Laser optical assembly for a flow cytometer

Abstract

A flow cytometer, a laser optical assembly for a flow cytometer, and a method of assembling a laser optical assembly for a flow cytometer are provided. The flow cytometer is able to produce consistent and accurate results despite exposure to adverse environmental conditions, such as, for example, temperature variations over a relatively wide temperature range and / or relatively large amounts of random axis mechanical vibrations. Additionally or alternatively, the flow cytometer of the present disclosure is relatively insensitive to actual or apparent shifts in the core stream, employs a beam that converges slowly along an axis perpendicular to the flow of the core stream, and provides the ability to accurately measure transit times.

Description

[0001] This application is a divisional application of IDEXX Laboratories, Inc., with application number 201980023244.2, entitled "Laser Optical Components for Flow Cytometers", filed on September 28, 2020, and entered the Chinese national phase (international application date: March 28, 2019, international application number: PCT / US2019 / 024568).

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 650,783, filed March 30, 2018, entitled “Flow Cytometer, Laser Optical Assembly of Flow Cytometer, and Method of Assembling Laser Optical Assembly of Flow Cytometer,” the entire contents of which are incorporated herein by reference. Technical Field

[0004] This disclosure relates to flow cytometry, and more specifically, to a flow cytometer, a laser optics assembly for a flow cytometer, and a method for assembling the laser optics assembly of a flow cytometer. Background Technology

[0005] Flow cytometry typically requires 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. The particles absorb and scatter the laser light based on their refractive index, size, shape, and other properties. For each particle, the intensity of absorbed and scattered light is measured. Absorption and scattering measurements are used to identify and quantify particle type and characteristics. Recently, transit time measurements have been used, either additionally or alternatively, to determine particle type and / or characteristics.

[0006] Understandably, to maintain accurate performance, flow cytometry must be performed consistently from one test to another. One way to ensure consistency is to eliminate as many environmental factors as possible, such as temperature variations, mechanical vibrations, etc., and / or to continuously calibrate the flow cytometer to ensure that environmental factors and / or other variables do not affect performance. However, while this may be a feasible solution in a precision laboratory, it is not feasible in many other situations, such as in a practitioner's office or in the field.

[0007] Therefore, it is desirable to provide a flow cytometer capable of withstanding adverse environmental conditions and relatively insensitive to other variables, as well as a flow cytometer laser optics assembly, so that the flow cytometer and laser optics assembly produce consistent and accurate results without the need for repeated alignment and / or calibration. A method for assembling the laser optics assembly of the flow cytometer is also desirable. Summary of the Invention

[0008] This disclosure provides a flow cytometer and its laser optics that produce consistent and accurate results despite exposure to adverse environmental conditions, such as temperature variations over a relatively wide temperature range and / or relatively large amounts of random axial mechanical vibration. The flow cytometer of this disclosure is also relatively insensitive to actual or apparent deviations of the core flow, can operate without the need for beam stops, employs a slowly converging beam along an axis perpendicular to the core flow, and provides the ability to accurately measure transit time. Methods for assembling the flow cytometer and laser optics are also provided. These and other aspects and features of this disclosure are described in detail below. To the extent consistent, any aspect and feature detailed herein may be used with or without any other aspect and feature detailed herein, whether described together or separately below.

[0009] According to one aspect of this disclosure, a laser optical assembly for a flow cytometer is provided. The laser optical assembly includes a substrate defining a tube, a collimating assembly at least partially disposed within the tube, a first lens at least partially disposed within the tube, a second lens at least partially disposed within the tube, and a third lens at least partially disposed within the tube. The collimating assembly, the first lens, the second lens, and the third lens are fixed relative to the substrate to withstand at least 30 seconds of random axial mechanical vibration of 10G.

[0010] In one aspect of this disclosure, at least one cover plate secures the collimation assembly, the first lens, the second lens, and the third lens relative to the substrate. At least one cover plate may be bolted to the substrate.

[0011] In another aspect of this disclosure, each of the collimating assembly, the first lens, the second lens, and the third lens is secured relative to the substrate by a separate cover plate. Each cover plate may be bolted to the substrate.

[0012] In another aspect of this disclosure, the collimation assembly includes a laser diode and a collimating lens aligned with the laser diode.

[0013] In another aspect of this disclosure, the tube of the substrate defines a first chamber, a second chamber, a third chamber, and a fourth chamber, the first chamber being configured to at least partially accommodate the collimating assembly, the second chamber being configured to at least partially accommodate the first lens, the third chamber being configured to at least partially accommodate the second lens, and the fourth chamber being configured to at least partially accommodate the third lens.

[0014] In another aspect of this disclosure, the first lens, the second lens, and the third lens are fixed within respective first lens holders, second lens holders, and third lens holders, which are at least partially disposed within the second, third, and fourth chambers of the tube of the substrate.

[0015] In another aspect of this disclosure, at least one of the first, second, or third lens holders includes a finger extending from it, the finger being configured to allow rotational adjustment of the at least one lens holder within a respective cavity during assembly. At least one of the first, second, or third lens holders may include a finger extending from it, the finger being configured to allow axial adjustment of the at least one lens holder within a respective cavity during assembly. Alternatively or additionally, at least one of the first, second, or third lens holders may include a finger extending from it, the finger being configured to allow both rotational and axial adjustment of the at least one lens holder within a respective cavity during assembly.

[0016] In another aspect of this disclosure, the first lens is a cylindrical positive lens, the second lens is a cylindrical negative lens, and the third lens is a cylindrical objective lens. In such an aspect, the first, second, and third lenses may be arranged sequentially along a tube extending from the collimating assembly.

[0017] A flow cytometer provided according to an aspect of this disclosure includes a lens subassembly comprising a plurality of lenses arranged along an axis, a flow unit located axially downstream of the lens subassembly, and a collimation subassembly located axially upstream of 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 of materials having mutually balanced coefficients of thermal expansion, thereby maintaining the predetermined axial distance under temperature variations up to 30°C.

[0018] In one aspect of this disclosure, the temperature change is from 10°C to 40°C.

[0019] In another aspect of this disclosure, the first support is formed of PEEK, and the second support is formed of brass.

[0020] In another aspect of this disclosure, the three supports configured to maintain the specified axial distance are formed of materials having mutually balanced coefficients of thermal expansion. In such an aspect, the first support may be formed of PEEK, the second support may be formed of brass, and the third support may be formed of aluminum.

[0021] In another aspect of this disclosure, the flow cytometer further includes a mounting platform having the lens subassembly, the collimation subassembly, and a housing that supports the flow unit mounted on the housing to maintain a predetermined axial distance between the flow unit and the lens subassembly. In these aspects, the housing and mounting platform are formed of materials having mutually balanced coefficients of thermal expansion, thereby maintaining the predetermined axial distance between the flow unit and the lens subassembly under temperature variations of up to 30°C.

[0022] In another aspect of this disclosure, the housing is formed of copolyester, and the mounting platform is formed of aluminum.

[0023] A method for assembling a laser optics assembly for a flow cytometer according to an aspect of this disclosure includes at least partially securing a collimating assembly within a tube of a substrate. The collimating assembly includes a laser diode and a collimating lens configured to generate a laser beam along an axis, wherein the laser beam has a first beam waist diameter in a first direction and a second beam waist diameter in a second direction. The method further includes: at least partially positioning a third lens within the tube of the substrate along the axis; rotatably adjusting the third lens about the axis to minimize the first beam waist diameter; fixing the third lens relative to the substrate; at least partially positioning a first lens within the tube of the substrate along the axis; rotatably adjusting the first lens about the axis to maintain the first beam waist diameter; fixing the first lens relative to the substrate; at least partially placing a second lens within the tube of the substrate along the axis; rotatably adjusting the second lens about the axis to maintain the first beam waist diameter; axially adjusting the second lens along the axis to set the second beam diameter to a desired value; and fixing the second lens relative to the substrate.

[0024] In one aspect of this disclosure, the third lens is positioned furthest from the collimating assembly, the first lens is positioned closest to the collimating assembly, and the second lens is positioned between the first lens and the third lens.

[0025] In another aspect of this disclosure, the third lens is a cylindrical objective lens, the first lens is a cylindrical positive lens, and the second lens is a cylindrical negative lens.

[0026] In another aspect of this disclosure, a third lens is located in a third chamber of the barrel, the third chamber being configured to axially constrain the third lens and allow rotation of the third lens before it is fixed; a first lens is located in a first chamber of the barrel, the first chamber being configured to axially constrain the first lens and allow rotation of the first lens before it is fixed; and a second lens is located in a second chamber of the barrel, the second chamber being configured to allow rotation and translation of the second lens before it is fixed.

[0027] In another aspect of this disclosure, the first waist has a 1 / e [width] of 6.7 μm to 9 μm. 2 diameter.

[0028] In another aspect of this disclosure, the second beam has a 1 / e [size] of 190 μm to 210 μm. 2 Diameter. More specifically, the second beam can have a diameter of 200 μm (1 / e). 2 diameter.

[0029] Another flow cytometer according to an aspect of this disclosure includes a flow unit 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 cylindrical positive lens disposed on the axis and configured to receive the laser beam from the collimation assembly; a cylindrical negative lens disposed on the axis and configured to receive the laser beam from the cylindrical positive lens; a cylindrical objective lens disposed on the axis and configured to receive the laser beam from the cylindrical negative lens and project the laser beam onto the flow unit such that the laser beam incident on the flow unit defines a first beam waist 1 / e of 6.7 μm to 9 μm in a direction parallel to the flow direction of the flow unit. 2 The diameter, and a second beam of 1 / e defined in a direction perpendicular to the flow direction of the flow unit, ranging from 190 μm to 210 μm. 2 diameter.

[0030] In one aspect of this disclosure, the first waist 1 / e 2 Diameter and second beam 1 / e 2 The diameter was chosen such that performance was not reduced despite the actual radial offset of the core flow within the flow unit being as high as 15 μm.

[0031] In another aspect of this disclosure, the first waist 1 / e 2 Diameter and second beam 1 / e 2 The diameter was chosen such that performance was not degraded despite the apparent radial shift of the core flow of up to 15 μm caused by the shift of the laser beam focus.

[0032] In another aspect of this disclosure, the first waist 1 / e2 The diameter is selected such that, with a change in flow velocity through the flow unit of less than or equal to 2%, the transit time measurement can distinguish the size of particles or cells within 1 μm. Attached Figure Description

[0033] This document describes, with reference to the accompanying drawings, various aspects and features of the currently disclosed flow cytometer and its laser optics components, wherein similar reference numerals denote similar or identical elements, and:

[0034] Figure 1 This is a perspective view of the laser optics, flow unit, and sensor module of a flow cytometer provided in this disclosure;

[0035] Figure 2 yes Figure 1 A longitudinal cross-sectional view of the module;

[0036] Figure 3 and Figure 4 They are Figure 1 Front and rear perspective views of the laser optical components of the module;

[0037] Figure 5 yes Figure 3 and Figure 4 A perspective partial cross-sectional view of the laser optical components;

[0038] Figure 6 and Figure 7 They are Figure 3 and Figure 4 Front and rear perspective views of the collimator sub-assembly of the laser optical component;

[0039] Figure 8 yes Figure 6 and Figure 7 Longitudinal cross-sectional view of the collimator component;

[0040] Figure 9 yes Figure 6 and Figure 7 A partial perspective cross-sectional view of the collimator component;

[0041] Figure 10 yes Figure 3 and Figure 4 A perspective view of the lens sub-assembly of the laser optical component;

[0042] Figure 11 yes Figure 3 and Figure 4 A cross-sectional view of the laser optical components. Figure 3 and Figure 4 It shows Figure 10 Lens subassemblies;

[0043] Figures 12 to 14 yes Figure 1 A side view schematic of the module shows the axial adjustment of the cylindrical negative lens of the laser optics assembly; and

[0044] Figures 15 to 17 yes Figure 1 The top view of the module shows the axial adjustment of the cylindrical negative lens of the laser optics component. Detailed Implementation

[0045] Turn Figure 1 and Figure 2 This disclosure provides a flow cytometer comprising a laser optics element, a flow unit, and a sensor module, generally identified by reference numeral 10. Although not shown, the flow cytometer may further include, for example, a housing enclosing the internal operable components of the flow cytometer, an electronic module configured to control module 10 and process test results received from module 10, a sample receiving module configured to receive a sample to be tested, a pump module configured to pump the sample and sheath fluid into the flow unit assembly 300, and a waste module configured to safely collect the sample and sheath fluid after testing. Alternatively or additionally, any other suitable modules, components, and / or features are contemplated for use with module 10 of the flow cytometer of this disclosure.

[0046] Continue to refer to Figure 1 and Figure 2 Module 10 includes a mounting platform 100, a laser optics assembly 200 fixed to the mounting platform 100, a flow unit assembly 300 fixed to the mounting platform 100 and operatively positioned relative to the laser optics assembly 200, and a sensor assembly 400 operatively positioned relative to both the laser optics assembly 200 and the flow unit assembly 300, the sensor assembly 400 being used for both forward scattering detection and side scattering detection. The laser optics assembly 200, the flow unit assembly 300, and the sensor assembly 400 are independently fastened to the mounting platform 100 using bolts 110 and / or any other suitable fastening structure to maintain the relative positions of these assemblies 200 to 400.

[0047] As described below, module 10 is configured such that the flow cytometer can operate over a wide temperature range, such as, for example, from 10°C to 40°C, and can withstand 30 seconds of random 10G axial vibration without performance degradation. Performance degradation is defined herein as a loss of intensity and / or sensitivity greater than 5%.

[0048] Additionally, as detailed below, module 10 is configured such that the flow cytometer is relatively insensitive to actual or apparent deviations of the core flow, such deviations being, for example, radial deviations of up to 15 μm relative to the previously aligned flow axis of the core flow; module 10 is configured such that the flow cytometer operates without the need for a beam stop to prevent unscattered laser light from reaching the forward-scattering sensor of sensor assembly 400; and module 10 is configured such that the flow cytometer employs a slowly converging beam along a flow axis perpendicular to the core flow, which in embodiments allows the beam to be set at 1 / e at the core flow. 2 width.

[0049] Furthermore, as described below, when the core flow rate is stabilized within 2%, module 10 also provides the flow cytometer with the ability to measure the transit time of particles with diameters ranging from 4 micrometers to 16 micrometers with an accuracy of 1 μm.

[0050] Reference Figures 2 to 5 The laser optics assembly 200 includes a clamp subassembly 210, a collimation subassembly 230, and a plurality of lens subassemblies 270, 280, and 290. The clamp subassembly 210 includes a base plate 212 that defines at least one pair (e.g., two pairs) of feet 214 along opposite sides of the base plate 212. Each foot 214 includes a hole 216 defining a hole through it to allow the laser optics assembly 200 to be securely mounted on the mounting platform 100 using bolts 110. The base plate 212 also defines a generally cylindrical cylinder 218 that extends along the base plate 212 between the feet 214. The cylinder 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 cylinder 218. Chambers 219, 221, 223, and 225 are configured to house the collimating sub-assembly 230 and lens sub-assemblies 270, 280, and 290, respectively, located therein. The clamping sub-assembly 210 also includes cover plates 220, 222, 224, and 226, configured to be securely mounted to the base plate 212 using bolts 228 to enclose and secure the collimating sub-assembly 230 and lens sub-assemblies 270, 280, and 290 relative to each other within chambers 219, 221, 223, and 225, respectively. The assembly and alignment of the collimating sub-assembly 230 and lens sub-assemblies 270, 280, and 290 within the clamping sub-assembly 210 are described in detail below.

[0051] Reference Figures 6 to 9The collimator assembly 230 includes a support plate 232, a support hub 234, an insert 236, and a spring washer 237. The support plate 232, support hub 234, insert 236, and spring washer 237 are configured to operably engage with each other and hold the collimating lens 238 of the collimator assembly 230 relative to the laser diode 240 of the collimator assembly 230.

[0052] More specifically, the support plate 232 defines an outer surface 242a and an inner surface 242b, and includes a central hole 244 and a plurality of radial holes 246. Figure 2 A plurality of radial holes 246 are defined to pass through the support disk 232 between its outer surface 242a and inner surface 242b, respectively. A central hole 244 defines an external opening on the outer surface side of the support disk 232, which is larger than the internal opening of the central hole 244 defined on the inner surface side of the support disk 232, such that the laser diode 240 can be inserted into the central hole 244 through the external opening but is prevented from passing through the internal opening. Thus, the laser diode 240 can be inserted into and positioned within the internal opening of the central hole 244 to secure the laser diode 240 relative to the support disk 232. The laser diode 240 includes a suitable electrical connector 241 that enables the laser diode 240 to be connected to a power supply and control electronics (not shown). The laser diode 240 can be configured to emit red light having a wavelength in the range of 630 nm to 665 nm, or in an embodiment, can be configured to emit red light having a wavelength in the range of 635 nm to 650 nm.

[0053] The support hub 234 defines a generally T-shaped configuration, which includes a disc portion 247 positioned to abut against the inner surface 242b of the support disc 232, and a body portion 248 extending from the disc portion 247 in a direction opposite to that of the support disc 232. A central cavity 250 extends through the disc portion 247 and the body portion 248, and a plurality of radial holes 252 ( Figure 2 It is confined within the disc portion 247. Thread 254 is provided on at least a portion of the inner surface of the defining cavity 250 of the support hub 234.

[0054] Insert 236 defines a generally cylindrical configuration that defines an internal channel 256 therethrough. Insert 236 also includes threads 258 disposed on at least a portion of its outer surface, the threads 258 being configured to engage with threads 254 of the support hub 234. More specifically, insert 236 is configured (e.g., using an adhesive) to hold collimating lens 238 within the channel 256 of insert 236, and insert 236 is configured to be positioned within a central cavity 250 of the support hub 234 in a threaded engagement manner. A spring washer 237 is configured to position within the central cavity 250 between insert 236 and support disc 232 to maintain tension between insert 236 and support disc 232.

[0055] Continue to refer to Figures 6 to 9 To assemble the collimator assembly 230, the laser diode 240 is fixed within the support disk 232, and the collimating lens 238 is fixed within the insert 236. The insert 236 is then threaded into the central cavity 250 of the support hub 234. With the laser diode 240 fixed within the support disk 232 and the insert 236 engaged within the support hub 234 (where the collimating lens 238 is fixed), the support disk 232 and the support hub 234 are positioned relative to each other such that the inner surface 242b of the support disk 232 abuts against the support hub 234, the central hole 244 of the support disk 232 is aligned with the central cavity 250 of the support hub 234, and the radial holes 246 of the support disk 232 align with the corresponding radial holes 252 of the support hub 234 (see...). Figure 2 Alignment. A fixing device (not shown) can be used to hold the support plate 232 and the support hub 234 in this position and facilitates the alignment of the support plate 232 and the support hub 234, as detailed below.

[0056] With the support plate 232 and support hub 234 positioned as described above, bolts 260 are inserted through radial holes 246 and engaged, for example via threaded connection, within radial holes 252 to secure the support plate 232 and support hub 234 relative to each other (see...). Figure 2 Position adjustments (such as vertical and / or horizontal adjustments) between the support plate 232 and the support hub 234 can be made before or after each bolt 260 is engaged via, for example, an adjustment knob (not shown) associated with the fixing device, to align the laser diode 240 relative to the collimating lens 238, such that the beam emitted from the laser diode 240 is well collimated and directed in a direction coaxial with the optical axis of the collimating lens 238. A reverse beam expander (not shown) associated with the fixing device can also be used to verify this alignment.

[0057] To adjust the axial distance between the collimating lens 238 and the laser diode 240, the insert 236 is screwed into (threaded engagement) or unscrewed out (thread disengagement) the central cavity 250 of the support hub 234, thereby moving the collimating lens 238 toward or away from the laser diode. A reverse beam expander (not shown) can again be used to ensure that the specified axial distance between the collimating lens 238 and the laser diode 240 is achieved. By threading the insert 236 into the appropriate position corresponding to the specified 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 the gap between the insert 236 and the support disk 232 and ensuring that the specified axial distance between the collimating lens 238 and the laser diode 240 is maintained despite, for example, mechanical vibration applied to the collimator assembly 230.

[0058] Once the beams and optical axes of the collimating lens 238 and the laser diode 240 are coaxial with each other and the beams are collimated, the bolts 260 can be properly tightened to lock the support disc 232 and the support hub 234 relative to each other, thereby maintaining the engagement and positioning between the support disc 232 and the support hub 234 despite the application of mechanical vibrations, for example, to the collimating subassembly 230.

[0059] The support disc 232 and support hub 234 described in detail above lock each other in order to ensure the horizontal, vertical and axial alignment of the collimating lens 238 and the laser diode 240 relative to each other.

[0060] Still refer to Figures 6 to 9 The collimator assembly 230 is configured to maintain a predetermined axial distance between the collimating lens 238 and the laser diode 240 despite changes in ambient temperature. More specifically, the collimator assembly 230 is configured to sufficiently maintain the predetermined axial distance between the collimating lens 238 and the laser diode 240 within a range of 30°C, such as from 10°C to 40°C, without performance degradation. In embodiments, this is achieved by forming the support hub 234 and the insert 236 from materials with different coefficients of thermal expansion, or by forming the support disk 232, support hub 234, and insert 236 from materials with different coefficients of thermal expansion, which maintain the predetermined axial distance between the laser diode 240 and the collimating lens 238 of the flow cytometer for operation in the range of 10°C to 40°C, thus without performance degradation. While other suitable materials with linear coefficients of thermal expansion could also be considered, which could conversely balance the flow cytometer's response to temperature fluctuations in the range of 10°C to 40°C, in embodiments, this is achieved by using materials with a coefficient of thermal expansion of 1.8 x 10⁻⁶. -5The linear thermal expansion coefficient of the brass-formed support hub 234 is (with a 4.5x10) -5 The linear coefficient of thermal expansion (CTE) of the flow cytometer is achieved through an insert 236 formed of PEEK (polyetheretherketone). This balance includes not only compensating for the CTE of certain components of the flow cytometer but also taking into account the temperature-dependent variation of the refractive index of the flow cytometer's optical components. As used herein, "prescribed axial distance" should be understood to include a range of distances to account for, for example, the temperature-dependent variation of the target axial distance between the collimating lens 238 and the laser diode 240. This range may include a variation of no more than 0.025% in the prescribed axial distance between the collimating lens 238 and the laser diode 240, or, in the embodiments, a variation of no more than 0.012%.

[0061] See also Figures 2 to 5 To assemble the collimator 230 with the fixture sub-assembly 210, the main body 248 of the support hub 234 of the collimator 230 is located within the first chamber 219 of the cylinder 218 of the base plate 212 of the fixture sub-assembly 210. A cover plate 220 is then positioned around the main body 248 of the support hub 234 and engaged with the base plate 212 on both sides of the support hub 234 via bolts 228. This encloses the main body 248 of the support hub 234 within the first chamber 219 and, under pressure, fixes the collimator 230 in place relative to the base plate 212. In this embodiment, the collimator 230 is assembled with the fixture sub-assembly 210 before assembling the lens sub-assemblies 270, 280, and 290. Before tightening the bolts 218, the collimator 230 is rotated as needed to ensure that the fast axis of the laser beam is aligned perpendicular to the bottom surface of the base plate 212.

[0062] Turn Figure 10 and Figure 11 , combined Figures 2 to 5As described above, the laser optical assembly 200 includes three lens subassemblies 270, 280, and 290. Each lens subassembly 270, 280, and 290 includes lens holders 272, 282, and 292 that define lens receiving portions 274, 284, and 294, respectively. The lens receiving portions 274, 284, and 294 are configured to securely hold corresponding lenses 276, 286, and 296 therein. Lens 276 is configured as a cylindrical positive lens and is configured as part of lens subassembly 270. Lens 276 is configured to be positioned within a second chamber 221 of the cylinder 218 of substrate 212 and secured in the second chamber 221 via a second cover plate 222, thereby positioning the cylindrical positive lens 276 closest to the collimating lens 238. Lens 286 is configured as a cylindrical negative lens and is part of lens subassembly 280. Lens 286 is configured to be positioned within the third chamber 223 of the tube 218 of substrate 212 and fixed in the third chamber 223 via a third cover plate 224, thereby placing the cylindrical negative lens 286 next to the cylindrical positive lens 276 on the opposite side of the collimating subassembly 230. Lens 296 is configured as a cylindrical objective lens and is part of lens subassembly 290. Lens 296 is configured to be positioned within the fourth chamber 225 of the tube 218 of substrate 212 and fixed in the fourth chamber 225 via a fourth cover plate 226, thereby placing the cylindrical objective lens 296 next to the cylindrical negative lens 286 on the opposite side of the cylindrical positive lens 276.

[0063] Each lens holder 272, 282, 292 includes fingers 278, 288, 298 extending radially outward therefrom. The fingers 278, 288, 298 are configured to extend through grooves (not explicitly shown) defined in the substrate 212 of adjacent chambers 221, 223, 225, such that the fingers 278, 288, 298 extend from the substrate 212 on the underside of the substrate 212.

[0064] To assemble lens subassemblies 270, 280, and 290 within fixture subassembly 210, lenses 276, 286, and 296 are respectively engaged within receiving portions 274, 284, and 294 of lens supports 272, 282, and 292, and lens supports 272, 282, and 292 are respectively positioned within chambers 221, 223, and 225. Supports 272 and 292 define a thickness that generally approximates the width of chambers 221 and 225, and / or supports 272 and 292 include complementary features to hold supports 272 and 292, and thus lenses 276 and 296, respectively, in fixed axial positions within the corresponding chambers 221 and 225. However, the fingers 278 and 298 of the lens supports 272 and 292 can be manipulated to rotate the lens supports 272 and 292 relative to the substrate 212, thereby rotating the lenses 276 and 296. On the other hand, the lens support 282 defines a thickness that is reduced relative to the width of the chamber 223, such that when the fingers 288 of the lens holder 282 are manipulated accordingly, the support 282 can be translated axially along the cylinder 218, and therefore the lens 286 can be translated axially along the cylinder 218. Alternatively, the fingers 288 can be manipulated to rotate the lens support 282 relative to the substrate 212, and thus rotate the lens 286. Since it has been found that the rotational alignment of the lenses 276, 286, and 296 and the axial positioning of the lens 286 are important alignments to ensure the accurate performance of the flow cytometer, the above-described detailed configuration that enables the rotational alignment of the lenses 276, 286, and 296 and the axial positioning of the lens 286 is advantageous.

[0065] During assembly, once the collimating sub-assembly 230 is installed, the lens sub-assembly 290 is inserted into the chamber 225. The lens sub-assembly 290 is rotated and adjusted using the finger 298, and secured using the cover plate 226 and bolts 228 to hold it in place relative to the substrate 212 under pressure. The substrate 212 is configured such that the lens sub-assembly 290 is mounted at a distance from the collimating lens 238, approximately equal to the sum of the focal lengths of the lenses 296 and 238. Once the lens sub-assembly 290 is installed, as described above, verification is performed to ensure that the flow parallel to the core flow through the flow unit 340 (see...) Figure 2 A waist 1 / e2 diameter of 6.7 μm to 9 μm was achieved along the direction of the beam.

[0066] After assembling and verifying the lens subassembly 290, the lens subassembly 270 is inserted into the chamber 221. The lens subassembly 270 is rotated and adjusted using the finger 278, and secured using the cover plate 222 and bolts 228 to fix it in place relative to the substrate 212 under pressure. The cylindrical positive lens 276 of the lens subassembly 270 is rotatably aligned such that the focal axis of the cylindrical positive lens 276 is perpendicular to the focal axis of the cylindrical objective lens 296, and this is confirmed by reaffirming that a waist of 6.7 μm to 9 μm is maintained in a direction parallel to the flow direction of the core flow. 2 The diameter was verified.

[0067] Next, the lens subassembly 280 is inserted into the chamber 223. The lens subassembly 270 is rotatably and / or axially adjusted using the finger 288, and the lens subassembly 270 is secured using the cover plate 224 and bolts 228 to fix the lens subassembly 280 in place relative to the substrate 212 under pressure. The cylindrical negative lens 286 of the lens subassembly 280 is rotatably aligned so that the focal axis of the cylindrical negative lens 286 is perpendicular to the focal axis of the cylindrical objective lens 296 and parallel to the focal axis of the cylindrical positive lens 276, and this is achieved by reconfirming that a waist of 6.7 μm to 9 μm is maintained in a direction parallel to the flow direction of the core flow. 2 The diameter was verified. The axial spacing of the cylindrical negative lens 286 was adjusted so that the flow perpendicular to the core flow passed through the flow unit 340 (see...). Figure 2 The direction of the beam 1 / e in the embodiment is 190μm to 210μm. 2 Width, or the 200μm beam 1 / e in the implementation examples. 2 width.

[0068] Suitable fasteners (not shown) can be used to hold the various components and to facilitate the manipulation of the fingers 278, 288, 298, enabling adjustments during assembly, such as any suitable test equipment used to measure the beam width during the aforementioned verification. Once fully assembled and verified as described above, the laser optics assembly 200 provides a beam waist of 6.7 μm to 9 μm along the parallel direction. 2 1 / e of the diameter and the vertical direction of 190μm to 210μm (or 200μm). 2 Bundle width.

[0069] Reference Figures 12 to 17 , Figures 12 to 14 The divergence range of different laser diodes relative to the fast (larger divergence) axis of laser diode 240 is shown. This fast axis is set parallel to the core flow in flow unit 340 and perpendicular to the bottom surface of substrate 212 as previously described. Figures 15 to 17The diagram illustrates the divergence range of different laser diodes relative to the slow (less divergent) axis of laser diode 240, which is configured to be perpendicular to the core flow in flow unit 340 and parallel to the bottom surface of substrate 212. In one embodiment, laser diode 240 (e.g., Ushio HL6363MG-A type laser diode) will have a fast axis divergence greater than its slow axis divergence, but these two divergences are otherwise independent of each other.

[0070] In a well-aligned system, the fast axis (parallel to the core flow in the flow unit 340) of the laser diode 240 diverges, controlling the waist of the flow at the core flow of the flow unit. (From the laser diode...) Figure 12 The maximally divergent beam (described in the text) provides a minimum beam waist of 6.7 μm at the core flow, and the laser diode's ( Figure 14 The minimally divergent beam (as shown) provides a maximum beam waist of 9.0 μm at the core flow. Furthermore, in a well-aligned system, and regardless of the axial orientation of the cylindrical negative lens 286 within its adjustment range, the beam waist parallel to the core flow is maintained.

[0071] However, a minute change in the axial position of the cylindrical negative lens 286 allows for a reduction of 1 / e of the beam of the laser diode (flowing perpendicular to the core flow) within the flow unit 340. 2 The width can be adjusted to a range of 190 μm to 210 μm, or to 200 μm as in the examples. These axial position variations of several hundred micrometers... Figures 15 to 17 It is imperceptible in the middle, but in all three figures, 1 / e 2 The width of the laser beam is 200 μm in the direction perpendicular to the centerline of the core flow and at the centerline of the core flow. Figure 15 The slow-axis laser beam with maximum divergence is depicted, and Figure 17 A slow-axis laser beam with minimal divergence is depicted. Figure 15 In the middle, the cylindrical negative lens 286 is positioned furthest from the laser diode 240, while... Figure 17 In this configuration, the cylindrical negative lens 286 is positioned as close as possible to the laser diode 240. It should also be noted that the position of the axial focal point of the slow axis varies with different degrees of divergence of the slow axis and the position of the cylindrical negative lens 286; the focal point of the slow axis is... Figure 15 The center is positioned closest to the laser diode at 240, and... Figure 17 The center is positioned 240 units away from the laser diode.

[0072] As detailed below, there is a bundle 1 / e in the direction perpendicular to the flow through the core flow of flow unit 340. 2The diameter of the laser optics 200 is advantageously insensitive to the (actual or apparent) radial offset of the core flow within a radius of 15 μm, so the (actual or apparent) radial offset of the core flow within a radius of 15 μm does not cause a performance degradation.

[0073] return Figures 1 to 5 The components of the laser optical assembly 200 described above not only facilitate assembly and alignment but also provide a configuration in which the collimator sub-assembly 230 and lens sub-assemblies 270 to 290 are respectively and independently fixed to the substrate 212. This configuration of the laser optical assembly 200 has shown that, at the beam waist 1 / e of the laser optical assembly 200... 2 The ability to withstand 10G random axial vibration for 30 seconds with a diameter variation not exceeding 5%. More specifically, using the Qualmark OVTT, available from North American ESPEC Inc. in Denver, Colorado, USA. TM Vibration testing was conducted using an 18-type all-axial vibration table system. The vibration test was performed by fixing the laser optics assembly 200 to the vibration table and setting the table to random axial mechanical vibration at 10G for at least 30 seconds. Omega was used. TM The HHVB82 accelerometer is a verification accelerometer available from Omega Engineering Ltd. in Norwalk, Connecticut, USA.

[0074] Reference Figure 1 and Figure 2 As described above, the flow unit assembly 300 is mounted on the mounting platform 100. More specifically, the flow unit assembly 300 includes an input end 310, a flow unit 340, and an output end 350. The input end 310 is coupled to a nozzle 320, which is defined by a housing 330. The input end 310 is used to deliver sample and sheath fluid to the nozzle 320. The flow unit 340 is connected downstream of the nozzle 320 to receive sample and sheath (not shown) fluid from the nozzle 320. The output end 350 is located downstream of the flow unit 340 to guide the sample and sheath fluid to a suitable collection container after testing. The housing 330 of the flow unit assembly 300 is located within an aperture 120 defined by the mounting platform 100 and is securely fixed to the mounting platform 100 using a plurality of bolts 110 to maintain a predetermined distance between the flow unit 340 and the cylindrical objective lens 296. This predetermined distance is an important control distance to ensure the accuracy of the flow cytometer.

[0075] The housing 330 and mounting platform 100 of the flow unit assembly 300 are configured to sufficiently maintain a predetermined distance between the flow unit 340 and the cylindrical objective lens 296 within a temperature range of, for example, 30°C from 10°C to 40°C without compromising performance. This is achieved by forming the housing 330 and mounting platform 100 of the flow unit assembly 300 with materials having different linear coefficients of thermal expansion, said materials being configured to maintain a predetermined axial distance between the objective lens 296 and the flow unit 340 within a temperature range of 10°C to 40°C. In an embodiment, this is achieved by using materials having 8.0 x 10⁻⁶... -5 Eastman Tritan's coefficient of thermal expansion TM The copolyester MX811 forms the housing 330 and is composed of (having 2.38x10) -5 The mounting platform 100 is formed from aluminum with a linear coefficient of thermal expansion. The housing 330 comes into direct contact with samples such as blood and sheath fluids; therefore, the housing 330 must also be suitable for this purpose. (Eastman Tritan) TM The copolyester MX811 is available from Eastman Chemical Company in Kingsport, Tennessee, USA, but other suitable combinations of materials with linear coefficients of thermal expansion may also be considered, which conversely balance the flow cytometer's response to temperature fluctuations in the range of 10°C to 40°C. This balance includes not only compensating for the linear coefficients of thermal expansion of certain components of the flow cytometer but also taking into account temperature-dependent variations in the refractive index of the optical components of the flow cytometer 10. Similarly, as described above, "prescribed axial distance" should be understood to include a range of distances to account for temperature-dependent variations in the target axial distance, for example, between objective lens 296 and flow unit 340. This range may include variations of no more than 0.01% in the prescribed axial distance between objective lens 296 and flow unit 340, or, in the embodiment, no more than 0.005%.

[0076] When the flow unit assembly 300 is mounted on the mounting platform 100, the surface of the flow unit 340 is not oriented parallel to the plane of the cylindrical objective lens 296, but rather offset by an angle of 5° relative to the plane of the cylindrical objective lens 296, in order to ensure that any specular reflections from the surface of the flow unit 340 do not connect back to the laser optics. For a similar purpose, the flow unit 340 is also coated with an anti-reflective coating.

[0077] Continue to refer to Figure 1 and Figure 2The sensor assembly 400 includes a forward scattering subassembly 410 and a side scattering subassembly 420. The forward scattering subassembly 410 includes a plate 412 and a sensor array 414, which includes an extinction sensor, a low-angle forward scattering sensor, and a high-angle forward scattering sensor. The side scattering subassembly 420 includes a lens mount 422. Figure 2 ), supported on lens mounting component 422 ( Figure 2 Lens 424 inside Figure 2 The side-scattering subassembly 420 has a center capture angle of 78° relative to the direction of the laser beam, instead of a right angle (i.e., 90°), to increase the side-scattering signal.

[0078] Generally refer to Figures 1 to 2 The insensitivity of module 10 to the (actual or apparent) radial offset of the core flow within the aforementioned 15 μm radius will be described in more detail below. Traditionally, a Cartesian coordinate system is defined, where the core flow flows along the positive y-axis and the laser beam flux points in the positive z-axis direction. This is achieved by controlling the beam waist 1 / e along the y-axis. 2 Diameter and bundle 1 / e along the x-axis 2 The diameter can be set within an elliptical region, within which the maximum intensity of the laser beam does not decrease by more than a predetermined amount. As described below, module 10 is configured to remain insensitive to (actual or apparent) radial offset of the core flow within a radius of 15 μm, thus maintaining performance (intensity reduction equal to or less than 5%) despite such radial offset of the core flow.

[0079] The laser beam from the laser optics assembly 200 is aligned with the core flow of the flow unit 340 flowing through the flow unit assembly 300, while simultaneously monitoring the scattered laser light from the particles flowing into the flow unit 340 and converting it into an electrical signal. The relative positions of the laser optics assembly 200 and the flow unit 340 are adjusted in the x and z directions to maximize these signals. Before completing this x-axis and z-axis alignment, the sensor of the sensor assembly 400 is horizontally aligned with the laser beam; further alignment in the y-direction is not required.

[0080] The flow cytometer disclosed herein measures based on the maximum scattered signal or the maximum area under the curve of the scattered signal. Therefore, the beam waist in the y-direction does not need to be considered, except that its effect on the intensity of the laser beam is a function of the distance along the z-axis. In the z-direction, the relative intensity is defined in Equation (1):

[0081]

[0082] Where I 0，zIt is the intensity at z = 0 (the position where the waist is aligned with the center of the core flow), and the z-position and Rayleigh range are defined in micrometers (μm). R Then, based on the constraints, formula (2) is provided:

[0083]

[0084] Among them, the waist 1 / e in the y direction 2 The diameter is ω 0，y The values ​​are specified in μm, and for the nominal laser wavelength, λ = 0.64 μm, and the beam quality factor is M. 2 =1.2.

[0085] Relatively large (e.g., 190 μm to 210 μm) x-direction beam 1 / e 2 The diameter is determined based on the distance along the z-axis to minimize its impact on the intensity of the laser beam. However, if the core stream is offset along the x-axis from its alignment position, the beam diameter in the x-direction does indeed affect how much beam intensity can be scattered. In the x-direction, the relative intensity is defined in equation (3):

[0086]

[0087] Among them, similar to equations (1) and (2), I 0，x It is the intensity at x = 0 (again, the center of the beam diameter aligned with the center of the core flow), and the beam 1 / e in the x position and x direction is defined in μm. 2 diameter ω x .

[0088] For a given waist 1 / e 2 diameter ω 0， y and bundle 1 / e 2 diameter ω x The product of equations (1) and (3) can be used to solve for the combination of x and z positions, which describes the outer limit of the reduction in bundle strength from the considered value, such as 5%. For example, equation (4):

[0089]

[0090] Since the beam diameter is defined along an orthogonal axis equivalent to the coordinate axes, equation (5) holds:

[0091] I0 = I 0，z =I 0，x

[0092] Based on the above, the beam diameter is selected to ensure that, when the radial offset of the core flow relative to the y-axis is up to 15 μm, the intensity at the offset center remains within 5% of the original center aligned with the system. These core flow offsets (in the case of radial movement of the core flow from its original center) can be real (in the case of a change in the focus of the laser optics due to the offset of one or more components) or apparent.

[0093] Taking into account the above, and also considering that the bundle 1 / e can be limited... 2 To mitigate reflections at the inner edges of the flow unit 340, an x-direction beam 1 / e ranging from 190 μm to 210 μm (or 200 μm) was selected in the x-direction range of the diameter. 2 Diameter. Taking into account the above, and also considering that the waist can be limited by 1 / e... 2 To improve the ability to perform relevant time-of-flight (TOF) measurements, a beam waist of 6.7 μm to 9 μm was selected in the y-direction range, as described below. 2 diameter.

[0094] Another important consideration for the two beam diameter components (x-axis and y-axis) is that a wider beam diameter distributes laser power over a larger area. In fact, the beam intensity along a given axis is inversely proportional to its beam diameter along the same axis. Module 10 provides a beam 1 / e in the x-direction of 190 μm to 210 μm (or 200 μm). 2 Diameter and waist 1 / e in the y-direction of 6.7μm to 9μm 2 The diameter is used to balance the aforementioned constraints. Therefore, a larger region in which the core flow is actually or apparently offset is achieved, while mitigating the contribution of stray light scattered from the sidewalls of the flow unit 340. Furthermore, these balanced constraints allow for accurate TOF measurements, as described below, and minimize the laser power requirements of module 10.

[0095] Regarding Time-of-Flight (TOF) measurements, as cells or particles flow through flow unit 340, they first encounter increasing laser intensity until they match the maximum laser intensity, after which they encounter decreasing laser intensity. Accordingly, as a general approximation, the scattering intensity from a given particle or cell is proportional to the overlap volume between the incremental intensity of the laser beam and the incremental volume of the particle's cross-section. Therefore, by considering how spherical particles of a certain diameter overlap with the laser beam, the relatively scaled width of different particle overlaps can be compared. Moreover, as long as the cell flow rate remains consistent, the TOF scales accordingly.

[0096] Based on the above, and using the estimated full width at half maximum (FWHM) variation as the maximum proportional change in the width of the scattering intensity curve, for a given particle or cell, it is possible to determine which y-axis beam widths will still classify the particle or cell diameter within ±1 μm of its actual diameter with approximately 95% confidence. However, flow rate variability limits the ability to accurately determine the diameter of particles or cells, and this must be taken into account.

[0097] As provided by currently available flow cytometers, a beam waist of 6.7 μm to 9 μm in the y-direction is used. 2 The diameter and flow rate variation controlled within 2% of the average value enable the differentiation of time-of-flight (TOF) between particles or cells (with diameters between 4 μm and 16 μm) having a diameter difference of at least ±1 μm. Furthermore, flow rate variations can be detected, for example, using a pressure sensor, and periodic flow rate variations can be compensated for by a correction based on this correction (from the pump module pumping the sample and sheath fluid through the flow unit 340) to reflect these variations.

[0098] It should be understood that any specific numerical indication herein includes a range of values ​​to account for material and manufacturing tolerances and / or error margins of measuring instruments generally accepted in the art.

[0099] Based on the foregoing and with reference to the accompanying drawings, those skilled in the art will understand that certain modifications may be made to this disclosure without departing from its scope. Although several embodiments of this disclosure have been shown in the drawings, they are not intended to be limited thereto, as the scope of the disclosure is intended to be as broad as permitted in the art, 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 examples of particular embodiments. Other modifications within the scope and spirit of the appended claims will be apparent to those skilled in the art.

Claims

1. A flow cytometer, comprising: Flow unit, the flow unit defining the flow direction; A collimation assembly, comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; A cylindrical positive lens, which is disposed on the axis downstream of the collimating assembly; A cylindrical negative lens, wherein the cylindrical negative lens is disposed downstream of the cylindrical positive lens on the axis; A cylindrical objective lens, positioned downstream of the cylindrical negative lens on the axis and configured to project the laser beam onto the flow unit, such that the laser beam incident on the flow unit defines a first beam waist 1 / e in a direction parallel to the flow direction of the flow unit. 2 The diameter and the second waist 1 / e defined in a direction perpendicular to the flow direction of the flow unit. 2 Diameter, wherein the second waist 1 / e 2 Its diameter is as high as 210 μm; A substrate defining a tube, wherein each of the collimating assembly, the cylindrical positive lens, the cylindrical negative lens, and the cylindrical objective lens is at least partially disposed within the tube; and At least one cover plate, wherein the at least one cover plate independently fixes the collimation assembly, the cylindrical positive lens, the cylindrical negative lens, and the cylindrical objective lens relative to the substrate.

2. The flow cytometer according to claim 1, wherein, The collimation component further includes: At least two supports are configured to maintain a predetermined axial distance between the laser diode and the collimating lens, wherein the at least two supports are formed of a material having a linear coefficient of thermal expansion, the material maintaining the predetermined axial distance between the laser diode and the collimating lens under temperature variations up to 30ºC.

3. The flow cytometer according to claim 2, wherein, The specified axial distance is maintained under temperature variations ranging from 10°C to 40°C.

4. The flow cytometer according to claim 2, wherein, The first support member is formed of a first material, and the second support member is formed of a different second material.

5. The flow cytometer according to claim 2, wherein, The at least two supports include three supports configured to maintain the specified axial distance, the three supports being formed of materials having a coefficient of thermal expansion that is balanced with each other.

6. The flow cytometer according to claim 5, wherein, The first support member is formed of a first material, the second support member is formed of a second material, and the third support member is formed of a third material, wherein at least two of the first, second, and third materials are different from each other.

7. The flow cytometer according to claim 2, further comprising: The mounting platform includes the cylindrical positive lens, the cylindrical negative lens, the cylindrical objective lens, the collimation assembly, and a housing. The housing supports the flow unit mounted on it to maintain a predetermined axial distance between the flow unit and the lens subassembly. The housing and the mounting platform are formed of a material with a coefficient of thermal expansion that maintains the specified axial distance between the flow unit and the lens subassembly under temperature variations of up to 30ºC.

8. The flow cytometer according to claim 1, wherein, First waist 1 / e 2 The diameter ranges from 6.7 μm to 9 μm.

9. The flow cytometer according to claim 1, wherein, The collimation assembly, the cylindrical positive lens, the cylindrical negative lens, and the cylindrical objective are cooperatively configured such that performance is not degraded even if at least one of the actual radial offset of the core flow within the flow unit is up to 15 μm or the apparent radial offset of the core flow due to the deflection of the laser beam focus is up to 15 μm.

10. The flow cytometer according to claim 1, wherein, The collimation assembly, the cylindrical positive lens, the cylindrical negative lens, and the cylindrical objective lens are cooperatively configured such that transit time measurements can distinguish particle or cell sizes within 1 μm, even when the change in flow velocity through the flow unit is less than or equal to 2%.

11. A flow cytometer, comprising: Flow unit, the flow unit defining the flow direction; A collimation assembly, comprising a laser diode and a collimating lens configured to generate a laser beam along an axis; Multiple lenses are arranged on the axis and cooperate to project the laser beam onto the flow unit, such that the laser beam incident on the flow unit has a first beam waist of 6.7 μm to 9 μm in a direction parallel to the flow direction of the flow unit. 2 The diameter and such that the laser beam incident on the flow unit has a second beam waist of up to 210 μm in a direction perpendicular to the flow direction of the flow unit. 2 diameter; A substrate defining a tube, wherein the collimating assembly and the plurality of lenses are at least partially disposed within the tube; as well as At least one cover plate, wherein the at least one cover plate independently fixes the collimation assembly and the plurality of lenses relative to the substrate.

12. The flow cytometer according to claim 11, wherein, The plurality of lenses includes at least one of the following: a cylindrical positive lens, a cylindrical negative lens, or a cylindrical objective lens.

13. The flow cytometer according to claim 11, wherein, The plurality of lenses includes each of a cylindrical positive lens, a cylindrical negative lens, and a cylindrical objective lens.

14. The flow cytometer according to claim 13, wherein, The cylindrical positive lens is positioned closer to the collimating assembly, the cylindrical objective lens is positioned closer to the flow unit, and the cylindrical negative lens is disposed between the cylindrical positive lens and the cylindrical objective lens.

15. The flow cytometer according to claim 11, further comprising: The mounting platform has the plurality of lenses, the collimation assembly, and a housing, the housing supporting the flow unit mounted on the housing to maintain a predetermined axial distance between the flow unit and the plurality of lenses.

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