Angled flow cells for flow cytometry

Abstract

Provided are devices and systems including angled flow cells enabling the collection of side and forward scatter and methods of using such angled flow cells. The flow cells including a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer, the opaque middle layer including a void and; a sealed chamber formed by the combined edges of the void, the first optically clear layer, and the second optically clear layer; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite the outer surface of the second optically clear layer; and at least one light collection lens.

Description

ANGLED FLOW CELLS FOR FLOW CYTOMETRYCROSS-REFERENCE TO RELATED APPLICATION

[0001] The following application claims benefit of U.S. Provisional Application No. 63 / 658,968 filed June 12, 2024, the contents of which are hereby incorporated by reference in its entirety.BACKGROUND

[0002] The field of flow cytometry has become the gold standard for high throughput particle analysis. Generally, samples are flowed through a flow cell and past an analysis point. In optical analysis, the analysis point includes a laser beam. The defined point of laser interrogation combined with efficient capture and analysis of scattered and fluorescent light enables a flow cytometer to identify properties of particles within the sample as they flow past at high linear velocities. The capturing and analysis of scatter and fluorescence properties provides information for many applications, allowing for the counting, identification, characterization, and sorting of cells in a sample.

[0003] As the magnitude of scattered light is highly dependent on the angle of collection, flow cytometers generally measure scattered light at both 90 degrees (side scatter) and the forward direction (forward scatter). However, many current flow cell fabrication methods, including those for acousto-fluidic flow cells, use a multilayered manufacturing approach. The differing refractive indexes of the materials within the layers may impact the light scatter, causing optical interference, and decreasing the accuracy of the collected data.SUMMARY

[0004] This Summary is provided to introduce a selection of concepts in a simplified form that is further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter.

[0005] The present disclosure describes devices including a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer, the opaque middle layer including a void; a sealed chamber formed by the combined edges of the void, the first optically clear layer, and the second optically clear layer; at least one hole formed in the first optically clear layer or the second optically clear layer that is fluidly connected to the sealed chamber to form a sample inlet to, or a sample outlet from, the sealed chamber; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite the outer surface of the second optically clear layer; and at least one light collection lens.

[0006] The present disclosure describes systems including, a flow cell including: a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer and including a space; a sealed chamber formed by the combined edges of the space, the first optically clear layer, and the second optically clear layer; at least one hole formed in the first optically clear layer or the second optically clear layer that is fluidly connected to the sealed chamber to form a sample inlet to, or a sample outlet from, the sealed chamber; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite the outer surface of the second optically clear layer; a laserconfigured to enter the front prism at an entry angle relative to a plane of a face of the front prism; and at least one light collection lens adjacent to the outer surface of the second optically clear layer and configured to collect light scattered by the interaction of the laser and a sample.

[0007] The present disclosure also describes methods of collecting light scatter from a sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGs. 1A-1C illustrate a conventional optical configuration of a flow cytometer. FIG. 1A shows a 3-D view of the entire flow cell assembly. FIG. 1 B shows a 3-D view of the 90-degree collection optics of the flow cell assembly in FIG. 1A. FIG. 1C shows a 3-D view of the intersection of the focused sample stream, the focusing point of the laser, and the optical collection volume.

[0009] FIG. 2 illustrates an exemplary layered optical flow cell.

[0010] FIGs. 3A-3D illustrate an exemplary angled layered flow cell with optics according to an embodiment. FIG. 3A shows a 3-D view of the entire flow cell assembly. FIG. 3B shows a zoomed-in 3-D view of the upper portion of the flow cell assembly in FIG. 3A. FIG. 3C shows a 2-D cross sectional view of the system in plane with the laser. FIG. 3D shows a zoomed in view of the two-dimensional cross-section from FIG. 3C.

[0011] FIG. 4 illustrates the intersection of a laser with the surfaces of the flow channel of FIG. 3. The arrow indicates the exit point of the laser from the flow channel.

[0012] FIG. 5 illustrates an exemplary angled layered flow cell with optics according to an embodiment.DETAILED DESCRIPTION

[0013] Flow cytometry is used to measure the physical and characteristics of cells. Optical flow cytometry uses laser light to analyze individual cells or particles in a sample as they flow through a flow cell. Analysis of the light scatter and fluorescence provides information about the cells and particles within the sample, allowing for the counting, identification, characterization including state and internal complexities of the cell, and sorting of cells. Scatter from cells and particles is known to be very angle dependent, which has made collection of light in precise geometric configuration very important for studies that use scatter.

[0014] Unwanted optical signals that interfere with the detection of fluorescence and light scatter from a sample can impact data collection and analysis. Generally, the refractive indexes of the flow cell material (typically something such as crown glass, quartz, or fused silica) is not perfectly matched to the fluid the particle is flowing in (most typically an aqueous solution). Aqueous solutions used in flow cells generally have a refractive index of about 1 .33, which is closer to that of most optical materials (1.5) than the refractive index of air (1.0). While closer, this difference in refractive index is the driving reason focusing approaches were initially developed for analysis of flowing particles. Crossland and Taylor (A Device for Counting Small Particles suspended in a Fluid through a Tube, Nature 71, 37-38 (1935)) realized in 1953 that particles were flowing close to the wall of their flow cells, causing significant optical background light scatter / reflection. Therefore, they invented hydrodynamic focusing to move the particles from the walls to one defined point away from the walls so that optical interrogation could occur with reduced background reflections and scatter. It has become common practice to use a focusing method such as hydrodynamic, acoustic, or inertial focusing to position particles for optimal optical analysis in flow cytometers.

[0015] Flow cells with geometric configurations that are optically clear on four sides have decreased optical noise but can be difficult to manufacture. Flow cell fabrication methods using a multilayer approach do not allow for four optically clear sides, but are easier to manufacture. However, flow cells that are optically clear on only two sides (front and back or top and bottom) can be optically challenging, as the light scatter off surfaces with differing refractive indexes generates a lot of 'stray' light that produces optical interference in light collection, decreasing the accuracy of the collected data. The approaches of the present disclosure allow for optimal collection and excitation from a sample in a flow cell that is optically clear on only two sides. In some aspects, the devices, systems and methods described herein allow for the simultaneous collection of forward scatter, side scatter, and fluorescence.

[0016] A typical optical configuration of a clow cytometer flow cell is shown in FIGs. 1A-1C. FIG. 1A shows a sample stream 102 introduced into flow cell assembly 100 via sample inlet 104, hydrodynamic sample focusing device 106 to focus the sample stream 102 into the flow channel 108, and exit line 110 to remove the sample after optical processing. FIG. 1 A also shows laser 112 interrogating the sample stream 102 and the laser 112 being blocked by a blocker bar 114. The blocker bar prevents the unscattered laser light from directly entering the lens or detector but allows scatter light to pass over it and into the detector. Light scattered by particles in the forward direction propagates above and below the bar 114 and may be measured by a detector. 90-degree collection optics 116 are shown, where side scatter collection is achieved via a gel coupled lens 118 positioned 90 degrees to the laser 112. In embodiments, the gel coupled lens 118 is a detector. Light collection volume 126 of the 90-degree collection optics 116 is also shown.

[0017] FIG. 1B depicts cut out 120 of FIG. 1A and shows a closer view of the flow channel 108, where a particle 122, flowing in the hydrodynamically focused sample stream 102, is being interrogated by the laser 112. The direction of flow of the particles 122 is shown by the arrow 124. FIG. 1 B includes a closer view of the side scatter collection of the light collection volume 126, via gel coupled lens 118 at 90 degrees to the laser 112.

[0018] FIG. 1C depicts cut out 128 of FIG. 1 B and shows interrogation volume 130, which is created by the intersection of the focused sample stream 102, the focusing point of the laser 112, and light collection volume 126 of the 90-degree collection optics 116. The 90-degree collection optics 116 are used to collect both the side scattered light and the fluorescence emitted from the particle 122 as it passes through the interrogation volume 130.

[0019] FIG. 2 illustrates an exemplary layered optical flow cell 200 including a first optically clear layer 210 and a second optically clear layer 212 with an opaque middle layer 208 sandwiched between the first optically clear layer 210 and the second optically clear layer 212. In some aspects, the middle layer may include a flow channel 202. The first or second optically clear layer may have an inlet 204 and / or an outlet 206 connected to the flow channel 202 (also referred to as a void or space), allowing for the introduction of a sample into a sealed chamber formed by the combined edges of the middle layer 208, the first optically clear layer 210, and the second optically clear layer 212. The first optically clear layer 210 may be referred to as a front / top layer and the second optically clear layer 212 may be referred to as the back / bottom layer.

[0020] These microfabricated devices are typically analyzed using an epifluorescence (epi) microscopy optical configuration (where the optical back scatter is collected back along the same light path as the incoming excitation light). Additionally, these flow cells enable simple transmission imaging (light emitted through the front / top and collected from the back / bottom). However, conventionally, these flow cells have not been used for simultaneouscollection of forward scatter, 90-degree side scatter, and fluorescence due to the geometric limitations of only having two optically clear sides and the differences in the refractive indices of layered construction.

[0021] The approaches of the present disclosure allow for optimal collection and excitation from a sample in a flow cell that is optically clear on only two sides. In some aspects, the devices, systems and methods described herein allow for the simultaneous collection of forward scatter, side scatter, and fluorescence. This approach is summarized in the exemplary embodiment of an angled layered flow cell 300 shown in FIGs. 3A-3D.

[0022] FIG. 3A shows a three-dimensional (3-D) zoomed-out view of a layered flow cell 300 that has been implemented with additional optical components to enable its integration into a conventional flow cytometer's optical path. FIG. 3A shows a laser 302 with the arrow indicating the direction of propagation of the laser 302. The layered flow cell 300 may have a sample inlet 304 and a sample outlet 306 connected by a flow channel 316, through which a sample can flow from the sample inlet 304 to the sample outlet 306 and pass through the laser 302.

[0023] FIG. 3B shows a zoomed-in 3-D view of the upper portion 308 of the flow cell assembly in FIG. 3A that emphasizes the optical elements of the layered flow cell 300. In some aspects, FIG. 3B includes a front prism 310 and a front optical layer 312 of the flow cell. In some aspects, the front prism 310 has the same refractive index as the optical material of the front optical layer 312 of the flow cell. The front optical layer 312 having an outer surface 336. A middle layer 314 of the flow cell contains the flow channel 316. A back optical layer 318 of the flow cell with an outer surface 338 and a back prism 320 are also shown. In some aspects, the back prism 320 has the same refractive index as the optical material of the back optical layer 318 of the flow cell. FIG. 3B also shows a collection lens for 90-degree scatter and fluorescence 322 and a blocker bar 324 that prevents the laser 302 from directly entering the lens or detector for collection of forward light scatter 326. In embodiments, collection lens for 90-degree scatter and fluorescence 322 is also referred to as a detector. Air space 334 is also shown. In embodiments, the laser 302 passes from air space 334 through the front prism 310, front optical layer 312 of the flow cell, the flow channel 316, the back optical layer 318 of the flow cell, the back prism 320, and back into air space 334.

[0024] FIG. 3C shows a two-dimensional (2-D) cross sectional view of the flow cell on the plane of the laser excitation as shown by lines A-A' in FIG. 3B. Angle a is shown, which is the relative angle of the laser 302 to the entering face 340 of the front prism 310 and exiting face 342 of the back prism 320. Angle O \s also shown, which is the angle between the laser 302 and the faces of the angled surfaces of the layered flow cell. FIG. 3D shows a zoomed in view of the two-dimensional cross-section 328 from FIG. 3C, that emphasizes the relative angle between the faces of the flow cell and the laser 302, which is denoted as O. This cross-section emphasizes the intersection of the laser 302 with the interfaces between the prisms (310 and 320), optical layers (312 and 318), and the solution in the flow channel 316. The center of the panel where the laser 302 intersects the middle layer 314 indicates the particle position and O is the angle between the laser 302 and the faces of the angled surfaces of the layered flow cell. The laser 302 is propagating from the lower left to the upper right of the diagram.

[0025] In flow cytometry analysis it is important to consider the scattering of light that occurs at the interface of two different materials due to the refractive index change between the materials. The schematic in FIG. 3C shows the laser 302 entering and exiting at angle a. As shown, where a is 90 degrees, the refractive index differences between air (1.0) and most optical materials (~1.5) results in significant back scatter and reflection at the entry surface as therefractive index of the medium that the laser is propagating through (air) is less than the optical material it is entering (e.g., borosilicate glass, quartz, or fused silica). This could be problematic, but the dimensions of the flow cell are kept such that the entry point of light into the prism is well outside the field of view of the collection optics. The entry angle (a) is also maintained at 90 degrees to the plane of the face of the entry and exit prisms to eliminate any potential angling of the laser as it passes through the prism entry and exit faces, though other entry angles may also be used.

[0026] FIGs. 30 and 3D show several interfaces as the laser 302 propagates from air to prism (FIG. 3C - 334 to 310), prism to optical layer (FIG. 3D - 310 to 312), optical layer to aqueous solution (FIG. 3D - 312 to 136), aqueous solution to optical layer (FIG. 3D - 316 to 318), optical layer to prism (FIG. 3D - 318 to 320), and from prism to air (FIG. 3C - 320 to 334). Reflection and scatter of light occurs to some extent any time that light is propagating from a medium with a lower index of refraction into a medium with a higher index of refraction. Therefore, interfaces between prisms and optical layers of the present disclosure can be engineered such that they have the same or very similar refractive indexes. The entry point of light into the prism should be kept physically distant (a mm or more) from the focal point of the collection optics (e.g., collection lens for 90-degree scatter and fluorescence 322). In this manner, the angled flow cell approach will minimize stray light reflection at the interfaces of air into the entry prism, prism into the optical layer, and optical layer into the exit prism. As the refractive index of the exit prism is greater than that of air, the interface from the exit prism to air is not of concern.

[0027] In some aspects, the interfaces between the optical layers and the aqueous solution are generally close (less than one mm) to the particle 330 under analysis. However, in some instances, the interfaces may be further apart, for example when analyzing larger particles such as 3D tissue models.

[0028] When considering the requirements of collection of light at 90 degrees relative to the laser and in the forward direction of the laser, it is necessary to angle the flow cell. As the refractive index of optical materials (~1.5) are expected to be greater than that of an aqueous solution (1 .33), scatter from the laser propagating from the entry optical layer into the aqueous solution is not of concern. However, due to scattering effects that result from the differences in refractive indexes between the materials, scatter as the light propagates from the aqueous solution to the optical layer of the flow cell (e.g., FIG. 3D - 316 to 318) is of concern. The embodiment shown in FIGs. 3A-3D can have relatively typical dimensions for a flow cytometer designed to analyze particles the size of cells. The laser 302 can have a width (W) of 80 microns while the dimensions of the flow channel can be 200 microns in depth (D) and 500 microns in height (H). In embodiments, the relative dimensions of the width of the laser 302 and the depth and the height of the flow channel can be proportional to 80 microns, 200 microns, and 500 microns respectively.

[0029] FIG. 4 shows a zoomed-in view of the intersection of the laser 302 with the interfaces between optical layers (312 and 318) and the solution in the flow channel 316. The dot 330 at the center of the flow channel 316 indicates the particle position where the laser 302 is expected to intersect with a particle in flow that has been focused to a position for analysis. O \s the angle that the laser 302 is propagating at relative to the flow cell surfaces. FIG. 4 is rotated 90 degrees from what is shown in FIG. 3D such that the laser 302 is propagating from the upper left to the lower right. The entry optical layer 312 is the top layer, the middle layer 314 has the flow channel 316 cut in it. In embodiments, the flow channel 316 can be filled with aqueous solution. The back optical layer 318 is shown as the layer on the bottom.

[0030] The angle of the laser relative the flow cell surfaces (0) is key to efficient optical excitation of the particle 330 without generating excessive scatter from the interface 332 of the back optical layer 318 and the solution in the flow channel 316 (indicated by an arrow in FIG. 4). When using a laser for excitation, it is common for the laser to emit highly polarized light. This enables the use of Brewster's angle to minimize the amount scatter from the interface of the bottom optical surface and the solution. As the solution is expected to be aqueous its refractive index will be about 1.33 and the optical surface will have a refractive index of about 1.5, as such Brewster's angle will be approximately 53 degrees. At this angle it is expected that the highly polarized light will primarily be refracted through the optical surface and scatter / reflection will be minimized.

[0031] While FIGs. 3A-3D show the use of prisms optically cemented to move air to optical surface interfaces away from the point of optical collection, it can also be seen that use of sufficiently thick optical layers, such as 1 mm thick borosilicate glass slides, will also move the point of air to optical material out of the point of collection. This is shown in FIG. 3D, where the laser enters well below the point of collection. As this point of entry will be out of the focal plane and (if 0 is 53 degrees and the optical layer 1 mm thick) more than 1 mm below the optical collection point, an entry prism may be desirable but not required for the entry side of the laser.

[0032] FIG. 4 shows an exemplary embodiment where the exit point of the laser from the flow channel can be within 100 to 200 microns of the collection point for a 0.2 mm deep by 0.5 mm wide flow channel. While flow channel dimensions could be expanded to move this point further away from the collection point, channel dimensions in the hundreds of microns are often fluidically advantageous. Therefore, one solution to minimize scatter is to collect from the bottom or back of the flow cell as light will primarily be scattered and reflected away from the point of collection. It will also be possible to collect light from above or the front of the flow cell, but this is anticipated to generate more background light scatter / reflection. It is for this reason that the schematics in FIG. 3 are drawn to show collection of light from the opposite surface that is opposite that of the entry direction of the exciting light of the flow cell system.

[0033] The exit point of the laser, given that the exit point of the laser from the flow channel is within 100 to 200 microns of the collection point for a 0.2 mm deep by 0.5 mm wide flow channel, is anticipated to be at least 1 mm above the collection point. It will also be out of the focal plane of collection. Therefore, the exit prism may not be necessary to eliminate scatter. However, the use of the prism in conjunction with a gel mounted lens would enable higher numerical aperture collection, which would result in more sensitive detection. While not wishing to be bound, it is theorized that in some aspects, the prism may be used to remove any air gaps in collection that would hamper numerical aperture of the collection lens. Therefore, it is anticipated that, for some applications, the exit prism would be highly desirable.

[0034] FIG. 5 shows a 3-D rendering of an embodiment of a layered flow cell 500 with many optimal features including prisms (510 & 520) having shapes such that they accommodate a 90-degree laser entry and exit as well as Brewster's angle at the prism to optical layer interfaces. FIG. 5 shows a laser 502 and the arrow indicates the direction of propagation of the laser 502. The layered flow cell 500 also has a sample inlet (not shown) and a sample outlet 506 connected by a flow channel 516, through which a sample can flow from the sample inlet to the sample outlet 506 and pass through the laser 502. FIG. 5 includes a front prism 510 and a front optical layer of the flow cell 512. In embodiments, the front prism 510 has the same refractive index as the optical material of the front optical layer of theflow cell 512. A middle layer 514 of the flow cell contains the flow channel 516. A back optical layer of the flow cell 518 and a back prism 520 are also shown. In embodiments, the back prism 520 has the same refractive index as the optical material of the back optical layer of the flow cell 518. FIG. 5 also shows a gel coupled lens 522 on the back side of the back prism 520 to enable efficient optical collection by eliminating air space 534 between the gel coupled lens 522 and the back prism 520. In embodiments, the gel coupled lens 522 is a detector. The blocker bar 524 is also shown, but forward scatter collection lenses are not shown as they are not always used or necessary (often a photodiode is placed right behind the blocker bar).

[0035] This flow cells described herein may be used with unfocused flowing particles, hydrodynamically focused particles, acoustically focused particles, inertially focused particles, dielectrophoretic focused particles or any flowing particle focused to a position by any means. In embodiments, the layered flow cell 500 is implemented with single stream acoustically focused flow cells.

[0036] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims.

[0037] The following statements of the invention are intended to characterize possible elements of the invention according to the foregoing description given in the specification and drawings. Because this application is a provisional application, these statements may be altered, deleted, or added to upon preparation and filing of a nonprovisional application. Such changes are not intended to affect the scope of equivalents according to the claims issuing from the nonprovisional application, if such changes occur. According to 35 U.S.C. § 111 (b), claims are not required for a provisional application. Consequently, the statements of the invention cannot be interpreted to be claims pursuant to 35 U.S.C. § 112.EXAMPLE CLAUSES

[0038] 1 . A flow cell including a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer, the opaque middle layer including a void; a sealed chamber formed by the combined edges of the void, the first optically clear layer, and the second optically clear layer; at least one hole formed in the first optically clear layer or the second optically clear layer that is fluidly connected to the sealed chamber to form a sample inlet to, or a sample outlet from, the sealed chamber; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite the outer surface of the second optically clear layer; and at least one light collection lens.

[0039] 2. The flow cell of clause 1 , wherein the first optically clear layer includes crown glass, borosilicate glass, quartz, or fused silica.

[0040] 3. The flow cell of clause 1 or 2, wherein a refractive index of the first optically clear layer is about 1 .5.

[0041] 4. The flow cell of any of clauses 1-3, wherein the front prism has a refractive index equivalent to a refractive index of the first optically clear layer.

[0042] 5. The flow cell of any of clauses 1-4, wherein the second optically clear layer includes crown glass, borosilicate glass, quartz, or fused silica.

[0043] 6. The flow cell of any of clauses 1-5, wherein a refractive index of the second optically clear layer is about 1.5.

[0044] 7. The flow cell of any of clauses 1-6, wherein the at least one light collection lens is adjacent to the outer surface of the second optically clear layer.

[0045] 8. The flow cell of any of clauses 1-7, wherein the at least one light collection lens is configured to collect light scattered from the sample at a 90-degree angle from a light source.

[0046] 9. The flow cell of any of clauses 1-8, wherein the at least one light collection lens is configured to collect light scattered from the sample at a Brewsters angle from a light source.

[0047] 10. The flow cell of any of clauses 1-9, further including a back prism disposed on a back side of the second optically clear layer.

[0048] 11 . The flow cell of clause 10, wherein the back prism has a refractive index equivalent to a refractive index of the second optically clear layer.

[0049] 12. The flow cell of clause 10 or 11, wherein the at least one light collection lens is disposed on the back prism.

[0050] 13. The flow cell of any of clauses 1-12, wherein the at least one light collection lens includes a forward scatter collection lens.

[0051] 14. The flow cell of clause 13, wherein the forward scatter collection lens is adjacent to the outer surface of the second optically clear layer.

[0052] 15. The flow cell of clause 13 or 14, wherein the forward scatter collection lens is configured to collect light scattered from the sample at a 0-degree angle to a light source.

[0053] 16. The flow cell of any of clauses 13-15, further including a blocker bar disposed between the second optically clear layer and the forward scatter collection lens.

[0054] 17. A system including, a flow cell including: a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer and including a void; a sealed chamber formed by the combined edges of the void, the first optically clear layer, and the second optically clear layer; at least one hole formed in the first optically clear layer or the second optically clear layer that is fluidly connected to the sealed chamber to form a sample inlet to, or a sample outlet from, the sealed chamber; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite the outer surface of the second optically clear layer; a laser configured to enter the front prism at an entry angle relative to a plane of a face of the front prism; and at least one light collection lens adjacent to the outer surface of the second optically clear layer and configured to collect light scattered by the interaction of the laser and a sample.

[0055] 18. The system of clause 17, wherein the at least one light collection lens is configured to collect light at a 90-degree angle from a light source.

[0056] 19. The system of clause 17 or 18, wherein the at least one light collection lens is configured to collect light at a Brewsters angle from a light source.

[0057] 20. The system of any of clauses 17-19, wherein the front prism has a refractive index equivalent to a refractive index of the first optically clear layer.

[0058] 21. The system of any of clauses 17-20, wherein the first optically clear layer includes a refractive index of about 1.5.

[0059] 22. The system of any of clauses 17-21 , wherein the second optically clear includes a refractive index of about 1.5.

[0060] 23. The system of any of clauses 17-22, further including a back prism disposed on the outer surface of the second optically clear layer.

[0061] 24. The system of clause 23, wherein the back prism has a refractive index equivalent to a refractive index of the second optically clear layer.

[0062] 25. The system of clause 23 or 24, wherein the at least one light collection lens is disposed on the back prism.

[0063] 26. The system of any of clauses 17-25, wherein the at least one light collection lens includes a side scatter collection lens and a forward scatter collection lens.

[0064] 27. The system of clause 26, wherein the side scatter collection lens is positioned at 90-degrees to the forward scatter collection lens.

[0065] 28. The system of clause 26 or 27, wherein the side scatter collection lens and a forward scatter collection lens are adjacent to the outer surface of the second optically clear layer.

[0066] 29. The system of any of clauses 26-28, wherein the side scatter collection lens is configured to collect light scattered from the sample at a 90-degree angle from a light source.

[0067] 30. The system of any of clauses 26-29, wherein the forward scatter collection lens is configured to collect light scattered from the sample at a 0-degree angle to a light source.

[0068] 31 . The system of any of clauses 26-30, further including a blocker bar disposed between the outer surface of the second optically clear layer and the forward scatter collection lens.

[0069] 32. A method of collecting light scatter from a sample.CLOSING PARAGRAPHS

[0070] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms "include” or "including” should be interpreted to recite: "comprise, consist of, or consist essentially of.” The transition term "comprise” or "comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase "consisting of' excludes any element, step, ingredient or component not specified. The transition phrase "consisting essentially of' limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect theembodiment. A material effect would cause a statistically significant reduction in the amount and / or purity of the target cell collection.

[0071] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term "about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0072] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0073] The terms "a,” "an,” "the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0074] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0075] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0076] Furthermore, numerous references have been made to patents, printed publications, journal articles, other written text, and web site content throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching(s), as of the filing date of the first application in the priority chain in which the specific reference was included. For instance, with regard to chemical compounds, nucleic acid, and amino acids sequences referenced herein that are available in a public database, the information in the database entry is incorporated herein by reference as of the date of an application in the priority chain in which the database identifier for that compound or sequence was first included in the text.

[0077] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0078] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0079] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and / or A Dictionary of Chemistry, 8thEdition (Ed. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

LISTING OF CLAIMSWhat is claimed is:

1. A flow cell comprising: a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer, the opaque middle layer comprising a void; a sealed chamber formed by the combined edges of the void, the first optically clear layer, and the second optically clear layer; at least one hole formed in the first optically clear layer or the second optically clear layer that is fluidly connected to the sealed chamber to form a sample inlet to, or a sample outlet from, the sealed chamber; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite an outer surface of the second optically clear layer; and at least one light collection lens.

2. The flow cell of claim 1 , wherein the first optically clear layer comprises crown glass, borosilicate glass, quartz, or fused silica.

3. The flow cell of claim 1 , wherein a refractive index of the first optically clear layer is about 1.5.

4. The flow cell of claim 1 , wherein the front prism has a refractive index equivalent to a refractive index of the first optically clear layer.

5. The flow cell of claim 1 , wherein the second optically clear layer comprises crown glass, borosilicate glass, quartz, or fused silica.

6. The flow cell of claim 1 , wherein a refractive index of the second optically clear layer is about 1 .5.

7. The flow cell of claim 1 , wherein the at least one light collection lens is adjacent to the outer surface of the second optically clear layer.

8. The flow cell of claim 1 , wherein the at least one light collection lens is configured to collect light scattered from the sample at a 90-degree angle from a light source.

9. The flow cell of claim 1 , wherein the at least one light collection lens is configured to collect light scattered from the sample at a Brewsters angle from a light source.

10. The flow cell of claim 1, further comprising a back prism disposed on an outer side of the second optically clear layer.11 . The flow cell of claim 10, wherein the back prism has a refractive index equivalent to a refractive index of the second optically clear layer.

12. The flow cell of claim 10, wherein the at least one light collection lens is disposed on the back prism.

13. The flow cell of claim 1, wherein the at least one light collection lens comprises a forward scatter collection lens.

14. The flow cell of claim 13, wherein the forward scatter collection lens is adjacent to the outer surface of the second optically clear layer.

15. The flow cell of claim 13, wherein the forward scatter collection lens is configured to collect light scattered from the sample at a 0-degree angle to a light source.

16. The flow cell of claim 13, further comprising a blocker bar disposed between the second optically clear layer and the forward scatter collection lens.

17. A system comprising: a flow cell comprising: a first optically clear layer and a second optically clear layer; an opaque middle layer disposed between the first optically clear layer and the second optically clear layer and comprising a void; a sealed chamber formed by the combined edges of the void, the first optically clear layer, and the second optically clear layer; at least one hole formed in the first optically clear layer or the second optically clear layer that is fluidly connected to the sealed chamber to form a sample inlet to, or a sample outlet from, the sealed chamber; a front prism disposed on an outer surface of the first optically clear layer, wherein the outer surface of the first optically clear layer is opposite the outer surface of the second optically clear layer; a laser configured to enter the front prism at an entry angle relative to a plane of a face of the front prism; and at least one light collection lens adjacent to the outer surface of the second optically clear layer, wherein the at least one light collection lens is configured to collect light scattered by an interaction of the laser and a sample.

18. The system of claim 17, wherein the at least one light collection lens is configured to collect light at a 90- degree angle from a light source.

19. The system of claim 17, wherein the at least one light collection lens is configured to collect light at a Brewsters angle from a light source.

20. The system of claim 17, wherein the front prism has a refractive index equivalent to a refractive index of the first optically clear layer.21 . The system of claim 17, wherein the first optically clear layer comprises a refractive index of about 1 .5.

22. The system of claim 17, wherein the second optically clear comprises a refractive index of about 1 .5.

23. The system of claim 17, further comprising a back prism disposed on the outer surface of the second optically clear layer.

24. The system of claim 23, wherein the back prism has a refractive index equivalent to a refractive index of the second optically clear layer.

25. The system of claim 23, wherein the at least one light collection lens is disposed on the back prism.

26. The system of claim 17, wherein the at least one light collection lens comprises a side scatter collection lens and a forward scatter collection lens.

27. The system of claim 26, wherein the side scatter collection lens is positioned at 90-degrees to the forward scatter collection lens.

28. The system of claim 26, wherein the side scatter collection lens and a forward scatter collection lens are adjacent to the outer surface of the second optically clear layer.

29. The system of claim 26, wherein the side scatter collection lens is configured to collect light scattered from the sample at a 90-degree angle from a light source.

30. The system of claim 26, wherein the forward scatter collection lens is configured to collect light scattered from the sample at a 0-degree angle to a light source.31 . The system of claim 26, further comprising a blocker bar disposed between the outer surface of the second optically clear layer and the forward scatter collection lens.

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