Flow cell for flow cytometer

Abstract

A high sensitivity flow cell for a flow cytometer includes a cuvette. The cuvette has a body including a body height, a body length, and a body width; and a flow channel centrally passing through the body along the height of the body and having an internal dimension through which the sample flows and intersects the excitation beam at an interrogation point, where the internal dimension has a specific aspect ratio.

Description

[0001] This application was filed as a PCT International Application on January 22, 2024, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 481,106, filed on January 23, 2023, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0002] Flow cytometry is used to study particles of various sizes by subjecting them to spectroscopic and other analytical methods while in motion. Sample particles can be suspended in a fluid stream and excited, charged, or impacted to produce fluorescence and / or scatter or emit light that is detected by the flow cytometer. Flow cytometry allows for simultaneous multi-parameter analysis of the physical and / or chemical properties of up to thousands of cells or particles per second. Samples can be characterized and, in some cases, sorted based on size, concentration, and phenotype. Flow cytometry can be applied to identify and characterize a variety of cellular subcomponents, such as vesicles, including extracellular vesicles, mitochondria, nanomedicines and other nanomaterials, proteins, lipids, and nucleic acids. Summary of the Invention

[0003] In general, the present disclosure relates to a flow cell for a flow cytometer. In some embodiments, and by way of non-limiting example, a flow cell is provided that includes a cuvette that increases the sensitivity of the flow cytometer, among other possible benefits and advantages.

[0004] Examples of the present disclosure relate to a high-sensitivity flow cell for a flow cytometer, comprising a cuvette. The cuvette comprises: a body comprising a body height, a body length, and a body width; and a flow channel extending centrally through the body along the body height and having an internal dimension through which a sample flows and intersects an excitation beam at an interrogation point, wherein the internal dimension has an aspect ratio of at least 2.39. In other examples presented herein, the cuvette further comprises: a curved mirror attached to the body and configured to reflect side scatter and fluorescence from the interrogation point; and a collection lens disposed across the width of the body opposite the curved mirror and configured to collect side scatter and fluorescence directly from the sample and reflected by the curved mirror.

[0005] In other examples presented herein, the aspect ratio is at least 3.30. In other examples presented herein, the aspect ratio is 3.33. In yet other examples presented herein, the internal dimensions have a channel length and a channel width, wherein the channel length is 0.6 mm and the channel width is 0.18 mm.

[0006] In other examples presented herein, the internal dimension has a channel length and a channel width, wherein the channel length is 1.3 mm and the channel width is 0.4 mm.In further examples presented herein, the excitation beams are provided by spatially separated lasers.

[0007] In other examples presented herein, the excitation beam is provided by a collinear laser. In other examples presented herein, the body length is at least 2.4 times greater than the body width. In further examples presented herein, the curved mirror has a mirror length and the collection lens has a lens length, each of which is substantially 96% of the body length, such that the curved mirror and the collection lens accommodate a wide collection angle of the generated side scatter. In other further examples presented herein, the curved mirror has a mirror length that is substantially 90% of the body length, such that the curved mirror avoids collecting noise scattered from the flow channel.

[0008] In other examples presented herein, the interior dimensions and the curved mirror are configured together to provide a numerical aperture of at least 1.24. In further examples presented herein, the interior dimensions and the curved mirror are configured together to provide a numerical aperture of substantially 1.24. In other examples presented herein, the interior dimensions and the curved mirror are configured together to provide a numerical aperture of no more than 1.27.

[0009] Other examples of the present disclosure relate to a cuvette for a flow cytometer. The cuvette includes: a body defining a flow channel having an interior dimension configured to maintain the size and velocity of a core flow of a sample in a sheath fluid; an interrogation point in the flow channel; and a curved mirror configured to capture fluorescent light emitted by the sample and light scattered by the sample at a collection angle and to reflect the emitted fluorescent light and scattered light to be collected, the curved mirror being configured such that the collection angle is greater than 55 degrees.

[0010] In other examples presented herein, the cuvette further includes a collection lens for collecting both the fluorescent light and the scattered light directly from the sample and reflected by the curved mirror. In other examples presented herein, the collection angle is greater than 60 degrees. In yet another example presented herein, the collection angle is greater than 70 degrees. In yet another example presented herein, the collection angle is substantially equal to 72 degrees.

[0011] Other examples of the present disclosure relate to a cuvette for a flow cytometer. The cuvette includes: a body defining a flow channel having an interior dimension configured to maintain the size and velocity of a core flow of a sample in a sheath fluid, the body including a body length; an interrogation point in the flow channel; and a curved mirror and a collection lens, the curved mirror and the collection lens together configured to capture fluorescent light emitted by the sample and light scattered by the sample, wherein each of the curved mirror and the collection lens has a length that is at least 90% of the body length. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a system diagram of an example flow cytometry system in which illumination / excitation and detection systems embodying aspects of the present disclosure may interrogate a sample using a fluidics system 104 .

[0013] Figure 2 yes Figure 1 Example optical illumination / excitation and detection systems of example flow cytometry systems and their interaction with a flow cell including a cuvette embodying aspects of the present disclosure.

[0014] Figure 3 is a perspective view of an example cuvette embodying aspects of the present disclosure, showing an example travel path of an excitation laser through the cuvette, with light collected at a forward scatter detector.

[0015] Figure 4 yes Figure 3 Another perspective view of a cuvette showing an example travel path of the excitation laser through the cuvette, where light and fluorescence are collected at side scatter and fluorescence detectors.

[0016] Figure 5 yes Figure 3 Cross-sectional view of the cuvette.

[0017] Figure 6 yes Figure 3 Top view of the cuvette.

[0018] Figure 7 yes Figure 3 Front view of the cuvette.

[0019] Figure 8 yes Figure 3 Side view of the cuvette.

[0020] Figure 9 yes Figure 3 Rear view of the cuvette.

[0021] Figure 10 yes Figure 3 Another side view of the cuvette.

[0022] Figure 11 yes Figure 3 Bottom view of the cuvette.

[0023] Figure 12 is a diagram of example side scatter and fluorescence travel paths in a cuvette embodying aspects of the present disclosure.

[0024] Figure 13 is a top view of a second example cuvette according to aspects of the present disclosure.

[0025] Figure 14 According to various aspects of the present disclosure Figure 13 Side view of a second example cuvette.

[0026] Figure 15 is a top view of a third example cuvette according to aspects of the present disclosure.

[0027] Figure 16 According to various aspects of the present disclosure Figure 15 Side view of the third example cuvette.

[0028] Figure 17 is a top view of a fourth example cuvette according to aspects of the present disclosure.

[0029] Figure 18 According to various aspects of the present disclosure Figure 17 Side view of the fourth example cuvette.

[0030] Figure 19 is a top view of a fifth example cuvette according to aspects of the present disclosure.

[0031] Figure 20 According to various aspects of the present disclosure Figure 19 Side view of the fifth example cuvette.

[0032] Figure 21 is a top view of a sixth example cuvette according to aspects of the present disclosure.

[0033] Figure 22 According to various aspects of the present disclosure Figure 21 Side view of the sixth example cuvette.

[0034] Figure 23 is a top view of a seventh example cuvette according to aspects of the present disclosure.

[0035] Figure 24 According to various aspects of the present disclosure Figure 23 Side view of the seventh example cuvette. DETAILED DESCRIPTION

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

[0037] Now refer to Figure 1, a schematic block diagram of the operating components of an example flow cytometry system 102 is shown. In this example, the flow cytometry system 102 includes a fluidics system 104 and a sample illumination / excitation and detection system 106. In some embodiments, the fluidics system 104 includes a sample delivery system 110, a sheath fluid delivery system 112, and an output collection system 114. The sample illumination / excitation and detection system 106 includes an illumination / excitation system 116, a flow cell 118 including a cuvette 124, and a detection instrument 120. A sample source 121 and a sheath fluid source 123 are also shown.

[0038] The flow cytometry system 102 includes a main housing for organizing the various components of the flow cytometry system 102, which may include some or all of the components of the fluidics system 104 and the illumination / excitation and detection system 106. The flow cytometry system 102 includes power and communication connections that provide power to the fluidics system 104 or the various components of the illumination / excitation and detection system 106. The flow cytometry system 102 also includes a communication path between the illumination / excitation and detection system 106 and a computing device (not shown). The flow cytometry system 102 can incorporate various components of the fluidics system 104, such as supply and waste containers for samples and sheath fluids, or externally accessible trays for loading one or more samples.

[0039] A computing device includes at least a processor and memory and can be any number of known computing devices or a special-purpose computing device. A computing device is a physically tangible device that processes data. Example types of computing devices include personal computers, stand-alone server computers, blade server computers, mainframe computers, handheld computers, smartphones, special-purpose computing devices, and other types of devices that process data.

[0040] A computing device typically includes at least one central processing unit ("CPU"), system memory, and a system bus that couples the system memory to the CPU. The system memory includes random access memory ("RAM") and read-only memory ("ROM"). A basic input / output system, containing basic routines that help transfer information between elements within the device, such as during startup, is stored in ROM. The device also includes a mass storage device. The mass storage device is capable of storing software instructions and data.

[0041] A mass storage device and its associated computer-readable data storage medium provide non-volatile, non-transitory storage for a device. Although the description of a computer-readable data storage medium contained herein refers to a mass storage device such as a hard disk or CD-ROM drive, it should be understood by those skilled in the art that a computer-readable data storage medium can be any usable non-transitory physical device or article from which a device can read data and / or instructions.

[0042] Computer-readable data storage media include volatile and non-volatile media, removable and non-removable media implemented in any method or technology for storing information such as computer-readable software instructions, data structures, program modules or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, CD-ROM, digital versatile disks ("DVD"), other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage devices or other magnetic storage devices, or any other medium that can be used to store the desired information and can be accessed by the device. In some embodiments, the computer-readable data storage medium includes non-transitory media.

[0043] The computing device may also include an input / output controller for receiving and processing input from a variety of other devices, including a keyboard, a mouse, a touch user interface display, or other types of input devices. Similarly, the input / output controller provides output to a touch user interface display, a printer, or other types of output devices.

[0044] In an embodiment, the computing device is fully integrated within the flow cytometer 102 and can be operated via buttons, keys, or one or more touch screens on the flow cytometer 102. In an example, the computing device can be computer software loaded onto any number of common or custom combinations of a processor and memory. The computing device can provide a conversion system for converting the output detection signals from the high-sensitivity detector into computer-processable data, or can receive already converted data. The computing device can enable analysis of the data generated by the interrogation and detection system 106, and can also provide accessible readout of the data and analysis of the data.

[0045] The flow cytometer 102 (including the fluidics system 104 and the illumination / excitation and detection system 106) and the computing device may all be separate components that operate remotely from one another (such as over a network), or may be fully integrated into a single housing. Figure 1 In the example embodiment of FIG, fluidics system 104 and interrogation and detection system 106 are depicted as being organized within the housing of flow cytometer 102, and computing device 106 is remotely connected to flow cytometer 102. Together, the components of system 100 provide for analysis of particles and data generated by interrogation of the particles.

[0046] The fluidic system 104 typically includes one or more source or collection containers and transfer components for storing and moving fluids necessary for the effective operation of the illumination / excitation and detection system 106. The fluidic system 104 typically includes at least a sample source and delivery system and a waste receiving container. In embodiments, the fluidic system 104 also includes a sheath fluid source and delivery system.

[0047] The sample delivery system 110 is a system configured to move a sample from a sample source 121 to the flow cell 118. An example of a sample source 121 is a test tube containing a sample. In other embodiments, the sample source 121 is a microplate containing one or more samples. In some embodiments, the sample delivery system 110 is controlled by a computing device.

[0048] In some embodiments, the fluidics system 104 includes a sheath fluid delivery system 112. The sheath fluid delivery system 112 is a system configured to deliver sheath fluid from a sheath fluid source 123 to the flow cell 118. In some embodiments, the sheath fluid delivery system 112 is controlled by a computing device. As described above, in some embodiments, the sheath fluid delivery system 112 supplies sheath fluid to the flow cell 118, where it is incorporated into the core flow. Other embodiments do not include a sheath fluid delivery system.

[0049] After the sample has been interrogated by the sample illumination / excitation and detection system 106, the waste collection system 114 receives fluid, including the sample and sheath fluid, from the flow cell 118. In some embodiments, the waste collection system 114 stores the sample for subsequent use or disposal. In some embodiments, the waste collection system 114 stores the sample in different locations based on the properties of the sample determined by the sample illumination / excitation and detection system 106. In some embodiments, the waste collection system 114 is a passive container for fluid passing through the flow cell 118. In other embodiments, the waste collection system 114 actively draws fluid from the flow cell 118.

[0050] The illumination / excitation and detection system 106 enables analysis of cells or particles moving in a fluid stream and typically includes a flow cell, wherein the illumination / excitation and detection system 106 is coupled to the fluid system 104. The sample illumination / excitation and detection system 106 is a system configured to illuminate and / or excite the sample in the flow stream and collect fluorescence and light scattered or emitted by the detection system 120. An example of an illumination / excitation system 116 is a laser and one or more optical components for delivering the illumination / excitation light beam. Other embodiments of the illumination / excitation system 116 are also possible. The illumination / excitation system 116 illuminates the sample as the sample passes through the cuvette 124 of the flow cell 118. The detection system 120 detects at least some of the light emitted, scattered and / or fluoresced by the sample. The light detected by the detection system 120 can be used to identify the sample or evaluate properties of the sample, including the identity or properties of individual particles in the sample.

[0051] In embodiments, either or both of the illumination / excitation system 116 and the detection system 120 communicate with a computing device. For example, the computing device operates to control the operation of the flow cytometry system 102 and analyze the contents of the sample. In some embodiments, the computing device may be instrument electronics specific to either or both of the illumination / excitation system 116 and the detection system 120.

[0052] The flow cell 118 includes a flow narrowing device and is configured to prepare a fluid containing a sample for entry into the cuvette 124 for interrogation with an illumination / excitation source. The flow cell 118 directs the fluid containing the sample into the flow narrowing device, thereby generating a core flow. In some embodiments, the core flow also includes a sheath fluid surrounding the sample.

[0053] As the core stream passes through the cuvette 124, the illumination / excitation system 116 illuminates the core stream. The detection system 120 detects light transmitted, scattered, and / or fluoresced by the sample to identify the sample and determine its properties. In some embodiments, the sample illumination / excitation and detection system 106 is controlled by a computing device. In addition, in some embodiments, the sample illumination / excitation and detection system 106 transmits electrical signals corresponding to the light transmitted, scattered, and / or fluoresced by the sample to the computing device.

[0054] In various possible embodiments, the principles described herein can be implemented in various types of flow cytometer systems 102. For example, some embodiments relate to sorting flow cytometers, while other embodiments relate to non-sorting flow cytometers. When implemented as a sorting flow cytometer, the flow cytometer system 102 typically includes sorting control electronics as part of a computing device, a vibration generator coupled to a fluid nozzle (e.g., which can be part of or disposed after the flow cell 118), and a sorting plate electrically coupled to a charge generator that generates an electric field between them to appropriately guide the droplets as they separate from the core stream into the output collection system 114. The flow cytometer system 102 is an example of a particle analyzer.

[0055] Now refer to Figure 2 , Figure 1 The example optical illumination / excitation and detection system 106 of the example flow cytometry detection system and its interaction with the flow cell 118 including the cuvette 124 embody aspects of the present disclosure.

[0056] The illumination / excitation and detection system 106 generally includes an illumination / excitation system 116, a flow cell 118, and a detection instrument 120. The illumination / excitation system 116 directs a light beam 122 toward the flow cell 118 and may include features to direct the light beam toward an appropriate point in a cuvette 124. The detection system 120 receives fluorescent light and light scattered or emitted by the sample in the flow cell 118 and generally includes a forward scatter detector 128 and a fluorescence and side scatter detector 130.

[0057] The cuvette 124 provides an intersection between the fluid system 104, which controls the movement of the sample, and the illumination / excitation and detection system, which enables the acquisition of data about the sample. The cuvette is an important part of the flow cytometer. The design of the cuvette defines the sensitivity and resolution of the entire instrument. Depending on the specific needs of a particular sample or experiment, the cuvette must be designed to accommodate different sizes and shapes. For flow cytometers, sensitivity is a key factor in the successful design of a cuvette for the instrument, and its effect on sensitivity is key to the successful analysis of any given sample.

[0058] Now refer to Figure 3 , shows a perspective view of a cuvette 124 embodying aspects of the present disclosure, illustrating the path of a light beam 122 through the cuvette 124 to produce forward scatter. The cuvette 124 includes a body 202 and a beam shaper lens 204. A flow channel 206 passes through the center of the body 202 and includes an interrogation point 208.

[0059] In the example cuvette 124, the body 202 comprises a generally rectangular shape, but other shapes, such as a cuboid or a circular body, are also contemplated. The body 202 typically comprises three dimensions, such as length, width, and height, or x, y, and z, which determine various characteristics of the flow cell. For example, in the cuvette 124, the path of the light beam 122 through the body 202 is along the y-axis, the flow channel 206 passes through the body 202 along the z-axis, and the x-axis is perpendicular to both the y-axis and the z-axis. In an embodiment, the body 202 is prismatic in shape. In an embodiment, the body 202 is manufactured using UV fused silica due to its low absorption, but can be manufactured from other materials such as glass, fused quartz, or optical grade plastics.

[0060] The beam shaper lens 204 generally acts as a focusing lens along the y-axis of the elliptical beam traveling through the interrogation point 208. The beam shaper lens 204 is attached to the body 202 and is positioned on the surface of the cuvette 124 at the entry point of the beam 122. In the exemplary cuvette 124, the beam shaper lens 204 is a cylindrical lens, specifically having a cylindrical axis parallel to the y-axis of the body 202. The cylindrical axis of the beam shaper lens 204 is oriented perpendicular to the direction of liquid sample flow through the flow channel 206. The beam shaper lens is formed from UV fused silica, but can be fabricated from other materials such as glass, fused quartz, or optical-grade plastic. In embodiments, the beam shaper lens 204 is fused or glued to the body 202.

[0061] The flow channel 206 provides a path along which sample particles can flow or be transported, for example, by sheath fluid, to intersect the light beam 122 from the illumination / excitation system 116. The flow channel 206 is disposed along a z-axis oriented centrally of the body 202 and is arranged perpendicular to the light beam 122. The flow channel 206 has a rectangular cross-sectional shape, although other cross-sectional shapes, such as circular or rectangular channels, are also contemplated. Sample particles can be transported along the flow channel 206 by fluid sample flow, sheath fluid flow, or a combination of sample and sheath flow.

[0062] The interrogation point 208 is located at the intersection between the flow channel 206 and the light beam 122 from the illumination / excitation system 116. The interrogation point 208 is the location of the collision between the sample and the light beam 122 and the point from which fluorescent and / or scattered or emitted light is collected. Figures 5 to 11 The components of the cuvette 124 are discussed in more detail.

[0063] The forward scatter detector 128 is any suitable detection instrument for detecting scattered or emitted light from a sample, and may be a photodiode or a photomultiplier tube, as examples.

[0064] The light beam 122 enters the body 202 via the beam shaper lens 204 and passes through the body 202 along the y-axis, and intersects the flow channel 206 perpendicularly at the interrogation point 208. The intersection and subsequent interaction between the light beam 122 and the sample in the flow channel 206 causes the light to be scattered or emitted by the sample, resulting in forward scattering that continues in the y-axis direction through the body 202. The forward scatter detector 128 communicates with a computing device, such as a computing device. In embodiments, the computing device performs measurement and analysis of the forward scatter detected by the forward scatter detector 128.

[0065] Now refer to Figure 4 , shows a perspective view of a cuvette 124 embodying aspects of the present disclosure, wherein the path of the light beam 122 through the cuvette 124 to produce fluorescence and / or side scatter is shown. Figure 3 In addition to the features discussed, the cuvette 124 also includes a curved mirror 210 and a collection lens 212 .

[0066] Curved mirror 210 is a concave mirror attached to the x-axis surface of body 202. Curved mirror 210 is configured to collect light scattered by the sample or fluorescent light emitted by the sample and reflect it toward collection lens 212 to be directed toward fluorescence and side scatter detector 130.

[0067] The collecting lens 212 is disposed opposite the curved mirror 210 and is attached to the opposite x-axis surface of the body 202. The collecting lens 212 is used to focus the side scattered and emitted fluorescence reflected by the curved mirror 210 toward the fluorescence and side scatter detector 130. In an embodiment, the collecting lens 212 may be an aspherical lens. Figures 5 to 11 The components of the cuvette 124 are discussed in more detail.

[0068] The fluorescence and side scatter detector 130 is any suitable detection instrument for detecting light scattered or emitted from a sample, and may be, for example, a photodiode or a photomultiplier tube, such as an avalanche PD.

[0069] The light beam 122 enters the body 202 via the beam shaper 204 and traverses the body 202, for example, along the y-axis, and intersects the flow channel 206 perpendicularly at the interrogation point 208. The intersection and subsequent interaction between the light beam 122 and the sample in the flow channel 206 causes light scattered by the sample or fluorescence emitted by the sample, generating fluorescence or side scatter, which travels away from the interrogation point 208 and is reflected by the curved mirror 210, which directs the scattered or fluorescence toward the collection lens 212. The collection lens 212 collects the side scatter and directs it to the fluorescence and side scatter detector 130. The collection lens 212 also receives the fluorescence and side scattered light directly from the interrogation point 208. In embodiments, the collection lens 212 may be an aspherical lens.

[0070] For nano-flow cytometry, the light scattering from nanoparticles is several orders of magnitude smaller than that from microparticles, so this paper discloses an optimized design of a flow cell and a cuvette for flow cytometry to provide higher sensitivity and effectiveness at a reduced scale. Conventional nano-flow assemblies suffer from low sensitivity from nanoparticles (e.g., extracellular vesicles) at slow sheath flow rates and low fluorescence sensitivity under narrow emission bins. Similarly, for spectral flow cytometry, the emission bin is narrower than that in conventional flow cytometers, so the fluorescence signal from the same sample is smaller, so the flow cell design disclosed herein is optimized to provide improved spectral performance.

[0071] The internal dimensions of the flow channel provide features for controlling the flow rates of the sample and sheath fluid and for interacting with the collection optics that focus the scattered light or emitted light or emitted fluorescence into the sample. The collection optics typically consist of a curved mirror (e.g., curved mirror 210) and a collection lens (e.g., collection lens 212) that are glued or fused together from both sides of a cuvette (e.g., cuvette 124). Together, the mirror and lens achieve efficient collection of scattered or emitted fluorescence and / or light at the interrogation point within the channel. The physical dimensions of the channel and curved mirror determine the numerical aperture of the assembly, which in turn enables calculation of the collection angle.

[0072] Now refer to Figure 5 , a cross-section of an example cuvette 124 is shown. Figure 5 The cross section provides a complete view of the flow channel 206. In addition to the interrogation point 208, the flow channel 206 also includes an entry point 214, a flow focusing region 216, and an exit point 218.

[0073] Various aspects of the present disclosure provide a flow cell that provides a wider collection angle for scattered / emitted light and enables increased sensitivity of the assembly. Due to changes in channel design to provide an increased collection angle, the average velocity of the sheath is increased. By adjusting the sheath and sample flow rates, an appropriate core flow size can be achieved at the illumination / excitation point. The core flow size should be similar to or smaller than the size of the excitation beam at the illumination / excitation point.

[0074] In various aspects of the present disclosure, the size of the flow channel is enlarged. This increase in the flow channel size opens up the collection angle and enables the collection of a larger amount of light. In addition, in order to provide sample speeds that allow for efficient illumination / excitation using a significantly wider channel size, the sheath flow rate is significantly increased. An advantageous consequence of the faster sheath flow rate is that it enables the separation of excitation pulses of several laser sources and thus creates a spatially separated design in which each laser can have a specified time delay. In order to focus scattered / emitted light or fluorescence, this aspect of the present disclosure can be further combined with an asymmetric design from both sides of the channel.

[0075] Now common reference Figures 6 to 11 , various views of an example cuvette 124 are shown. The cuvette 124 generally includes a body 202, a beam shaper lens 204, a flow channel 206, a curved mirror 210, and a collection lens 212.

[0076] The dimensions of the body 202 are determined by the fluidics requirements and the effective focusing of the excitation light scattered or emitted from the interrogation point 208. The body 202 generally includes three dimensions, such as length, width, and height, or x, y, and z, which determine various characteristics of the cuvette. The body 202 generally has a rectangular shape, wherein the y and z dimensions are substantially equal and both are 2 to 3 times, 2.4 to 2.9 times, and 2.43 to 2.87 times larger than the x dimension. In embodiments, each of the y and z dimensions can be substantially 2.44 or 2.86 times the y dimension. In embodiments, the y dimension is between 8 mm and 12 mm, between 9 mm and 11 mm, or approximately 10 mm; the x dimension is between 3.00 mm and 5.00 mm, between 3.40 mm and 4.50 mm, between 3.50 mm and 4.10 mm, or substantially equal to 3.50 mm or 4.10 mm; and the z dimension is between 8 mm and 12 mm, between 9 mm and 11 mm, or approximately 10 mm.

[0077] The beam shaper lens 204 may be flat aspheric and made of an optically transparent material, which may have a refractive index similar to that of the body 202. Optical coupling of the beam shaper lens 204 to the body 202 may be achieved, for example, by index matching gel, optical adhesive, or direct optical bonding.

[0078] The flow channel 206 guides the sample and sheath fluid when in use and provides internal dimensions to determine the characteristics of the core flow. The flow channel 206 has internal dimensions in three dimensions (e.g., length, width, and height, or x, y, and z). Although the height (z dimension) of the flow channel 206 generally follows the height of the body 202, the length and width of the flow channel 206 can be adjusted to provide a desired velocity and core flow size within the flow channel. The flow of sample and sheath fluid is adjusted to provide a single file line of sample particles along the center of the flow channel 206, and this line of sample particles constitutes the core flow. The core flow size varies based on the internal dimensions of the channel and the flow rates of the sample and sheath fluid. The velocity of the sample and sheath fluid also depends on the internal dimensions of the flow channel 206, making the internal dimensions of the flow channel key to achieving effective flow in the cuvette 124. In an embodiment, the core flow can be adjusted to be substantially equal to or less than the illumination / excitation beam size.

[0079] Increasing the internal dimensions of the flow channel 206 along the Y-axis has the beneficial effect of providing a wider collection angle and increasing the sensitivity of the flow cytometer. However, it also affects the velocity and size of the core stream and may have concurrent effects that require fine-tuning to achieve the necessary fluid properties at the desired collection angle. In embodiments, the flow channel velocity can be increased sufficiently to allow for the splitting of excitation pulses from multiple lasers or other illumination / excitation instruments, each with a specified time delay, thereby producing spatially separated patterns in the detected scatter.

[0080] The internal dimensions of the flow channels 206 can be characterized by their aspect ratio, which is the ratio of the width of the channel to its height. Flow cell assemblies embodying aspects of the present disclosure can have an aspect ratio greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, or greater than 3.40. Example flow cell assemblies embodying aspects of the present disclosure have an aspect ratio substantially equal to 3.25, 3.30, 3.33, or 3.40.

[0081] The interrogation point 208 provides the intersection point between the flow channel 206 and the excitation beam entering the cuvette 124. The interrogation point 208 is the point from which forward scatter and side scatter are emitted. Detection and direction elements such as the beam shaper lens 204, curved mirror 210, and collection lens 212 receive the light scattered or emitted from the interrogation point 208 or fluorescent light.

[0082] In embodiments, the interrogation point 208 can be moved, for example, by moving each of the beam shaper lens 204, the curved mirror 210, and the collection lens 212 upward (toward the surface of the body 202 where sample entry occurs) to increase light collection efficiency. In embodiments, the beam shaper lens 204, the curved mirror 210, and the collection lens 212 can each be moved upward or extended by 1 mm in the height or z-dimension to increase light collection efficiency from the bottom of the cuvette 124 (in the direction of the surface of the body 202 where the sample exits the flow channel 206).

[0083] The curved mirror 210 can be a plano-concave back surface mirror made of an optically transparent material such as glass, quartz, or optical quality plastic, which can have a refractive index similar to that of the body 202. The curved mirror 210 can have a flat front surface that is optically coupled to an adjacent flat surface of the body 202 to minimize optical losses. The optical coupling of the curved mirror 210 to the body 202 can be achieved, for example, by an index matching gel, an optical adhesive, or direct optical bonding.

[0084] The physical size of the curved mirror can limit light collection and prevent detection of light from the corners of the channel, which can potentially generate additional noise / background. As described in conjunction with various example embodiments disclosed herein, the expanded flow channel 206 can introduce additional noise due to the wider collection angle collecting light impinging on the edges of the flow channel. Limiting the size of the curved mirror redirects light to avoid these potential noise sources and improves the separation between noise and particle signals.

[0085] The physical dimensions of the curved mirror 210 are configured to limit the collection of light and prevent detection of light scattered from the corners of the channel rather than from the sample, which could potentially generate additional noise or background. The physical dimensions of the curved mirror 210 and the internal dimensions of the flow channel 206 together determine the numerical aperture of the cuvette 124. In embodiments, the numerical aperture can be configured to provide a wide collection angle and reduce noise generated by scattering from the edges of the flow channel 206. The numerical aperture can be less than 1.28, less than 1.27, less than 1.26, or less than 1.25. The numerical aperture can be substantially equal to 1.24.

[0086] A collection lens 212 is disposed across the body 202 of the cuvette 124 opposite the curved mirror 210. Optical coupling of the collection lens 212 to the body 202 can be achieved, for example, by an index matching gel, an optical adhesive, or direct optical bonding. Together, the curved mirror 210 and the collection lens 212 provide collection optics for focusing side scattered light into the detection fiber. In embodiments, the curved mirror and the collection lens can each have a length substantially equal to 80%, 85%, 90%, 95%, 96%, 97%, or 100% of the length of the body. In embodiments, the curved mirror 210 can have a reduced length compared to the collection lens 212, rather than being substantially equal to the length of the collection lens. For example, the collection lens 212 can have a length approximately 96% of the length of the body 202, and the curved mirror 210 can have a length approximately 90% of the length of the body 202.

[0087] Now refer to Figure 12 , a diagram of an example side scatter travel path in a cuvette embodying aspects of the present disclosure, including the collection angle. For reference, interrogation point 208 and curved mirror 210 are labeled.

[0088] As discussed throughout, the excitation beam 122 enters the body 202 and intersects the sample at the interrogation point 208. Due to this interaction between the sample and the illumination / excitation beam 122, fluorescence and light are scattered or emitted from the interrogation point 208, and the light scattered to the side is reflected by the curved mirror 210. The angle between the illumination / excitation beam and the scattered light that encounters the curved mirror 210 is the collection angle of the scattered or emitted light. In embodiments, a collection angle greater than 54 degrees may be desirable to provide a wider collection of scatter from the interrogation point 208 and increase the sensitivity of the flow cytometer. In embodiments, a collection angle greater than 55 degrees, 60 degrees, 65 degrees, 70 degrees, or 75 degrees may be desirable. In embodiments, the collection angle is configured to be substantially equal to 72 degrees.

[0089] Now refer to Figures 13 to 24 Various example flow cell assemblies are described.

[0090] Now refer to Figure 13 and Figure 14 , the measurement results according to Table 1 below show an example cuvette assembly 324:

[0091] Table 1: Dimensions of an Example Cuvette Assembly 324

[0092] size Length (Y) (mm) Width (X) (mm) Height (Z) (mm) Flow cell body 10.00 4.10 10.00 Channel size 0.60 0.18 10.00 curved mirror 9.60 1.90 8.00 Collecting lens 9.60 1.83 8.00 Beam shaper lenses 2.50 4.10 7.00

[0093] The cuvette assembly 324 has an extended y-axis dimension relative to the x-dimension in both the body and the flow channel, thereby providing a wide collection angle for scattered / emitted light from the sample. The cuvette assembly 324 also has an extended z-dimension relative to the x-dimension to provide stability for the core flow when the sample and / or sheath flow rates are low. Each of the curved mirror and collection lens of the cuvette assembly 324 also extends in the y-dimension and the z-dimension to accommodate the wide collection angle of scattered / emitted light from the sample. In the example cuvette assembly 324, the curved mirror and collection lens each substantially occupy 96% of the length of the surface of the body to which they are attached. The physical dimensions of the channel and the curved mirror together determine the numerical aperture of the assembly. The example cuvette assembly 324 has a numerical aperture equivalent to approximately 1.08, wherein the collection angle is approximately 72 degrees.

[0094] Now refer to Figure 15 and Figure 16 , another example cuvette assembly 424 is shown according to the measurement results of Table 2 below:

[0095] Table 2: Dimensions of Example Cuvette Assembly 424

[0096] size Length (Y) (mm) Width (X) (mm) Height (Z) (mm) Flow cell body 10.00 4.10 10.00 Channel size 0.60 0.18 10.00 curved mirror 9.00 1.90 8.00 Collecting lens 9.60 1.83 8.00 Beam shaper lenses 2.50 4.10 7.00

[0097] Compared to cuvette assembly 324, cuvette assembly 424 has a shortened y-dimension of the curved mirror, which enables the mirror to avoid collecting scattered light from the corners of the channel, which may contribute additional noise to the system.

[0098] Now refer to Figure 17 and Figure 18 , another example cuvette assembly 524 is shown according to the measurement results of Table 3 below:

[0099] Table 3: Dimensions of Example Cuvette Assembly 524

[0100] size Length (Y) (mm) Width (X) (mm) Height (Z) (mm) Flow cell body 10.00 4.10 10.00 Channel size 0.60 0.18 10.00 curved mirror 9.00 1.90 9.00 Collecting lens 9.60 1.83 9.00 Beam shaper lenses 2.50 4.10 9.00

[0101] Compared to cuvette assemblies 324 and 424, cuvette assembly 524 moves the interrogation point by 1 mm by extending the z-dimension of the curved mirror, collection lens, and beam shaper lens. This shift in the interrogation point provides a wider collection angle relative to the bottom of the flow cell assembly. In an embodiment, cuvette assembly 524 raises each of the curved mirror, collection lens, and beam shaper lens upward by 1 mm (toward the surface of the cuvette including the sample entry point) to increase light collection efficiency from the bottom of the flow cell.

[0102] Now refer to Figure 19 and Figure 20 , another example cuvette assembly 624 is shown according to the measurement results of Table 4 below:

[0103] Table 4: Dimensions of Example Cuvette Assembly 624

[0104] size Length (Y) (mm) Width (X) (mm) Height (Z) (mm) Flow cell body 10.00 3.50 10.00 Channel size 1.30 0.40 10.00 curved mirror 9.60 1.90 8.00 Collecting lens 9.60 1.83 8.00 Beam shaper lenses 2.50 3.00 8.00

[0105] Cuvette assembly 624 has a main y- and z-dimension, and the flow channel is further extended relative to the x-dimension compared to cuvette assemblies 324, 424, and 524 to further accommodate a larger collection angle of scattered / emitted light and the fluidics required to provide a more stable core flow for low sample and sheath fluid flow rates. This may be achieved by extending one or both of the y- and z-dimensions, or by reducing the x-dimension to focus scattered light and fluorescence.

[0106] Although the cuvette assembly 624, which includes a body, flow channel, curved mirror, and collection lens, is generally larger than the cuvette assemblies 324, 424, and 524, it is noteworthy that all four assemblies have a similar aspect ratio of approximately 3.25. The cuvette assembly 624 can have an aspect ratio substantially equal to 3.25, while other cuvette assemblies embodying aspects of the present disclosure can have aspect ratios greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, or greater than 3.40. An example flow cell assembly embodying aspects of the present disclosure has an aspect ratio substantially equal to 3.33.

[0107] Cuvette assembly 624 may have a different fluidic configuration than cuvette assemblies 324, 424, 524. The expanded channel size of cuvette assembly 624 enables accelerated sheath flow rates and allows the configuration of the instrument to be changed from a collinear laser design to a spatially separated laser design.

[0108] Now refer to Figure 21 and Figure 22 , another example cuvette assembly 724 is shown according to the measurement results of Table 5 below:

[0109] Table 5: Dimensions of Example Cuvette Assembly 724

[0110] size Length (Y) (mm) Width (X) (mm) Height (Z) (mm) Flow cell body 10.00 3.50 10.00 Channel size 1.30 0.40 10.00 curved mirror 9.00 1.90 8.00 Collecting lens 9.60 1.83 8.00 Beam shaper lenses 2.50 3.00 8.00

[0111] Cuvette assembly 724 is substantially similar to cuvette assembly 624 with a reduced y-dimension of the curved mirror to provide better control and avoid collecting light scatter from the corners of the channel instead of the sample, which can cause system noise.

[0112] Now refer to Figure 23 and Figure 24 , another example cuvette assembly 824 is shown according to the measurement results of Table 6 below:

[0113] Table 6: Dimensions of Example Cuvette Assembly 824

[0114] size Length (Y) (mm) Width (X) (mm) Height (Z) (mm) Flow cell body 10.00 3.50 10.00 Channel size 1.30 0.40 10.00 curved mirror 9.00 1.90 9.00 Collecting lens 9.60 1.83 9.00 Beam shaper lenses 2.50 3.00 9.00

[0115] The cuvette assembly 824 moves the interrogation point by increasing the z-dimension of the curved mirror, collection lens, and beam shaper lens. This movement of the interrogation point increases the collection angle from the bottom surface of the flow cell assembly. The example assemblies discussed herein and other flow cells embodying aspects of the present disclosure provide optimized flow cell designs and corresponding collection optics designs.

[0116] Illustrative examples of the systems and methods described herein are provided below.Implementations of the systems or methods described herein may include any one or more of the following, and any combination of the following.

[0117] Item 1. A cuvette for a flow cytometer, comprising: a body defining a flow channel having internal dimensions configured to maintain the size and velocity of a core flow of a sample in a sheath fluid; an interrogation point in the flow channel; and a curved mirror for capturing fluorescence emitted by the sample and light scattered by the sample at a collection angle and reflecting the emitted fluorescence and scattered light to be collected, the curved mirror being configured such that the collection angle is greater than 55 degrees.

[0118] Item 2. The cuvette according to Item 1, further comprising a collecting lens for collecting fluorescent light and scattered light directly from the sample and reflected by the curved mirror.

[0119] Item 3. The cuvette of Item 1 , wherein the collection angle is greater than 60 degrees.

[0120] Item 4. The cuvette of Item 1 , wherein the collection angle is greater than 70 degrees.

[0121] Clause 5. The cuvette of clause 1, wherein the collection angle is substantially equal to 72 degrees.

[0122] Item 6. A cuvette for a flow cytometer, comprising: a body defining a flow channel having internal dimensions configured to maintain the size and velocity of a core flow of a sample in a sheath fluid, the body comprising a body length; an interrogation point in the flow channel; and a curved mirror and a collection lens, the curved mirror and the collection lens together configured to capture fluorescence emitted by the sample and light scattered by the sample, wherein each of the curved mirror and the collection lens has a length of at least 90% of the length of the body.

[0123] The various embodiments described above are provided by way of illustration only and should not be construed as limiting the claims appended hereto. Those skilled in the art will readily appreciate that various modifications and variations may be made without following the example embodiments and applications shown and described herein, and without departing from the full scope of the appended claims.

Claims

1. A high-sensitivity flow cell for a flow cytometer, comprising: A cuvette, the cuvette having: a body, the body comprising a body height, a body length, and a body width; as well as A flow channel extends through the body centrally along its height and has an interior dimension through which a sample flows and intersects the excitation beam at an interrogation point, wherein the interior dimension has an aspect ratio of at least 2.

39.

2. The high-sensitivity flow cell according to claim 1, further comprising: a curved mirror attached to the body and configured to reflect side scatter and fluorescence from the interrogation point; as well as A collection lens is disposed across the width of the body opposite the curved mirror and configured to collect side scatter and fluorescence directly from the sample and reflected by the curved mirror.

3. The high-sensitivity flow cell according to claim 1, wherein The aspect ratio is at least 3.

30.

4. The high-sensitivity flow cell according to claim 3, wherein The aspect ratio is 3.

33.

5. The high-sensitivity flow cell according to claim 1, wherein The internal dimensions include a channel length and a channel width, wherein the channel length is 0.6 mm and the channel width is 0.18 mm.

6. The high-sensitivity flow cell according to claim 1, wherein The internal dimensions include a channel length and a channel width, wherein the channel length is 1.3 mm and the channel width is 0.4 mm.

7. The high-sensitivity flow cell according to claim 1, wherein The excitation beams are provided by spatially separated lasers.

8. The high-sensitivity flow cell according to claim 1, wherein The excitation light beam is provided by a collinear laser.

9. The high-sensitivity flow cell according to claim 1, wherein The body length is at least 2.4 times greater than the body width.

10. The high-sensitivity flow cell according to claim 9, wherein The curved mirror has a mirror length and the collecting lens has a lens length, each of which is substantially 96% of the body length, such that the curved mirror and the collecting lens accommodate a wide collection angle of the generated side scatter.

11. The high-sensitivity flow cell according to claim 9, wherein The curved mirror has a mirror length that is substantially 90% of the body length so that the curved mirror avoids collecting noise scattered from the flow channel.

12. The high-sensitivity flow cell according to claim 1, wherein The interior dimensions and the curved mirror together are configured to provide a numerical aperture of at least 1.

24.

13. The high-sensitivity flow cell according to claim 12, wherein The internal dimensions and the curved mirror together are configured to provide a numerical aperture of substantially 1.

24.

14. The high-sensitivity flow cell according to claim 12, wherein The internal dimensions and the curved mirror together are configured to provide a numerical aperture of no more than 1.27.

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