensity light of the second portion. By changing the second characteristic of the second portion 404 relative to the first characteristic of the first portion 402, the suppression element 400 can modulate the intensity of the excitation beam.

[0053] The first portion 402 has an elliptical shape, such as an oval shape formed by a closed curve. Figure 4 In the example shown, the first portion 402 has a circular shape. Figure 4 As further shown in , the first portion 402 has a diameter D1. In some examples, the diameter D1 ranges from about 1 mm to about 5 mm. In some other examples, the diameter D1 ranges from about 2 mm to about 3 mm. It is conceivable that the shape and size of the first portion 402 can be adjusted to conform to various shapes of the excitation beam emitted by the light emitting unit 110. In some examples, the first portion 402 has a substrate thickness ranging from about 0.5 mm to about 1.0 mm. In other examples, the first portion 402 is made of UV fused silica and provides a wavelength coverage of about 350 nm to about 800 nm.

[0054] Although Figure 4 The second portion 404 is shown as having an annular shape surrounding the first portion 402, but the shape of the second portion 404 may vary. For example, the second portion 404 may have a rectangular or square shape surrounding the first portion 402.

[0055] Figure 6 A first embodiment 602 is shown in which a first characteristic of the first portion 402 is a first material thickness T 1 , and the second characteristic of the second portion 404 is the second material thickness T 2 In the first embodiment 602, the second material thickness T 2 Different from the first material thickness T 1 , so that the difference (ΔT = T 1 -–T 2 ) causes a phase shift between the high intensity portion and the low intensity portion of the excitation light beam. For example, the high intensity portion of the excitation light beam passes through the first material thickness T of the first portion 402. 1 , and the low-strength portion passes through the second material thickness T of the second portion 404 2 , which causes a phase shift between the high-intensity part and the low-intensity part.

[0056] First material thickness T 1 With the second material thickness T 2 The optimal difference (ΔT) between the first and second portions 402 and 404 of the suppressor element 400 may vary depending on the wavelength of the excitation light beam and the refractive index of the first and second portions 402 and 404 of the suppressor element 400. 1 With the second material thickness T 2 The phase shift generated by the difference between can be determined based on equation (1),

[0057] (1)

[0058] where Δψ is the phase shift between the high-intensity and low-intensity parts of the excitation beam, λ is the wavelength of the excitation beam, and T 1 is the first material thickness, T 2 is the second material thickness, and N is the refractive index of suppression element 400 .

[0059] In some examples, the first portion 402 and the second portion 404 are made of the same material. In some examples, the first material thickness T 1 With the second material thickness T 2 The difference (ΔT) between the two is obtained by the etching inhibition element 400 to have a first material thickness T 1 and the second material thickness T 2 to achieve.

[0060] As long as the first material thickness T 1 and the second material thickness T 2 Different, the first material thickness T 1 and the second material thickness T 2 The relative thickness of Figure 6 In the example shown, the first material thickness T 1 Than the second material thickness T 2 In an alternative example, the second material thickness T 2 Can be thicker than the first material T 1 thick.

[0061] First material thickness T 1 The difference between the second material thickness (ΔT) T 1 -–T 2 The optical path is adjusted or a phase shift is caused between the first part and the second part of the excitation light beam. In some examples, the first material thickness T 1 The difference between the second material thickness (ΔT) T 1 -–T 2 A phase shift of π / 2 or any odd multiple thereof is caused between the first part and the second part of the excitation light beam. For example, the first material thickness T 1 With the second material thickness T 2 The difference (ΔT) between the first and second parts of the excitation beam can cause a phase shift of 3π / 2, 5π / 2, or 7π / 2. The first material thickness T is selected 1 With the second material thickness T 2 The difference (ΔT) between them is determined to induce a phase shift that provides maximum contrast between the low-intensity light fringes and the high-intensity central portion of the excitation beam.

[0062] Figure 6 A second embodiment 604 is further shown, in which the first characteristic of the first portion 402 is a first refractive index N 1, and the second characteristic of the second portion 404 is a second refractive index N 2 In the second embodiment 604, the second refractive index N 2 Different from the first refractive index N 1 , so that the difference (ΔN) causes a phase shift between the high intensity portion and the low intensity portion of the excitation light beam. For example, the high intensity portion of the excitation light beam passes through the first refractive index N of the first portion 402. 1 , while the low intensity portion passes through the second refractive index N of the second portion 404 2 , which causes a phase shift between the high-intensity part and the low-intensity part.

[0063] The first refractive index N 1 With the second refractive index N 2 The optimal difference (ΔN) between the two can be determined based on the wavelength of the excitation beam and the second refractive index N 2 The thickness of the second portion 404 (eg, T 2 -–T 1 ) varies. 1 With the second refractive index N 2 The phase shift generated by the difference between can be determined based on equation (2),

[0064] (2)

[0065] where Δψ is the phase shift between the high-intensity and low-intensity parts of the excitation beam, λ is the wavelength of the excitation beam, and N 2 is the second refractive index of the second portion 404 of the suppressor element 400, N 1 is the first refractive index of the first portion 402 of the suppressor element 400, T 2 is the second material thickness of the second portion 404 of the suppression element 400 (at Figure 6 In the example shown, T 2 including a second refractive index N 2 The thickness of the evaporated material and the first refractive index N 1 The thickness of the substrate), and T 1 is the first material thickness of the first portion 402 of the suppression element 400 in the second embodiment 604 (in Figure 6 In the example shown, T 1 is a first refractive index N 1 The thickness of the substrate). As long as the first refractive index N 1 and the second refractive index N 2 Different, the first refractive index N 1 With the second refractive index N 2 In some examples, the second refractive index N 2 Higher than the first refractive index N1 In an alternative example, the second refractive index N 2 Lower than the first refractive index N 1 .

[0066] In some examples, first portion 402 and second portion 404 are made of different materials having different refractive indices. As an illustrative example, second portion 404 can include one or more coatings.

[0067] In some examples, by 1 The different refractive indexes between the first portion 402 and the second portion 404 are obtained by evaporating one or more coatings on the substrate. Figure 6 In some other examples, by doping or implanting one or more materials into a layer having a thickness T 1 Different refractive indices can be obtained in the substrate to change the relative optical properties and refractive index of the first portion 402 and the second portion 404 of the inhibitory element 400.

[0068] In some examples, the first refractive index N 1 With the second refractive index N 2 The difference (ΔN) between the first and second parts of the excitation light beam has an optical path difference or a phase shift of π / 2 or any odd multiple thereof. 1 With the second refractive index N 2 The difference (ΔN) between the first and second parts of the excitation beam can cause the first and second parts of the excitation beam to have a phase shift of 3π / 2, 5π / 2, or 7π / 2. 1 With the second refractive index N 2 The difference (ΔN) between them is calculated to induce a phase shift that provides maximum contrast between the low-intensity light fringes and the high-intensity central portion of the excitation beam.

[0069] In some examples, the first characteristic of the first portion 402 is a first combination of material thickness and refractive index, and the second characteristic of the second portion 404 is a second combination of material thickness and refractive index. In such examples, one or more differences between the first combination of material thickness and refractive index and the second combination (e.g., different material thicknesses, or different refractive indices, or different combinations of material thickness and refractive index) cause different optical path lengths of the first portion and the second portion of the excitation beam, which produces a phase shift that suppresses light fringes of the excitation beam by destructive interference. In some additional examples, the first portion 402 is empty, such that the first portion 402 has no material thickness, and the refractive index of the first portion is equal to the refractive index of air.

[0070] In some examples, first portion 402 of suppression element 400 defines a hole, and second portion 404 of suppression element 400 includes a solid material surrounding the hole of first portion 402. In such examples, the first material thickness T of first portion 402 is 1 is zero, and the second material thickness T 2 can be any value greater than zero. In such an example, the first refractive index N of the first portion 402 1 is equal to the environment (eg, air) inside the detection system 100, and the second refractive index N of the second portion 404 is 2 Based on solid material surrounding the hole.

[0071] Now return to reference Figure 1 , the suppressing element 400 can be positioned in the optical path of each light source 111a to 111d between the first optical section and the second optical section of the beam expander 113a to 113d, as shown in the first position 406. Alternatively, the suppressing element 400 can be positioned in the common path of the excitation light beams and before the focusing lens 119, as shown in the second position 408. In both cases, the first position 406 and the second position 408 for the placement of the suppressing element 400 are before the focusing lens 119 of the detection system 100 so as to pre-condition the excitation light beam before it reaches the focusing lens 119 to suppress the light fringe pattern.

[0072] In both embodiments of the suppressor element 400 described herein, the first portion 402 and the second portion 404 are transmissive. Thus, the second portion 404 of the suppressor element 400 is not a physical or mechanical barrier, since it would otherwise induce an additional light fringe pattern in the excitation beam transmitted to the interrogation point of the detection system 100.

[0073] Figure 5 An example of a method 500 for detecting nanoparticles in a detection system 100 is schematically shown. Figure 5 As shown, the method 500 includes an operation of emitting an excitation light beam 502. According to the above example, the excitation light beam may be emitted by the light emitting unit 110 in the detection system 100.

[0074] Next, method 500 includes an operation 504 of passing the excitation light beam through the suppression element 400. According to the above example, a first portion of the excitation light beam having high intensity light passes through the first portion 402 of the suppression element 400, and a second portion of the excitation light beam having low intensity light (e.g., stripes) passes through the second portion 404 of the suppression element 400. The first feature of the first portion 402 and the second feature of the second portion 404 induce a phase shift between the low intensity light (e.g., stripes) and the high intensity light of the excitation light beam. In some examples, the phase shift is π / 2 or any odd multiple thereof.

[0075] In one embodiment, operation 504 comprises providing a material having a first material thickness T 1 The first portion 402 has a second material thickness T different from the first material thickness 2 The suppression element 400 of the second portion 404 produces a phase shift. In this embodiment, the high-intensity central portion of the excitation beam passes through the first material thickness T 1 , and the low-intensity light stripe of the excitation beam passes through the second material thickness T 2 .

[0076] In another embodiment, operation 504 comprises providing a first refractive index N 1 The first portion 402 and the second refractive index N different from the first refractive index 2 The suppression element 400 of the second portion 404 of the excitation beam produces a phase shift. In this embodiment, the high-intensity central portion of the excitation beam passes through the first refractive index N 1 , and the low-intensity light stripe of the excitation beam passes through the second refractive index N 2 .

[0077] Next, method 500 includes focusing the excitation beam at an interrogation point in detection system 100 (eg, in detection channel 18 of cuvette 15) for high scattered intensity detection of nanoparticles 506. The excitation beam may be focused by focusing lens 119 according to the above example.

[0078] Next, the method 500 includes an operation 508 of generating destructive interference at the interrogation point in the detection system 100. The destructive interference is caused by a phase shift between a first portion of the excitation beam having high intensity light, which passes through the first portion 402 of the suppression element 400, and a second portion of the excitation beam having low intensity light fringes, which passes through the second portion 404 of the suppression element 400. The destructive interference suppresses the low intensity light fringes of the second portion of the excitation beam and generates a maximum contrast between the high intensity light of the first portion of the excitation beam and the low intensity light fringes of the second portion.

[0079] In one illustrative example, method 500 includes using suppression element 400 to suppress diffraction fringes in detection system 100 without losing resolution by defocusing the excitation light beam emitted from light emission unit 110. Instead, method 500 uses suppression element 400 to cause a phase shift between low-intensity light fringes and a high-intensity central portion of the excitation light beam, which causes destructive interference between the two portions of the excitation light beam at the interrogation point. This suppresses the low-intensity light fringes, causing only the high-intensity portion of the excitation light beam to scatter light from the nanoparticles, and thereby reduces errors in nanoparticle detection.

[0080] Next, method 500 includes an operation 510 of detecting nanoparticles based on scattering of a high intensity portion of the excitation light beam. According to the above example, the nanoparticles can be detected by the light collection unit 120. In one illustrative example, operation 510 includes detecting nanoparticles having a size (e.g., diameter, maximum size, or average size) less than or equal to 80 nm.

[0081] The various embodiments described above are provided by way of illustration only and should not be construed as limiting in any way. Various modifications may be made to the embodiments described above without departing from the true spirit and scope of the present disclosure.

Claims

1. A detection system for a flow cytometer, comprising: a light source configured to generate an excitation light beam having a first portion and a second portion; An inhibitory element, the inhibitory element comprising: a first portion having a first characteristic; as well as a second portion surrounding the first portion, the second portion of the suppression element having a second characteristic, the second characteristic causing a phase shift between the first portion and the second portion of the excitation light beam; a focusing lens configured to focus the excitation beam for high scattered intensity detection of particles; as well as A light collecting unit is configured to detect the particles.

2. The detection system according to claim 1, wherein: One or more differences between the first feature and the second feature cause different optical path lengths of the first portion and the second portion of the excitation light beam, thereby producing the phase shift that suppresses light fringes of the excitation light beam.

3. The detection system according to claim 1, wherein: A difference between the first feature and the second feature causes the phase shift to suppress light fringes of the excitation light beam by destructive interference between the first portion and the second portion of the excitation light beam.

4. The detection system according to claim 1, wherein: The first characteristic is a first material thickness, the second characteristic is a second material thickness, and the second material thickness is different from the first material thickness.

5. The detection system according to claim 1, wherein: The first characteristic is a first refractive index, the second characteristic is a second refractive index, and the second refractive index is different from the first refractive index.

6. A detection system according to any one of the preceding claims, wherein: The phase shift is π / 2 or any odd multiple thereof.

7. A detection system according to any one of the preceding claims, wherein: The suppression element is positioned in the beam path of the excitation light beam and before the focusing lens.

8. A detection system according to any one of the preceding claims, wherein: The light collecting unit detects the particles having a size less than or equal to 80 nanometers.

9. The detection system according to claim 1, wherein: The first portion of the inhibitory element defines an aperture, and the second portion of the inhibitory element includes a solid material surrounding the aperture of the first portion of the inhibitory element.

10. An inhibitory element for a flow cytometer configured to detect nanoparticles, the inhibitory element comprising: a first portion having a first characteristic; as well as A second portion surrounds the first portion, the second portion having a second feature, wherein the second feature causes a phase shift between the low intensity light fringes and the high intensity central portion of the excitation light beam.

11. The inhibitory element according to claim 10, wherein: One or more differences between the first feature and the second feature cause different optical path lengths of the first portion and the second portion of the excitation light beam, thereby producing the phase shift that suppresses the low-intensity light fringes of the excitation light beam.

12. The inhibitory element according to claim 10, wherein: The first characteristic is a first material thickness, the second characteristic is a second material thickness, and the second material thickness is different from the first material thickness.

13. The inhibitory element according to claim 12, wherein: The difference between the first material thickness and the second material thickness causes the phase shift, which suppresses the low-intensity light fringes of the excitation light beam by causing destructive interference with the high-intensity central portion of the excitation light beam.

14. The inhibitory element according to claim 13, wherein: The phase shift is π / 2 or any odd multiple thereof.

15. The inhibitory element according to any one of claims 12 to 14, wherein The first portion and the second portion are made of the same material.

16. The inhibitory element according to any one of claims 12 to 15, wherein The first portion has an elliptical shape.

17. The inhibitory element according to any one of claims 12 to 16, wherein The first portion has a diameter ranging from about 1 mm to about 5 mm.

18. The inhibitory element according to claim 10, wherein: The first characteristic is a first refractive index, the second characteristic is a second refractive index, and the second refractive index is different from the first refractive index.

19. The inhibitory element according to claim 18, wherein: The difference between the first refractive index and the second refractive index causes the phase shift, which suppresses the low-intensity light fringes of the excitation light beam by causing destructive interference with the high-intensity central portion of the excitation light beam.

20. The inhibitory element according to claim 19, wherein: The phase shift is π / 2 or any odd multiple thereof.

21. The inhibitory element according to any one of claims 18 to 20, wherein The first portion and the second portion include different materials having different optical properties.

22. The inhibitory element according to any one of claims 18 to 21, wherein The second refractive index of the second portion is provided by doping or implanting the substrate with one or more materials.

23. The inhibitory element according to any one of claims 18 to 22, wherein The first portion has an elliptical shape.

24. The inhibitory element according to any one of claims 18 to 23, wherein The first portion has a diameter ranging from about 1 mm to about 5 mm.

25. The inhibitory element according to claim 10, wherein: The first portion of the inhibitory element defines an aperture, and the second portion of the inhibitory element includes a solid material surrounding the aperture of the first portion of the inhibitory element.

26. A method for detecting nanoparticles in a flow cytometer, comprising: emitting an excitation light beam having a first portion including a high-intensity central portion and a second portion including a low-intensity light stripe; passing the excitation light beam through an inhibitory element, wherein the first portion of the excitation light beam passes through a first portion of the inhibitory element and the second portion of the excitation light beam passes through a second portion of the inhibitory element; focusing the excitation beam for high scattering intensity detection of nanoparticles; as well as The nanoparticles were detected to have a size of 80 nanometers or less.

27. The method according to claim 26, wherein: The phase shift suppresses the low-intensity light fringes of the excitation light beam by causing destructive interference between the first portion and the second portion of the excitation light beam.

28. The method according to claim 27, wherein: The phase shift comprises π / 2 or any odd multiple thereof.

29. The method according to claim 27 or 28, wherein: The phase shift is produced by providing the suppression element with a first material thickness and a second material thickness different from the first material thickness, and wherein the high-intensity central portion of the excitation beam passes through the first material thickness and the low-intensity light stripes of the excitation beam pass through the second material thickness.

30. The method according to claim 27 or 28, wherein: The phase shift is produced by providing the suppression element with a first refractive index and a second refractive index different from the first refractive index, and wherein the high-intensity central portion of the excitation beam passes through the first refractive index and the low-intensity light fringes of the excitation beam pass through the second refractive index.

31. The method according to any one of claims 26 to 30, wherein: The light fringes are caused by at least one of: a diffraction limitation of a focusing lens, irregularities in the shape of the excitation beam relative to a Gaussian distribution, and aberrations of one or more optical components in the flow cytometer.

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