Flow cytometer, laser optical assembly thereof, and method for assembling the laser optical assembly

Abstract

Flow cytometer, its laser optical assembly, and method of assembling the laser optical assembly. The disclosed flow cytometer can provide consistent and accurate results even when exposed to adverse environmental conditions, such as temperature variations over a relatively wide temperature range and / or relatively large random-axis mechanical vibrations. Additionally or alternatively, the disclosed flow cytometer is relatively unaffected by actual or apparent core stream shifts and utilizes a beam that slowly converges along an axis perpendicular to the core stream flow, allowing time-of-flight to be accurately measured.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 650,783, filed March 30, 2018, entitled “FLOW CYTOMETER, LASER OPTICS ASSEMBLY THEREOF, AND METHODS OF ASSEMBLING THE SAME.”

[0002] FIELD OF THE DISCLOSURE This disclosure relates to flow cytometry, and in particular to a flow cytometer, a laser optics assembly for a flow cytometer, and a method for assembling a laser optics assembly. [Background technology]

[0003] Flow cytometers typically require a laser beam to pass through a relatively narrow sample core stream so that particles flowing through the sample core stream are illuminated by the laser beam and absorb and scatter the laser light according to the particle's refractive index, size, shape, and other properties. For each particle, the absorbed and scattered light intensity is measured. The absorption and scattering measurements are used to identify and quantify particle types and particle properties. More recently, time-of-flight measurements have been utilized additionally or alternatively to determine particle types and / or properties.

[0004] As can be appreciated, to maintain accurate performance, a flow cytometer must perform consistently from test to test. One way to ensure consistency is to eliminate as many environmental factors as possible (e.g., temperature changes, mechanical vibrations, etc.) and / or continuously calibrate the flow cytometer to ensure that environmental factors and / or other variables do not affect performance. However, while this may be a practical solution in a precision measurement laboratory, it is not practical in many other environments, such as, for example, a doctor's office or in the field.

[0005] It would therefore be desirable to provide a flow cytometer and its laser optics assembly that can withstand adverse environmental conditions and is relatively unaffected by other variables, thereby providing consistent and accurate results without the need for repeated alignment and / or calibration. It would also be desirable to provide a method for assembling the laser optics assembly. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure provides a flow cytometer and its laser optics assembly that can provide consistent and accurate results even when exposed to adverse environmental conditions, such as, for example, temperature variations within a relatively wide temperature range and / or relatively large random axis mechanical vibrations. The flow cytometer of the present disclosure is further relatively immune to real or apparent core stream shifts, operates without the need for beam stoppers, and utilizes a beam that slowly converges along an axis perpendicular to the flow of the core stream, allowing time of flight to be accurately measured. Additionally, methods of assembly of the flow cytometer and laser optics assembly are provided. These and other aspects and features of the present disclosure are detailed below. Consistently, any of the aspects and features detailed within this specification may or may not be utilized with any of the other aspects and features detailed herein, regardless of whether they are described below together or separately. [Means for solving the problem]

[0007] According to an aspect of the present disclosure, provided is a laser optics assembly for a flow cytometer including a base plate defining a barrel, a collimation assembly at least partially disposed within the barrel, a first lens at least partially disposed within the barrel, a second lens at least partially disposed within the barrel, and a third lens at least partially disposed within the barrel, wherein the collimation assembly, the first lens, the second lens, and the third lens are fixed relative to the base plate to withstand 10 G random axis mechanical vibration for at least 30 seconds without resulting in movement of the collimation assembly, the first lens, the second lens, or the third lens relative to the base plate.

[0008] In one aspect of the present disclosure, the at least one cover plate secures the collimating assembly, the first lens, the second lens, and the third lens to the base plate. The at least one cover plate can be bolted to the base plate.

[0009] In another aspect of the disclosure, a separate cover plate secures each of the collimating assembly, the first lens, the second lens, and the third lens to the base plate. Each of the cover plates can be bolted to the base plate.

[0010] In yet another aspect of the present disclosure, a collimation assembly includes a laser diode and a collimating lens positioned in alignment with the laser diode.

[0011] In yet another aspect of the disclosure, the barrel of the base plate defines a first chamber configured to at least partially receive the collimation assembly, a second chamber configured to at least partially receive the first lens, a third chamber configured to at least partially receive the second lens, and a fourth chamber configured to at least partially receive the third lens.

[0012] In yet another aspect of the present disclosure, the first lens, the second lens, and the third lens are secured within respective first lens cradles, second lens cradles, and third lens cradles that are at least partially disposed within the second chamber, the third chamber, and the fourth chamber of the barrel of the base plate, respectively.

[0013] In another aspect of the disclosure, at least one of the first lens cradle, the second lens cradle, or the third lens cradle includes fingers extending therefrom configured to allow rotational adjustment of the lens cradle in the corresponding chamber upon assembly. At least one of the first lens cradle, the second lens cradle, or the third lens cradle may include fingers extending therefrom configured to allow axial adjustment of the lens cradle in the corresponding chamber upon assembly. Additionally or alternatively, at least one of the first lens cradle, the second lens cradle, or the third lens cradle may include fingers extending therefrom configured to allow both rotational and axial adjustment of the lens cradle in the corresponding chamber upon assembly.

[0014] In yet another embodiment of the present disclosure, the first lens is a convex cylindrical lens, the second lens is a concave cylindrical lens, and the third lens is a cylindrical objective lens. The first lens, the second lens, and the third lens, in such an embodiment, may be arranged in sequence along a barrel extending from the collimation assembly.

[0015] A flow cytometer provided according to an aspect of the present disclosure includes a lens subassembly including a plurality of lenses arranged along an axis, a flow cell arranged axially down from the lens subassembly, and a collimation subassembly positioned axially up from the lens subassembly. The collimation subassembly includes a laser diode configured to emit a beam, a collimating lens configured to collimate the beam, and at least two supports configured to maintain a predetermined axial distance between the laser diode and the collimating lens. The at least two supports are formed from materials having thermal expansion coefficients that balance each other such that the predetermined axial distance is maintained through a temperature change of up to 30°C.

[0016] In one embodiment of the present disclosure, the temperature change is from 10°C to 40°C.

[0017] In another embodiment of the present disclosure, the first support is formed from PEEK and the second support is formed from brass.

[0018] In yet another aspect of the present disclosure, the three supports configured to maintain a predetermined axial distance are formed from materials having thermal expansion coefficients that balance each other. In such an aspect, the first support can be formed from PEEK, the second support can be formed from brass, and the third support can be formed from aluminum.

[0019] In yet another aspect of the present disclosure, the flow cytometer further includes a mounting platform having a lens subassembly, a collimation subassembly, and a housing that supports the attached flow cell and maintains a predetermined axial distance between the flow cell and the lens subassembly. In such an aspect, the housing and the mounting platform are formed from materials having coefficients of thermal expansion that balance each other such that the predetermined axial distance between the flow cell and the lens subassembly is maintained through a temperature change of up to 30° C.

[0020] In yet another aspect of the present disclosure, the housing is formed from a copolyester and the mounting platform is formed from aluminum.

[0021] A method of assembling a laser optics assembly of a flow cytometer provided according to an aspect of the present disclosure includes securing a collimation assembly at least partially within a barrel of a base plate, the collimation assembly including a laser diode and a collimating lens configured to generate a laser beam along an axis, the laser beam having a first beam waist diameter in a first direction and a second beam waist diameter in a second direction. The method includes the steps of positioning a third lens at least partially within a barrel of a base plate on an axis, rotatably adjusting the third lens about the axis so that a first beam waist diameter is minimized, fixing the third lens relative to the base plate, positioning a first lens at least partially within the barrel of the base plate on an axis, rotatably adjusting the first lens about the axis so that the first beam waist diameter is maintained, fixing the first lens relative to the base plate, positioning a second lens at least partially within the barrel of the base plate on an axis, rotatably adjusting the second lens about the axis so that the first beam waist diameter is maintained, axially adjusting the second lens along the axis so that the second beam diameter is set at a desired value, and fixing the second lens relative to the base plate.

[0022] In one aspect of the disclosure, the third lens is positioned furthest from the collimation assembly, the first lens is positioned proximate to the collimation assembly, and the second lens is positioned between the first lens and the third lens.

[0023] In yet another aspect of the present disclosure, the third lens is a cylindrical objective lens, the first lens is a convex cylindrical lens, and the second lens is a concave cylindrical lens.

[0024] In yet another aspect of the present disclosure, a third lens is positioned within a third chamber of the barrel configured to axially constrain the third lens and allow rotation of the third lens prior to fixing the third lens, a first lens is positioned within a first chamber of the barrel configured to axially constrain the first lens and allow rotation of the first lens prior to fixing the first lens, and a second lens is positioned within a second chamber of the barrel configured to allow rotation and translation of the second lens prior to fixing the second lens.

[0025] In yet another aspect of the present invention, the first beam waist has a 1 / e 2 It has a diameter.

[0026] In yet another aspect of the invention, the second beam has a 1 / e 2 More specifically, the second beam has a 1 / e diameter of 200 μm. 2 The diameter of the first and second electrodes may be 1 to 5 mm.

[0027] A flow cytometer provided according to an embodiment of the present disclosure includes a flow cell defining a flow direction, a collimation assembly including a laser diode and a collimating lens configured to generate a laser beam along an axis, a convex cylindrical lens disposed on the axis and configured to receive the laser beam from the collimation assembly, a concave cylindrical lens disposed on the axis and configured to receive the laser beam from the convex cylindrical lens, and a laser beam incident on the flow cell having a first beam waist 1 / e of 6.7 μm to 9 μm in a direction parallel to the flow direction of the flow cell. 2 The second beam 1 / e of 190 μm to 210 μm in the direction perpendicular to the flow direction of the flow cell 2 and a cylindrical objective lens configured to project the laser beam onto the flow cell to define a diameter.

[0028] In one embodiment of the present disclosure, the first beam waist 1 / e 2 diameter and 1 / e of the second beam 2 The diameter is chosen so that actual radial core stream shifts within the flow cell of up to 15 μm do not degrade performance.

[0029] In another aspect of the present disclosure, the first beam waist 1 / e 2 diameter and 1 / e of the second beam 2 The diameter is chosen so that no degradation in performance occurs in the event of an apparent radial core stream shift due to a shift in the focus of the laser beam of up to 15 μm.

[0030] In another aspect of the present disclosure, the first beam waist 1 / e 2 The diameter is chosen so that time-of-flight measurements can distinguish particle or cell sizes down to 1 μm or less with a change in flow rate through the flow cell of 2% or less.

[0031] Various aspects and features of the disclosed flow cytometer and its laser optics assembly are described herein with reference to the drawing figures, in which like reference numbers indicate similar or identical elements. [Brief description of the drawings]

[0032] [Figure 1] 1 is a perspective view of the laser optics, flow cell, and sensor module of a flow cytometer provided in accordance with the present disclosure. [Diagram 2] FIG. 2 is a longitudinal cross-sectional view of the module of FIG. [Diagram 3-4] 2A-2C are front and rear perspective views of a laser optics assembly of the module of FIG. 1. [Diagram 5] FIG. 5 is a perspective, partial cross-sectional view of the laser optics assembly of FIGS. 3 and 4. [Figure 6-7] 5A and 5B are front and rear perspective views of a collimation subassembly of the laser optics assembly of FIGS. 3 and 4. [Figure 8]FIG. 8 is a longitudinal cross-sectional view of the collimation subassembly of FIGS. 6 and 7. [Figure 9] FIG. 8 is a perspective, partial cross-sectional view of the collimation subassembly of FIGS. 6 and 7. [Figure 10] FIG. 5 is a perspective view of a lens subassembly of the laser optics assembly of FIGS. 3 and 4. [Figure 11] 11 is a cross-sectional view of the laser optics assembly of FIGS. 3 and 4, showing the lens subassembly of FIG. 10. [Figure 12-14] 2 is a schematic side view of the module of FIG. 1 showing axial adjustment of a concave cylindrical lens of the laser optics assembly. [Figure 15-17] 2 is a schematic top view of the module of FIG. 1 showing axial adjustment of the concave cylindrical lens of the laser optics assembly. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] 1 and 2, the present disclosure provides a flow cytometer including a laser optics, flow cell, and sensor module, generally designated by reference numeral 10. Although not shown, the flow cytometer may further include, for example, an outer housing that encloses the internal operable components of the flow cytometer, an electronics module configured to control the module 10 and process test results received therefrom, a sample receiving module configured to receive a test sample, a pump module configured to pump sample and sheath fluid into the flow cell assembly 300, and an exhaust module configured to allow safe collection of the sample and sheath fluid after testing. Alternatively or additionally, any other suitable modules, components, and / or features are contemplated for use with the module 10 of the flow cytometer of the present disclosure.

[0034] 1 and 2, module 10 includes a mounting platform 100, a laser optics assembly 200 secured to mounting platform 100, a flow cell assembly 300 secured to mounting platform 100 and operatively positioned relative to laser optics assembly 200, and a sensor assembly 400 operatively disposed relative to laser optics assembly 200 and flow cell assembly 300 for forward and side scatter detection. Laser optics assembly 200, flow cell assembly 300, and sensor assembly 400 are each independently fastened onto mounting platform 100 using bolts 110 and / or any other suitable fastening structure to maintain the relative positions of these assemblies 200-400.

[0035] As described in more detail below, module 10 is configured to enable the flow cytometer to operate over a wide temperature range, e.g., 10° C.-40° C., and to withstand random axial vibrations of 10 G for 30 seconds without degradation in performance, where degradation in performance is defined herein as a loss of intensity and / or sensitivity of greater than 5%.

[0036] Additionally, as described in more detail below, module 10 allows the flow cytometer to be relatively insensitive to actual or apparent core stream shifts, such as, for example, a radial shift of up to 15 μm of the core stream relative to a previously aligned flow axis, and operates without the need for a beam stopper to block unscattered laser light from reaching the forward scatter sensor of sensor assembly 400, and in embodiments, directs the beam to 1 / e of the core stream. 2 It is configured to use a beam that slowly converges along an axis perpendicular to the flow of the core stream allowing it to be set to a width.

[0037] Additionally, and as described in more detail below, module 10 provides a flow cytometer capable of measuring time of flight to within 1 μm accuracy for particles ranging from 4 microns to 16 microns in size when the core stream flow rate is stable within 2%.

[0038] 2-5, the laser optics assembly 200 includes a clamp subassembly 210, a collimation subassembly 230, and a plurality of lens subassemblies 270, 280, 290. The clamp subassembly 210 includes a base plate 212 that defines at least a pair (e.g., two pairs) of legs 214 along both sides thereof that include an aperture 216 defined therethrough such that the laser optics assembly 200 can be securely mounted on the mounting platform 100 using bolts 110. The base plate 212 further defines a generally cylindrical barrel 218 extending along the base plate 212 between the legs 214. The barrel 218 defines a first chamber 219, a second chamber 221, a third chamber 223, and a fourth chamber 225 aligned along the length of the barrel 218. The chambers 219, 221, 223, and 225 are configured to receive the collimation subassembly 230 and the lens subassemblies 270, 280, 290, respectively. The clamp subassembly 210 further includes cover plates 220, 222, 224, 226 configured to be securely mounted onto the base plate 212 using bolts 228 to enclose and secure the collimation subassembly 230 and the lens subassemblies 270, 280, 290, respectively, within the chambers 219, 221, 223, 225. The assembly of the collimation subassembly 230 and the lens subassemblies 270, 280, 290 within the clamp subassembly 210 and their alignment are described in more detail below.

[0039] 6-9, the collimation subassembly 230 includes a support disk 232, a support hub 234, an insert 236, and a spring washer 237 configured to operably engage with each other and hold the collimating lens 238 of the collimation subassembly 230 in position relative to the laser diode 240 of the collimation subassembly 230.

[0040] The support disk 232, more specifically, defines an outer surface 242a and an inner surface 242b, and includes a central aperture 244 and a plurality of radial apertures 246 (FIG. 2) defined therethrough between the outer surface 242a and the inner surface 242b, respectively. The central aperture 244 defines an outer opening on the outer surface side of the support disk 232, which is larger than an inner opening of the central aperture 244 defined on the inner surface side of the support disk 232, such that the laser diode 240 can be inserted into the central aperture 244 through the outer opening, but cannot pass through the inner opening. Thus, the laser diode 240 can be inserted through the inner opening of the central aperture 244 and seated within the central aperture 244 to secure the laser diode 240 relative to the support disk 232. The laser diode 240 includes a suitable electrical connector 241 that allows connection to power electronics and control electronics (not shown). Laser diode 240 may be configured to emit red light having a wavelength in the range of 630 nm to 665 nm, or in an embodiment, in the range of 635 nm to 650 nm.

[0041] The support hub 234 defines a generally T-shaped shape including a disk portion 247 positioned to abut the inner surface 242b of the support disk 232 and a body portion 248 extending from the disk portion 247 in a direction away from the support disk 232. A central lumen 250 extends through both the disk portion 247 and the body portion 248, and a plurality of radial bores 252 (FIG. 2) are defined in the disk portion 247. Threads 254 are disposed on at least a portion of the inner surface of the support hub 234 that defines the lumen 250.

[0042] The insert 236 defines a generally cylindrical shape that defines an internal passageway 256. The insert 236 further includes threads 258 disposed on at least a portion of an outer surface, the threads 258 configured to engage with the threads 254 of the support hub 234. The insert 236 is more specifically configured to retain the collimating lens 238 within the passageway 256, for example using an adhesive, and is configured to be positioned within the central lumen 250 of the support hub 234 in a threadedly engaged state. A spring washer 237 is configured to be positioned within the central lumen 250 between the insert 236 and the support disk 232 to maintain tension between the insert 236 and the support disk 232.

[0043] 6-9, to assemble the collimation subassembly 230, the laser diode 240 is secured within the support disk 232 and the collimating lens 238 is secured within the insert 236. The insert 236 is then threaded into engagement within the central lumen 250 of the support hub 234. With the laser diode 240 secured within the support disk 232 and the insert 236 (with the collimating lens 238 secured therein) engaged within the support hub 234, the support disk 232 and the support hub 234 are positioned relative to one another such that the inner surface 242b of the support disk 232 abuts the support hub 234, the central aperture 244 of the support disk 232 is aligned with the central lumen 250 of the support hub 234, and the radial apertures 246 of the support disk 232 are aligned with corresponding radial bores 252 (see FIG. 2) of the support hub 234. As described in more detail below, fasteners (not shown) may be utilized to maintain the support disk 232 and support hub 234 in this position and to facilitate their alignment.

[0044] With the support disk 232 and support hub 234 positioned as detailed above, bolts 260 are inserted through radial apertures 246 and engaged (e.g., by threaded engagement) within radial bores 252 to secure the support disk 232 and support hub 234 relative to one another (see FIG. 2). Positional adjustments (e.g., vertical and / or horizontal adjustments) between the support disk 232 and support hub 234 may be made before or after engagement of each bolt 260, for example, via adjustment knobs (not shown) associated with the fixture, to align the laser diode 240 with the collimating lens 238 so that the beams emitted from the laser diode 240 are both sufficiently collimated and oriented coaxially with the optical axis of the collimating lens 238. An inverting beam expander (not shown) associated with the fixture may be utilized to verify this alignment.

[0045] To adjust the axial distance between the collimating lens 238 and the laser diode 240, the insert 236 is threaded in and out of the central lumen 250 of the support hub 234 to move the collimating lens 238 toward or away from the laser diode. To ensure that the predetermined axial distance between the collimating lens 238 and the laser diode 240 is achieved, an inverted beam expander (not shown) may again be utilized. When the insert 236 is threaded in the proper position corresponding to the predetermined axial distance between the collimating lens 238 and the laser diode 240, the spring washer 237 maintains tension between the insert 236 and the support disk 232, thereby eliminating play between the insert 236 and the support disk 232 and maintaining the predetermined axial distance between the collimating lens 238 and the laser diode 240, for example, even when the collimation subassembly 230 is subjected to mechanical vibrations.

[0046] Once the beam and optical axes of the collimating lens 238 and the laser diode 240 are coaxial with each other and the beam is collimated, the bolts 260 are appropriately tightened to lock the support disk 232 and the support hub 234 relative to each other, thereby maintaining engagement and positioning between the support disk 232 and the support hub 234, even if, for example, the collimation subassembly 230 is subjected to mechanical vibrations.

[0047] The locking of the support disk 232 and the support hub 234 relative to one another as detailed above secures the horizontal, vertical and axial alignment of the collimating lens 238 and the laser diode 240 relative to one another to ensure the alignment described above.

[0048] 6-9, the collimation subassembly 230 is configured to maintain a predetermined axial distance between the collimating lens 238 and the laser diode 240 regardless of temperature changes in the environment. More specifically, the collimation subassembly 230 is configured to adequately maintain a predetermined axial distance between the collimating lens 238 and the laser diode 240 at or below 30° C., for example within a range of 10° C.-40° C., without degrading performance. This is accomplished by forming the support hub 234 and insert 236, or in an embodiment, the support disk 232, support hub 234, and insert 236, from materials having different thermal expansion coefficients that maintain a predetermined axial distance between the laser diode 240 and the collimating lens 238 for a flow cytometer operating over a range of 10° C.-40° C., and thus without degrading performance. In an embodiment, this is achieved ... from materials having different thermal expansion coefficients that do not degrade performance. -5 The support hub 234 is made of PEEK (polyether ether ketone) (4.5×10 -5This is accomplished by forming the insert 236 from a quartz material having a linear thermal expansion coefficient of 0.025% or less, although other suitable materials having a linear thermal expansion coefficient that balances the response of the flow cytometer to temperature variations in the range of 10° C. to 40° C. are contemplated. This balancing not only includes compensating for the linear thermal expansion coefficients of some of the components of the flow cytometer, but also takes into account temperature dependent changes in the refractive index of the optical elements of the flow cytometer. As used herein, a "predetermined axial distance" is understood to encompass a range of distances to account for, for example, temperature dependent changes in a target axial distance between the collimating lens 238 and the laser diode 240. This range may include a variation from the predetermined axial distance between the collimating lens 238 and the laser diode 240 of 0.025% or less, or in an embodiment, 0.012% or less.

[0049] 2-5, to assemble the collimation subassembly 230 with the clamp subassembly 210, the body portion 248 of the support hub 234 of the collimation subassembly 230 is seated within the first chamber 219 of the barrel 218 of the base plate 212 of the clamp subassembly 210, and then the cover plate 220 is positioned around the body portion 248 of the support hub 234 and engaged with the base plate 212 on either side of the support hub 234 via bolts 228 to enclose the body portion 248 of the support hub 234 within the first chamber 219 and secure the collimation subassembly 230 in position relative to the base plate 212 under compression. In an embodiment, the collimation subassembly 230 is assembled with the clamp subassembly 210 prior to assembly of the lens subassemblies 270, 280, 290. Before tightening the bolts 218 , the collimation subassembly 230 is rotated if necessary to ensure that the fast axis of the laser beam is aligned perpendicular to the bottom surface of the base plate 212 .

[0050] 2-5, as well as FIG. 10 and FIG. 11, as described above, the laser optics assembly 200 includes three lens subassemblies 270, 280, 290. Each lens subassembly 270, 280, 290 includes a lens cradle 272, 282, 292, respectively, that defines a lens pocket 274, 284, 294, respectively, configured to securely hold a respective lens 276, 286, 296. The lens 276 is configured as a convex cylindrical lens and, as part of the lens subassembly 270, is configured to be positioned within the second chamber 221 of the barrel 218 of the base plate 212 and secured within the second chamber 221 via the second cover plate 222, such that the convex cylindrical lens 276 is positioned proximate to the collimating lens 238. The lens 286 is configured as a concave cylindrical lens and is configured as part of the lens subassembly 280 to be positioned within the third chamber 223 of the barrel 218 of the base plate 212 and secured within the third chamber 223 via the third cover plate 224 such that the concave cylindrical lens 286 is positioned adjacent to the convex cylindrical lens 276 on an opposite side to the convex cylindrical lens 276 relative to the collimating subassembly 230. The lens 296 is configured as a cylindrical objective lens and is configured as part of the lens subassembly 290 to be positioned within the fourth chamber 225 of the barrel 218 of the base plate 212 and secured within the fourth chamber 225 via the fourth cover plate 226 such that the cylindrical lens objective lens 296 is positioned adjacent to the concave cylindrical lens 286 on an opposite side to the convex cylindrical lens 276 relative to the convex cylindrical lens 276.

[0051] Each lens cradle 272, 282, 292 includes fingers 278, 288, 298 extending radially outward therefrom. The fingers 278, 288, 298 are configured to extend through slots (not explicitly shown) defined in the base plate 212 adjacent the chambers 221, 223, 225, respectively, so as to extend from the base plate 212 at a bottom surface thereof.

[0052] To assemble the lens subassemblies 270, 280, 290 within the clamp subassembly 210, the lenses 276, 286, 296 are engaged within the pockets 274, 284, 294 of the lens cradles 272, 282, 292, respectively, which are positioned within the chambers 221, 223, 225, respectively. The cradles 272, 292 each define a thickness that generally approximates a width of the chambers 221, 225 and / or include complementary features to maintain the cradles 272, 292, and thus the lenses 276, 296, respectively, at a constant axial position within the respective chambers 221, 225 during positioning. However, the fingers 278, 298 of the lens cradles 272, 292 may be manipulated to rotate the lens cradles 272, 292, and thus the lenses 276, 296, respectively, relative to the base plate 212. Meanwhile, the lens cradle 282 defines a thickness that is small relative to the width of the chamber 223 such that the cradle 282, and thus the lens 286, may translate axially along the barrel 218 upon corresponding manipulation of the fingers 288 of the lens cradle 282. The fingers 288 may further be manipulated to rotate the lens cradle 282, and thus the lens 286, relative to the base plate 212. The configuration detailed above that allows for the rotational alignment of the lenses 276, 286, 296 and the axial positioning of the lens 286 is advantageous as these have been found to be critical alignments to ensure accurate performance of a flow cytometer.

[0053] During assembly, once the collimation subassembly 230 is installed, the lens subassembly 290 is then inserted into the chamber 225, rotatably adjusted using fingers 298, and secured using cover plate 226 and bolts 228 to secure the lens subassembly 290 in a fixed position relative to the base plate 212 under compression. The base plate 212 is configured such that the lens subassembly 290 is located at a distance from the collimating lens 238 approximately equal to the sum of the focal length of the lens 296 and the focal length of the collimating lens 238. As detailed above, once the lens subassembly 290 is installed, it has a beam waist 1 / e of 6.7 μm to 9 μm in a direction parallel to the flow direction of the core stream through the flow cell 340 (see FIG. 2 ). 2 A verification is performed to ensure that the diameter has been achieved.

[0054] After assembly and verification of lens subassembly 290, lens subassembly 270 is inserted into chamber 221, rotatably adjusted using fingers 278, and secured using cover plate 222 and bolts 228 to secure lens subassembly 270 in a fixed position relative to base plate 212 under compression. Convex cylindrical lens 276 of lens subassembly 270 is rotationally aligned such that its optical power axis is perpendicular to the optical power axis of cylindrical objective lens 296, which defines a beam waist 1 / e of 6.7 μm to 9 μm parallel to the flow direction of the core stream. 2 This is verified by checking again that the diameter is maintained.

[0055] Lens subassembly 280 is then inserted into chamber 223, rotatably and / or axially adjusted using fingers 288, and secured using cover plate 224 and bolts 228 to secure lens subassembly 280 in position relative to base plate 212 under compression. The concave cylindrical lens 286 of lens subassembly 280 is rotationally aligned such that its refractive axis is perpendicular to the refractive axis of cylindrical objective lens 296 and parallel to the refractive axis of convex cylindrical lens 276, which results in a beam waist 1 / e of 6.7 μm to 9 μm parallel to the flow direction of the core stream. 2 The axial spacing of the concave cylindrical lenses 286 is in the direction perpendicular to the flow direction of the core stream through the flow cell 340 (see FIG. 2) and is in the range of 190 μm to 210 μm in an embodiment, or 200 μm in an embodiment, for a beam 1 / e 2 is adjusted to achieve the desired width.

[0056] Just as any suitable test equipment may be utilized to measure beam width during the verification described above, suitable fixtures (not shown) may be utilized to hold the various components to allow adjustments during assembly and to facilitate manipulation of the fingers 278, 288, 298. As detailed above, when fully assembled and verified, the laser optics assembly 200 has a beam waist 1 / e of 6.7 μm to 9 μm in the parallel direction. 2 diameter, and 1 / e of 190μm to 210μm (or 200μm) in the vertical direction 2 Generates beam width.

[0057] 12-17, FIGS. 12-14 show the divergence ranges of different laser diodes for the fast (large divergence) axis of laser diode 240, which is oriented parallel to the core stream in flow cell 340 and perpendicular to the bottom surface of base plate 212 as previously described. FIGS. 15-17 show the divergence ranges of different laser diodes for the slow (small divergence) axis of laser diode 240, which is oriented perpendicular to the core stream in flow cell 340 and parallel to the bottom surface of base plate 212. In one embodiment, laser diode 240 (e.g., Ushio HL6363MG-A laser diode) has a larger fast axis divergence than the slow axis divergence, but these two divergences are otherwise independent of each other.

[0058] The fast axis divergence of the laser diode 240 (parallel to the core stream in the flow cell 340) affects the beam waist of the flow cell core stream in a well aligned system. The most divergent beam from the laser diode (shown in FIG. 12) produces a minimum beam waist of 6.7 μm in the core stream, and the laser diode with the least divergent beam (shown in FIG. 14) produces a maximum beam waist of 9.0 μm in the core stream. Furthermore, in a well aligned system, the beam waist parallel to the core stream is maintained regardless of the axial placement of the concave cylindrical lens 286 within its adjustment range.

[0059] However, small changes in the axial position of the concave cylindrical lens 286 can result in a 1 / e shift in the laser diode beam (perpendicular to the core stream flow) within the flow cell 340. 2 This allows the width to be adjusted from 190 μm to 210 μm, and in an embodiment, 200 μm. These axial position variations of several hundred microns are not seen in Figures 15-17, but in all three figures, the axial position of the laser beam 1 / e at the core stream centerline is perpendicular to the core stream centerline. 2The width is 200 μm. FIG. 15 shows the maximum divergence slow axis laser beam and FIG. 17 shows the minimum divergence slow axis laser beam. In FIG. 15, the concave cylindrical lens 286 is located furthest from the laser diode 240, while in FIG. 17, the concave cylindrical lens 286 is located closest to the laser diode 240. Note also that the slow axis axial focus location changes with different slow axis divergence and placement of the concave cylindrical lens 286. The slow axis focus is closest to the laser diode 240 in FIG. 15 and furthest from the laser diode 240 in FIG. 17.

[0060] As described in more detail below, the beam 1 / e is directed perpendicular to the flow of the core stream through the flow cell 340 (FIG. 2). 2 The laser optics assembly 200 having a diameter advantageously is not affected by radial core stream shifts (actual or apparent) within a radius of 15 μm, and therefore radial core stream shifts (actual or apparent) within a radius of 15 μm do not result in degradation in performance.

[0061] 1-5, the above detailed assembly of the laser optics assembly 200 not only facilitates assembly and alignment, but also provides a configuration in which the collimation subassembly 230 and the lens subassemblies 270-290 are individually and independently secured to the base plate 212. This configuration of the laser optics assembly 200 reduces the beam waist 1 / e of the laser optics assembly 200. 2It has been shown that the laser optics assembly 200 can withstand 10G random axial vibration for 30 seconds without a change in diameter of more than 5%. More specifically, vibration testing was performed using a Qualmark OVTT® 18 Omni-Axial Vibration Table Top System available from ESPEC North America, Inc. (Denver, Colorado, USA). The vibration testing was performed by mounting the laser optics assembly 200 on a vibration table and setting the table to a random axial mechanical vibration of 10G for at least 30 seconds. Acceleration was verified using an Omega® HHVB82 Accelerometer available from Omega Engineering, Inc. (Norwalk, Connecticut, USA).

[0062] 1 and 2, as described above, the flow cell assembly 300 is mounted on the mounting platform 100. More specifically, the flow cell assembly 300 includes an input 310 coupled to a nozzle 320 defined by a housing 330 for delivering sample and sheath fluids to the nozzle 320, a flow cell 340 connected downstream of the nozzle 320 for receiving sample and sheath (not shown) fluids from the nozzle 320, and an output 350 disposed downstream of the flow cell 340 for directing the sample and sheath fluids to an appropriate collection reservoir after testing. The housing 330 of the flow cell assembly 300 is seated within an aperture 120 defined through the mounting platform 100 and secured to the mounting platform 100 using a number of bolts 110 to maintain a predetermined distance between the flow cell 340 and the cylindrical objective lens 296, which is an important distance to control to ensure the accuracy of the flow cytometer.

[0063] The housing 330 and mounting platform 100 of the flow cell assembly 300 are configured to adequately maintain a predetermined distance between the flow cell 340 and the cylindrical objective lens 296 within a 30° C. range (e.g., 10° C.-40° C.) without degrading performance. This is accomplished by forming the housing 330 and mounting platform 100 of the flow cell assembly 300 from materials having different linear thermal expansion coefficients that are configured to maintain a predetermined axial distance between the objective lens 296 and the flow cell 340 over a range of 10° C.-40° C. In an embodiment, it is the housing 330 that is in direct contact with the sample (e.g., blood) and sheath fluid, and therefore needs to be suitable for such purpose as well, and is made of Eastman Tritan® Copolyester MX811 (8.0×10 .0 mm) available from Eastman Chemical Company, Inc. (Kingsport, Tennessee, USA). -5 10), and by forming the mounting platform 100 from aluminum (2.38×10 -5 This is accomplished by forming the objective lens 296 and the flow cell 340 from a material having a linear thermal expansion coefficient of 0.01%, although other suitable material combinations that balance the response of the flow cytometer to temperature variations between 10° C. and 40° C. are contemplated. This balancing not only includes compensating for the linear thermal expansion coefficients of some of the components of the flow cytometer, but also takes into account temperature dependent changes in the refractive index of the optical elements of the flow cytometer 10. As noted above, the "predetermined axial distance" is understood to encompass a range of distances to account for temperature dependent changes in a target axial distance between the objective lens 296 and the flow cell 340, for example. This range may include a variation of 0.01% or less, or in embodiments, 0.005% or less, from the predetermined axial distance between the objective lens 296 and the flow cell 340.

[0064] With the flow cell assembly 300 mounted on the mounting platform 100, the face of the flow cell 340 is not oriented parallel to the plane of the cylindrical objective lens 296, but is offset by an angle of 5° to ensure that specular reflections from the cylindrical objective lens 296 do not couple back into the laser optics. The flow cell 340 is further coated with an anti-reflective coating for the same purpose.

[0065] 1 and 2, the sensor assembly 400 includes a forward scatter subassembly 410 and a side scatter subassembly 420. The forward scatter subassembly 410 includes a board 412 and a sensor array 414 including a light extinction sensor, a forward scatter low angle sensor, and a forward scatter high angle sensor. The side scatter subassembly 420 includes a lens mount 422 (FIG. 2), a lens 424 (FIG. 2) supported within the lens mount 422 (FIG. 2), and a side scatter sensor (not shown). The central capture angle of the side scatter subassembly 420 is 78° from the laser beam direction, rather than perpendicular (i.e., 90°), to increase the side scatter signal.

[0066] The insensitivity of the module 10 to radial core stream shifts (real or apparent) within a 15 μm radius as mentioned above will be described in more detail below, generally with reference to FIGS. 1-2. As is conventional, a Cartesian coordinate system is defined in which the core stream flows in the positive y-axis direction and the laser beam bundle is oriented in the positive z-axis direction. The beam waist 1 / e 2 The diameter is then increased along the y axis by 1 / e 2 By controlling the radius along the x-axis, it is possible to establish an elliptical region within which the maximum intensity of the laser beam does not decrease by more than a prescribed amount. As described in more detail below, module 10 is configured to remain immune to radial core stream shifts (real or apparent) within a 15 μm radius, and therefore maintains performance (less than 5% reduction in intensity) in the event of such radial core stream shifts.

[0067] The laser beam from the laser optics assembly 200 is aligned with the core stream flowing through the flow cell 340 of the flow cell assembly 300 while scattered laser light from particles flowing within the core stream is monitored and converted to an electrical signal. The relative positions of the laser optics assembly 200 and the flow cell 340 are adjusted in the x and z directions to maximize these signals. Before this x and z axis alignment is completed, the sensor of the sensor assembly 400 is aligned horizontally with respect to the laser beam and no further y direction alignment is required.

[0068] The disclosed flow cytometer bases its measurements on either the maximum scatter signal or the maximum area in the scatter signal profile, and therefore the y-direction beam waist need not be considered, except for its effect on the intensity of the laser beam as a function of distance along the z-axis. In the z-direction, relative intensity is defined as Equation (1):

number

number

[0069] Relatively large (e.g., 190 μm to 210 μm) x-direction beam 1 / e 2The diameter has minimal effect on the laser beam intensity as a function of distance along the z-axis. However, if the core stream shifts from its aligned position along the x-axis, the x-direction beam diameter affects how much beam intensity can be scattered. In the x-direction, the relative intensity is defined by equation (3):

number

[0070] Given beam waist 1 / e 2 Diameter ω 0,y and beam 1 / e 2 Diameter ω x For , the product of equation (1) and equation (3) is solved for the combination of x and z positions that exhibits the lowest limit of beam intensity reduction of a considered value, such as 5%. For example, equation (4):

number

[0071] Since the beam diameter is defined along the same orthogonal axes as the coordinate axes, equation (5) holds as follows:

number

[0072] In accordance with the above, the beam diameter is selected to ensure that for radial core stream shifts of up to 15 μm relative to the y-axis, the intensity at that shifted center remains within 5% of the original center to which the system was aligned. These core stream shifts can be real shifts (where the core stream moves radially from its original center) or apparent shifts (where the focus of the laser optics changes due to the shift of one or more components).

[0073] Considering the above, and also the beam 1 / e 2 Considering that the diameter x-direction spread can be limited to reduce reflections from the inner edge of the flow cell 340, the beam 1 / e in the x-direction is 190 μm to 210 μm (or 200 μm). 2 The diameter is selected taking into account the above and the beam waist 1 / e 2 A beam waist 1 / e in the y direction of 6.7 μm to 9 μm, taking into account that the radial y-spread can be limited to enhance the ability to measure the associated time-of-flight (TOF), as detailed below. 2 The diameter is selected.

[0074] Another important consideration for both beam diameter components (x-axis and y-axis) is that a wider beam diameter spreads the laser output over a larger area. In fact, the beam intensity along a given axis is inversely proportional to that beam diameter along that same axis. Module 10 has a beam 1 / e in the x-direction of 190 μm to 210 μm (or 200 μm). 2 diameter and y-direction beam waist 1 / e of 6.7μm to 9μm 2 The formation of a diameter balances the constraints detailed above, thus providing a large area over which the core stream may actually or apparently shift, while mitigating the contribution of stray light scattering from the side walls of the flow cell 340. In addition, these balanced constraints allow for accurate TOF measurements and minimize the laser power requirements of the module 10, as described in more detail below.

[0075] For TOF measurements, as the cells or particles flow through the flow cell 340, the cells or particles are first subjected to increasing laser intensity until it coincides with the maximum laser intensity, after which the particles are subjected to decreasing laser intensity. Thus, as a general approximation, the scattering intensity from a given particle or cell is proportional to the overlap volume between the incremental laser beam intensity and the incremental volume of the particle cross section. Thus, by considering how spherical particles of a range of sizes overlap with the laser beam, the relative scaling width of overlap for different particles can be compared. And, as long as the cell flow rate remains consistent, the TOF will scale accordingly.

[0076] Based on the above, for a given particle or cell, the change in full width at half maximum (FWHM), estimated as the maximum proportional change in the width of the scattering intensity curve, can be used to determine what y-axis beam width will consistently classify the particle or cell's diameter to within ±1 μm of its actual diameter, to approximately a 95% confidence level. However, flow rate variability must be taken into account, as it limits the ability to accurately determine the particle or cell's diameter.

[0077] As realized by the flow cytometer of the present disclosure, the beam waist 1 / e in the y direction of 6.7 μm to 9 μm 2 Using the diameter to control the flow rate variability to within about 2% of the mean allows TOF discrimination between particles or cells (4 μm to 16 μm in diameter) that differ by at least ±1 μm. Furthermore, periodic flow rate variability can be compensated for, for example, by using a pressure sensor to detect the flow rate variability and, based thereon, correcting for changes in the pulsatile flow (from a pump module that pumps sample and sheath fluids through the flow cell 340).

[0078] Any reference to a particular numerical value within this specification should be understood to encompass a range of values ​​to account for material and manufacturing tolerances generally accepted in the art, and / or errors in measuring equipment generally accepted in the art.

[0079] From the above description and with reference to the various drawings, those skilled in the art will understand that certain modifications can be made to the present disclosure without departing from the scope of the present disclosure. Although several embodiments of the present disclosure are shown in the drawings, the present disclosure is not intended to be limited to those embodiments, as the scope of the present disclosure is as broad as the art permits and the specification is intended to be read in the same manner. Therefore, the above description should not be construed as limiting, but merely as exemplifications of certain embodiments. Those skilled in the art will envision other modifications that fall within the scope and spirit of the claims appended hereto.

Claims

1. 1. A flow cytometer comprising a flow cell, a collimation assembly, a convex cylindrical lens, a concave cylindrical lens, and a cylindrical objective lens, the flow cell defines a flow direction; the collimation assembly comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; the convex cylindrical lens is disposed on the axis downstream from the collimation assembly; the concave cylindrical lens is disposed on the axis downstream from the convex cylindrical lens, The cylindrical objective lens is disposed on the axis downstream from the concave cylindrical lens, and the cylindrical objective lens is configured such that the laser beam incident on the flow cell has a first beam waist 1 / e in a direction parallel to the flow direction of the flow cell. ２ a second beam 1 / e defining a diameter and perpendicular to the flow direction of the flow cell; ２ The second beam 1 / e diameter is up to 210 μm. ２ configured to project the laser beam onto the flow cell to define a diameter The collimation assembly, the convex cylindrical lens, the concave cylindrical lens, and the cylindrical objective lens are cooperatively configured to avoid performance degradation in the event of at least one of an actual radial core stream shift in the flow cell of up to 15 μm or more, or an apparent radial core stream shift due to a shift in the focus of the laser beam of up to 15 μm or more. Flow cytometer.

2. The first beam waist 1 / e ２ The diameter is 6.7 μm to 9 μm.

2. The flow cytometer of claim 1.

3. A flow cytometer comprising a flow cell, a collimation assembly, a convex cylindrical lens, a concave cylindrical lens, and a cylindrical objective lens, the flow cell defines a flow direction; the collimation assembly comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; the convex cylindrical lens is disposed on the axis downstream from the collimation assembly; the concave cylindrical lens is disposed on the axis downstream from the convex cylindrical lens, the cylindrical objective lens is disposed on the axis downstream from the concave cylindrical lens, and the cylindrical objective lens is configured to project the laser beam onto the flow cell such that the laser beam incident on the flow cell defines a first beam waist 1 / e2 diameter in a direction parallel to the flow direction of the flow cell and a second beam 1 / e2 diameter in a direction perpendicular to the flow direction of the flow cell, the second beam 1 / e2 diameter being up to 210 μm; The collimation assembly, the convex cylindrical lens, the concave cylindrical lens, and the cylindrical objective lens are cooperatively configured such that time-of-flight measurements can distinguish particle or cell sizes down to 1 μm or less if the change in flow rate through the flow cell is 2% or less. Flow cytometer.

4. A flow cytometer comprising a flow cell, a collimation assembly, a convex cylindrical lens, a concave cylindrical lens, and a cylindrical objective lens, the flow cell defines a flow direction; the collimation assembly comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; the convex cylindrical lens is disposed on the axis downstream from the collimation assembly; the concave cylindrical lens is disposed on the axis downstream from the convex cylindrical lens, the cylindrical objective lens is disposed on the axis downstream from the concave cylindrical lens, and the cylindrical objective lens is configured to project the laser beam onto the flow cell such that the laser beam incident on the flow cell defines a first beam waist 1 / e2 diameter in a direction parallel to the flow direction of the flow cell and a second beam 1 / e2 diameter in a direction perpendicular to the flow direction of the flow cell, the second beam 1 / e2 diameter being up to 210 μm; The collimation assembly further comprises at least two supports configured to maintain a predetermined axial distance between the laser diode and the collimating lens, the at least two supports being formed from a material having a linear thermal expansion coefficient that maintains the predetermined axial distance between the laser diode and the collimating lens through a temperature change of up to 30°C. Flow cytometer.

5. The predetermined axial distance is maintained through a temperature change of 10° C. to 40° C.

5. The flow cytometer of claim 4.

6. The at least two supports include a first support and a second support, the first support being formed from a first material and the second support being formed from a second material different from the first material.

5. The flow cytometer of claim 4.

7. The at least two supports include three supports configured to maintain the predetermined axial distance and formed from materials having mutually balancing coefficients of thermal expansion.

5. The flow cytometer of claim 4.

8. The three supports include a first support, a second support, and a third support, the first support being formed from a first material, the second support being formed from a second material, and the third support being formed from a third material, and the first, second, and third materials being different from one another.

8. The flow cytometer of claim 7.

9. The flow cytometer further comprises a mounting platform having the convex cylindrical lens, the concave cylindrical lens, the cylindrical objective lens, the collimation assembly, and a housing configured to support the mounted flow cell and maintain a predetermined axial distance between the flow cell and the lens subassembly; The housing and the mounting platform are formed from a material having a coefficient of thermal expansion that maintains the predetermined axial distance between the flow cell and the lens subassembly through temperature changes of up to 30°C.

5. The flow cytometer of claim 4.

10. 1. A flow cytometer comprising a flow cell, a collimation assembly, and a plurality of lenses, the flow cell defines a flow direction; the collimation assembly comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; The plurality of lenses are arranged on the axis, and the laser beam incident on the flow cell has a first beam waist 1 / e in a direction parallel to the flow direction of the flow cell. ２ a first beam waist 1 / e diameter of 6.7 μm to 9 μm; ２ a second beam 1 / e defining a diameter and perpendicular to the flow direction of the flow cell; ２ The second beam 1 / e diameter is up to 210 μm ２ cooperating to project the laser beam onto the flow cell to define a diameter. The collimation assembly and the lenses are cooperatively configured to avoid performance degradation in the event of actual radial core stream shifts in the flow cell of up to 15 μm or more. A flow cytometer in which the collimation assembly and the multiple lenses are cooperatively configured to prevent performance degradation even when an apparent radial core stream shift occurs due to a shift in the focus of the laser beam of up to 15 μm or more.

11. A flow cytometer comprising a flow cell, a collimation assembly, and a plurality of lenses, the flow cell defines a flow direction; the collimation assembly comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; the plurality of lenses are disposed on the axis and cooperate to project the laser beam onto the flow cell such that the laser beam incident on the flow cell defines a first beam waist 1 / e diameter in a direction parallel to the flow direction of the flow cell, the first beam waist 1 / e diameter being between 6.7 μm and 9 μm, and a second beam 1 / e diameter in a direction perpendicular to the flow direction of the flow cell, the second beam 1 / e diameter being up to 210 μm; The collimation assembly and the plurality of lenses are cooperatively configured such that time-of-flight measurements can distinguish particle or cell sizes down to 1 μm or less with a change in flow rate through the flow cell of 2% or less. Flow cytometer.

12. The plurality of lenses includes a convex cylindrical lens, a concave cylindrical lens, and a cylindrical objective lens.

11. The flow cytometer of claim 10.

13. A flow cytometer comprising a flow cell, a collimation assembly, and a plurality of lenses, the flow cell defines a flow direction; the collimation assembly comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; the plurality of lenses are disposed on the axis and cooperate to project the laser beam onto the flow cell such that the laser beam incident on the flow cell defines a first beam waist 1 / e diameter in a direction parallel to the flow direction of the flow cell, the first beam waist 1 / e diameter being between 6.7 μm and 9 μm, and a second beam 1 / e diameter in a direction perpendicular to the flow direction of the flow cell, the second beam 1 / e diameter being up to 210 μm; The collimation assembly further comprises at least two supports configured to maintain a predetermined axial distance between the laser diode and the collimating lens, the at least two supports being formed from a material having a linear thermal expansion coefficient that maintains the predetermined axial distance between the laser diode and the collimating lens through a temperature change of up to 30°C. Flow cytometer.

14. The predetermined axial distance is maintained through a temperature change of 10° C. to 40° C.

14. The flow cytometer of claim 13.

15. The at least two supports include a first support and a second support, the first support being formed from a first material and the second support being formed from a second material different from the first material.

14. The flow cytometer of claim 13.

16. The at least two supports include three supports configured to maintain the predetermined axial distance and formed from materials having mutually balancing coefficients of thermal expansion.

14. The flow cytometer of claim 13.

17. The three supports include a first support, a second support, and a third support, the first support being formed from a first material, the second support being formed from a second material, and the third support being formed from a third material, and the first, second, and third materials being different from one another.

17. A flow cytometer as claimed in claim 16.

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