Patent Publication Number: US-11662542-B2

Title: Flow cytometer, laser optics assembly thereof, and methods of assembling the same

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 16/367,816, filed on Mar. 28, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/650,783, titled “FLOW CYTOMETER, LASER OPTICS ASSEMBLY THEREOF, AND METHODS OF ASSEMBLING THE SAME” and filed on Mar. 30, 2018. The entire contents of each of the above-noted applications are hereby incorporated herein by reference. 
    
    
     BACKGROUND 
     Technical Field 
     The present disclosure relates to flow cytometry and, more particularly, to a flow cytometer, a laser optics assembly for a flow cytometer, and methods of assembling the same. 
     Background of Related Art 
     Flow cytometers typically require a laser beam to pass through a relatively narrow sample core stream such that particles flowing through the sample core stream are illuminated by the laser beam, absorbing and scattering the laser light in accordance with the refractive indices, sizes, shapes, and other properties of the particles. For each particle, the light intensities absorbed and scattered are measured. The absorption and scattering measurements are used to identify and quantify particle types and particle characteristics. More recently, time-of-flight measurements have been additionally or alternatively utilized to determine particle types and/or characteristics. 
     As can be appreciated, in order 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 to continuously calibrate the flow cytometer to ensure that environmental factors and/or other variables are not effecting performance. However, while this may be a practical solution in a precision laboratory, it is not practical in many other settings such as, for example, in a practitioner&#39;s office or out in the field. 
     It would therefore be desirable to provide a flow cytometer and laser optics assembly thereof that are capable of withstanding adverse environmental conditions and are relatively insensitive to other variables, such that the flow cytometer and laser optics assembly yield consistent and accurate results without requiring repeated alignment and/or calibration. Methods of assembling the same would also be desirable. 
     SUMMARY 
     The present disclosure provides a flow cytometer and laser optics assembly thereof capable of yielding consistent and accurate results despite exposure to adverse environmental conditions such as, for example, temperature changes within a relatively wide temperature range and/or a relatively large amount of random-axis mechanical vibration. The flow cytometer of the present disclosure is also relatively insensitive to real or apparent core stream shifts, operates without the need for a beam stopper, employs a slowly converging beam along the axis perpendicular to core stream flow, and provides the ability to precisely measure time-of-flight. Methods of assembling the flow cytometer and laser optics assembly are also provided. These and other aspects and features of the present disclosure are detailed below. To the extent consistent, any of the aspects and features detailed herein may be utilized with or without any of the other aspects and features detailed herein, regardless of whether such aspects and features are described together or separately hereinbelow. 
     Provided in accordance with aspects of the present disclosure is a laser optics assembly of 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. The collimation assembly, the first lens, the second lens, and the third lens are secured relative to the base plate to withstand 10 G&#39;s of random-axis mechanical vibration for at least 30 seconds without effecting movement of the collimation assembly, the first lens, the second lens, or the third lens relative to the base plate. 
     In an aspect of the present disclosure, at least one cover plate secures the collimation assembly, the first lens, the second lens, and the third lens relative to the base plate. The at least one cover plate may be bolted to the base plate. 
     In another aspect of the present disclosure, a separate cover plate secures each of the collimation assembly, the first lens, the second lens, and the third lens relative to the base plate. Each of the cover plates may be bolted to the base plate. 
     In still another aspect of the present disclosure, the collimation assembly includes a laser diode and a collimation lens disposed in alignment with the laser diode. 
     In yet another aspect of the present 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. 
     In still yet another aspect of the present disclosure, the first, second, and third lenses are secured within respective first, second, and third lens cradles at least partially disposed within the second, third, and fourth chambers of the barrel of the base plate, respectively. 
     In another aspect of the present disclosure, at least one of the first, second, or third lens cradles includes a finger extending therefrom configured to permit rotational adjustment of the lens cradle within the corresponding chamber during assembly. At least one of the first, second, or third lens cradles may include a finger extending therefrom configured to permit axial adjustment of the lens cradle within the corresponding chamber during assembly. Additionally or alternatively, at least one of the first, second, or third lens cradles may include a finger extending therefrom configured to permit both rotational and axial adjustment of the lens cradle within the corresponding chamber during assembly. 
     In yet another aspect of the present disclosure, the first lens is a positive cylindrical lens, the second lens is a negative cylindrical lens, and the third lens is a cylindrical objective lens. The first, second, and third lens, in such aspects, may be arranged in order along the barrel extending from the collimation assembly. 
     A flow cytometer provided in accordance with aspects of the present disclosure includes a lens sub-assembly including a plurality of lenses arranged along an axis, a flow cell positioned down-axis from the lens sub-assembly, and a collimation sub-assembly positioned up-axis from the lens sub-assembly. The collimation sub-assembly 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 prescribed axial distance between the laser diode and the collimating lens. The at least two supports are formed from materials having coefficients of thermal expansion that balance each other such that the prescribed axial distance is maintained through a temperature variation of up to 30° C. 
     In an aspect of the present disclosure, the temperature variation is from 10° C. to 40° C. 
     In another aspect of the present disclosure, the first support is formed from PEEK and the second support is formed from brass. 
     In still another aspect of the present disclosure, three supports configured to maintain the prescribed axial distance are formed from materials having coefficients of thermal expansion that balance each other. In such aspects, the first support may be formed from PEEK, the second support may be formed from brass, and the third support may be formed from aluminum. 
     In yet another aspect of the present disclosure, the flow cytometer further includes a mounting platform having the lens sub-assembly, the collimation sub-assembly, and a housing supporting the flow cell mounted thereon to maintain a prescribed axial distance between the flow cell and the lens sub-assembly. In such aspects, the housing and the mounting platform are formed from materials having coefficients of thermal expansion that balance each other such that the prescribed axial distance between the flow cell and the lens sub-assembly is maintained through a temperature variation of up to 30° C. 
     In still yet another aspect of the present disclosure, the housing is formed from a copolyester and the mounting platform is formed from aluminum. 
     A method of assembling a laser optics assembly of a flow cytometer provided in accordance with aspects of the present disclosure includes securing a collimation assembly at least partially within a barrel of a base plate. The collimation assembly includes a laser diode and a collimating lens configured to produce 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 positioning a third lens at least partially within the barrel of the base plate on the axis, rotationally adjusting the third lens about the axis such that the first beam waist diameter is minimized, securing the third lens relative to the base plate, positioning a first lens at least partially within the barrel of the base plate on the axis, rotationally adjusting the first lens about the axis such that the first beam waist diameter is maintained, securing the first lens relative to the base plate, positioning a second lens at least partially within the barrel of the base plate on the axis, rotationally adjusting the second lens about the axis such that the first beam waist diameter is maintained, axially adjusting the second lens along the axis such that the second beam diameter is set at a desired value, and securing the second lens relative to the base plate. 
     In an aspect of the present disclosure, the third lens is positioned farthest from the collimation assembly, the first lens is positioned closest to the collimation assembly, and the second lens is positioned between the first and third lenses. 
     In another aspect of the present disclosure, the third lens is a cylindrical objective lens, the first lens is a positive cylindrical lens, and the second lens is a negative cylindrical lens. 
     In still another aspect of the present disclosure, the third lens is positioned within a third chamber of the barrel that is configured to axially constrain the third lens and permit rotation of the third lens prior to securing the third lens, the first lens is positioned within a first chamber of the barrel that is configured to axially constrain the first lens and permit rotation of the first lens prior to securing the first lens, and the second lens is positioned within a second chamber of the barrel that is configured to permit rotation and translation of the second lens prior to securing the second lens. 
     In yet another aspect of the present disclosure, the first beam waist has a 1/e 2  diameter of 6.7 μm to 9 μm. 
     In still yet another aspect of the present disclosure, the second beam has a 1/e 2  diameter of 190 μm to 210 μm. More specifically, the second beam may have a 1/e 2  diameter of 200 μm. 
     Another flow cytometer provided in accordance with aspects of the present disclosure includes a flow cell defining a flow direction, a collimation assembly including laser diode and a collimating lens configured to produce a laser beam along an axis, a positive cylindrical lens disposed on the axis and configured to receive the laser beam from the collimation assembly, a negative cylindrical lens disposed on the axis and configured to receive the laser beam from the positive cylindrical lens, a cylindrical objective lens disposed on the axis and configured to receive the laser beam from the negative cylindrical lens and 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 2  diameter in a direction parallel to the flow direction of the flow cell of 6.7 μm to 9 μm and a second beam 1/e 2  diameter in a direction perpendicular to the flow direction of the flow cell of 190 μm to 210 μm. 
     In an aspect of the present disclosure, the first beam waist 1/e 2  diameter and second beam 1/e 2  diameter are selected such that performance is not degraded despite an actual radial core stream shift within the flow cell of up to 15 μm. 
     In another aspect of the present disclosure, the first beam waist 1/e 2  diameter and second beam 1/e 2  diameter are selected such that performance is not degraded despite an apparent radial core stream shift resulting from a shift of a focal point of the laser beam of up to 15 μm. 
     In another aspect of the present disclosure, the first beam waist 1/e 2  diameter is selected such that time of flight measurements are capable of distinguishing particle or cell size to within 1 μm given a flow rate variation through the flow cell of less than or equal to 2%. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Various aspects and features of the presently disclosed flow cytometer and laser optics assembly thereof are described herein with reference to the drawings wherein like reference numerals identified similar or identical elements and: 
         FIG.  1    is a perspective view of a laser optics, flow cell, and sensor module of a flow cytometer provided in accordance with the present disclosure; 
         FIG.  2    is a longitudinal, cross-sectional view of the module of  FIG.  1   ; 
         FIGS.  3  and  4    are respective front and rear perspective views of a laser optics assembly of the module of  FIG.  1   ; 
         FIG.  5    is a perspective, partial cross-sectional view of the laser optics assembly of  FIGS.  3  and  4   ; 
         FIGS.  6  and  7    are respective front and rear perspective views of a collimation sub-assembly of the laser optics assembly of  FIGS.  3  and  4   ; 
         FIG.  8    is a longitudinal, cross-sectional view of the collimation sub-assembly of  FIGS.  6  and  7   ; 
         FIG.  9    is a perspective, partial cross-sectional view of the collimation sub-assembly of  FIGS.  6  and  7   ; 
         FIG.  10    is a perspective view of a lens sub-assembly of the laser optics assembly of  FIGS.  3  and  4   ; 
         FIG.  11    is a transverse, cross-sectional view of the laser optics assembly of  FIGS.  3  and  4    illustrating the lens sub-assembly of  FIG.  10   ; 
         FIGS.  12 - 14    are side view schematic diagrams of the module of  FIG.  1    illustrating axial adjustment of a negative cylindrical lens of the laser optics assembly; and 
         FIGS.  15 - 17    are top view schematic diagrams of the module of  FIG.  1    illustrating axial adjustment of the negative cylindrical lens of the laser optics assembly. 
     
    
    
     DETAILED DESCRIPTION 
     Turning to  FIGS.  1  and  2   , the present disclosure provides a flow cytometer including a laser optics, flow cell, and sensor module shown generally identified by reference numeral  10 . Although not shown, the flow cytometer may also include, for example, an outer housing enclosing the internal operable components of the flow cytometer, an electronics module configured to control module  10  and process test results received therefrom, a sample receiving module configured to receive a sample to be tested, a pump module configured to pump the sample and a sheath fluid into the flow cell assembly  300 , and a waste module configured to enable safe collection of the sample and sheath fluid after testing. Alternatively or additionally, any other suitable modules, components, and/or features for use with module  10  of the flow cytometer of the present disclosure are also contemplated. 
     Continuing with reference to  FIGS.  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 operably positioned relative to laser optics assembly  200 , and a sensor assembly  400  operably positioned relative to laser optics assembly  200  and flow cell assembly  300  for both 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 structures to maintain the relative positions of these assemblies  200 - 400 . 
     As detailed below, module  10  is configured such that the flow cytometer is capable of operating over a wide temperature range such as, for example, 10° C. to 40° C., and to withstand 10 G&#39;s of random-axis vibration for 30 seconds without degradation of performance. Degradation of performance is defined herein as an intensity and/or sensitivity loss of greater than 5%. 
     In addition, as also detailed below, module  10  is configured such that the flow cytometer is: relatively insensitive to real or apparent core stream shifts of, for example, up to a 15 μm radial shift relative to the previously aligned flow axis of the core stream; operates without the need for a beam stopper to block non-scattered laser light from reaching the forward scattering sensors of sensor assembly  400 ; and employs a slowly converging beam along the axis perpendicular to core stream flow that permits the beam to be set at, in embodiments, a 1/e 2  width at the core stream. 
     Further still and, again, as detailed below, module  10  provides the flow cytometer with the ability to measure time-of-flight with a precision of 1 μm for particles in the range of 4 to 16 microns in diameter when the flow rate of the core stream is stable within 2%. 
     Referring to  FIGS.  2 - 5   , laser optics assembly  200  includes a clamp sub-assembly  210 , a collimation sub-assembly  230 , and a plurality of lens sub-assemblies  270 ,  280 ,  290 . Clamp sub-assembly  210  includes a base plate  212  defining at least one pair, e.g., two pairs, of feet  214  along opposed side thereof that including apertures  216  defined therethrough to enable laser optics assembly  200  to be securely mounted onto mounting platform  100  using bolts  110 . Base plate  212  further defines a generally cylindrical barrel  218  that extends along base plate  212  between feet  214 . Barrel  218  defines first, second, third, and fourth chambers  219 ,  221 ,  223 , and  225  aligned along a length of barrel  218 . Chambers  219 ,  221 ,  223 , and  225  are configured to receive collimation sub-assembly  230  and lens sub-assemblies  270 ,  280 ,  290 , respectively, therein. Clamp sub-assembly  210  further includes cover plates  220 ,  222 ,  224 ,  226  configured to be securely mounted onto base plate  212  using bolts  228  to enclose and secure collimation sub-assembly  230  and lens sub-assemblies  270 ,  280 ,  290  within chambers  219 ,  221 ,  223 , and  225 , respectively, and relative to one another. The assembly of collimation sub-assembly  230  and lens sub-assemblies  270 ,  280 ,  290  within clamp sub-assembly  210 , and the alignment thereof, is detailed below. 
     With reference to  FIGS.  6 - 9   , collimation sub-assembly  230  includes a support disc  232 , a support hub  234 , an insert  236 , and a spring washer  237  that are configured to operably engage one another and retain a collimating lens  238  of collimation sub-assembly  230  in position relative to a laser diode  240  of collimation sub-assembly  230 . 
     Support disc  232 , more specifically, defines an outer face  242   a  and an inner face  242   b , and includes a central aperture  244  and a plurality of radial apertures  246  ( FIG.  2   ) defined therethrough between the outer and inner faces  242   a ,  242   b , respectively, thereof. Central aperture  244  defines an outer opening on the outer face side of support disc  232  that is greater than an inner opening of central aperture  244  defined on the inner face side of support disc  232  such that laser diode  240  may be inserted through the outer opening into central aperture  244  but is inhibited from passing though the inner opening. As such, laser diode  240  may be inserted through inner opening of central aperture  244  and seated therein to fix laser diode  240  relative to support disc  232 . Laser diode  240  includes suitable electrical connectors  241  that enable connection thereof to power and control electronics (not shown). Laser diode  240  may be configured to emit red light having a wavelength in the range of 630-665 nm, or, in embodiments, in the range of 635-650 nm. 
     Support hub  234  defines a generally T-shaped configuration including a disc portion  247  positioned to abut inner face  242   b  of support disc  232  and a body portion  248  extending from disc portion  247  in an opposite direction from support disc  232 . A central lumen  250  extends through both disc portion  247  and body portion  248  and a plurality of radial bores  252  ( FIG.  2   ) are defined within disc portion  247 . Threading  254  is disposed on at least a portion of the internal surface of support hub  234  that defines lumen  250 . 
     Insert  236  defines a generally cylindrical configuration defining an internal passage  256  therethrough. Insert  236  further includes threading  258  disposed on at least a portion of the external surface thereof that is configured to engage threading  254  of support hub  234 . Insert  236 , more specifically, is configured to retain collimating lens  238  within passage  256  thereof, e.g., using an adhesive, and is configured for positioning within central lumen  250  of support hub  234  in threaded engagement therewith. Spring washer  237  is configured for positioning within central lumen  250  between insert  236  and support disc  232  to maintain tension therebetween. 
     Continuing with reference to  FIGS.  6 - 9   , to assemble collimation sub-assembly  230 , laser diode  240  is secured within support disc  232  and collimating lens  238  is secured within inset  236 . Insert  236  is then threaded into engagement within central lumen  250  of support hub  234 . With laser diode  240  secured within support disc  232  and insert  236  (securing collimating lens  238  therein) engaged within support hub  234 , support disc  232  and support hub  234  are positioned relative to one another such that inner face  242   b  of support disc  232  abuts support hub  234 , central aperture  244  of support disc  232  is aligned with central lumen  250  of support hub  234 , and radial apertures  246  of support disc  232  are aligned with corresponding radial bores  252  of support hub  234  (see  FIG.  2   ). A fixture (not shown) may be utilized to maintain support disc  232  and support hub  234  in this position and to facilitate alignment thereof, as detailed below. 
     With support disc  232  and support hub  234  positioned as detailed above, bolts  260  are inserted through radial apertures  246  and into engagement within radial bores  252 , e.g., via threaded engagement, to secure support disc  232  and support hub  234  relative to one another (see  FIG.  2   ). Position adjustments, e.g., vertical and/or horizontal adjustment, between support disc  232  and support hub  234  may be made before or after engagement of each bolt  260  via, for example, adjustment knobs (not shown) associated with the fixture, in order to align laser diode  240  relative to collimating lens  238  such that a beam emitted from laser diode  240  is both well-collimated and pointing in a direction co-axial with the optical axis of collimating lens  238 . A reversed beam expander (not shown) associated with the fixture may also be utilized to verify this alignment. 
     In order to adjust the axial distance between collimating lens  238  and laser diode  240 , insert  236  is threaded into or out of central lumen  250  of support hub  234 , thereby moving collimating lens  238  towards or away from laser diode. The reversed beam expander (not shown) may again be utilized to ensure the prescribed axial distance between collimating lens  238  and laser diode  240  is achieved. With insert  236  threaded to the appropriate position, corresponding to the prescribed axial distance between collimating lens  238  and laser diode  240 , spring washer  237  maintains tension between insert  236  and support disc  232 , thus eliminating any play therebetween and ensuring the prescribed axial distance between collimating lens  238  and laser diode  240  is maintained despite, for example, mechanical vibrations applied to collimation sub-assembly  230 . 
     Once the beam and optical axis of collimating lens  238  and laser diode  240 , respectively, are coaxial with one another, and the beam is collimated, bolts  260  may be appropriately tightened to lock support disc  232  and support hub  234  relative to one another, thereby maintaining the engagement and positioning between support disc  232  and support hub  234  despite, for example, mechanical vibrations applied to collimation sub-assembly  230 . 
     The above-detailed locking of support disc  232  and support hub  234  relative to one another fixes the horizontal, vertical, and axial alignment of collimating lens  238  and laser diode  240  relative to one another to ensure the above-noted alignment. 
     Referring still to  FIGS.  6 - 9   , collimation sub-assembly  230  is configured to maintain the prescribed axial distance between collimating lens  238  and laser diode  240  despite environmental temperature changes. More specifically, collimation sub-assembly  230  is configured to sufficiently maintain the prescribed axial distance between collimating lens  238  and laser diode  240  within a 30° C. range such as, for example, from 10° C. to 40° C., without performance degradation. This is accomplished by forming support hub  234  and insert  236  or, in embodiments, support disc  232 , support hub  234 , and insert  236 , from materials having different coefficients of thermal expansion that maintain the prescribed axial distance between laser diode  240  and collimating lens  238  for the flow cytometer operating across the 10° C. to 40° C. range and, thus, do not degrade performance. In embodiments, this is accomplished by forming support hub  234  from brass (having a linear coefficient of thermal expansion of 1.8×10 −5 ) and insert  236  from PEEK (polyetheretherketone) (having a linear coefficient of thermal expansion of 4.5×10 −5 ), although other suitable materials having linear coefficients of thermal expansion that, in opposition, balance the response of the flow cytometer to temperature fluctuations in the 10° C. to 40° C. range are also contemplated. This balancing includes not only compensating for the linear coefficients of thermal expansion of some of the components of the flow cytometer, but also accounts for temperature-dependent changes in the refractive indices of the optical components of the flow cytometer. “Prescribed axial distance,” as utilized herein, is understood to encompass a range of distances so as to take into account, for example, temperature-dependent changes in the target axial distance between collimating lens  238  and laser diode  240 . This range may include variations from the prescribed axial distance between collimating lens  238  and laser diode  240  of no greater than 0.025% or, in embodiments, no greater than 0.012%. 
     With additional reference to  FIGS.  2 - 5   , in order to assemble collimation sub-assembly  230  with clamp sub-assembly  210 , body portion  248  of support hub  234  of collimation sub-assembly  230  is seated within first chamber  219  of barrel  218  of base plate  212  of clamp sub-assembly  210  and, thereafter, cover plate  220  is positioned about body portion  248  of support hub  234  and engaged with base plate  212  on either side of support hub  234  via bolts  228  to enclose body portion  248  of support hub  234  within first chamber  219  and secure collimation sub-assembly  230  in position relative to base plate  212  under compression. In embodiments, collimation sub-assembly  230  is assembled with clamp sub-assembly  210  prior to assembly of lens sub-assemblies  270 ,  280 ,  290 . Prior to tightening bolts  218 , collimation subassembly  230  is rotated as necessary to ensure that the fast axis of the laser beam is aligned perpendicular to the bottom surface of base plate  212 . 
     Turning to  FIGS.  10  and  11   , in conjunction with  FIGS.  2 - 5   , as noted above, laser optics assembly  200  includes three lens sub-assemblies  270 ,  280 ,  290 . Each lens sub-assembly  270 ,  280 ,  290  includes a lens cradle  272 ,  282 ,  292 , respectively, defining a lens pocket  274 ,  284 ,  294 , respectively, configured to fixedly retain a respective lens  276 ,  286 ,  296  therein. Lens  276  is configured as a positive cylindrical lens and, as part of lens-sub assembly  270 , is configured to be positioned within second chamber  221  of barrel  218  of base plate  212  and secured therein via second cover plate  222  such that positive cylindrical lens  276  is positioned closest to collimation lens  238 . Lens  286  is configured as a negative cylindrical lens and, as part of lens-sub assembly  280 , is configured to be positioned within third chamber  223  of barrel  218  of base plate  212  and secured therein via third cover plate  224  such that negative cylindrical lens  286  is positioned next to positive cylindrical lens  276  on an opposite side thereof relative to collimation sub-assembly  230 . Lens  296  is configured as a cylindrical objective lens and, as part of lens-sub assembly  290 , is configured to be positioned within fourth chamber  225  of barrel  218  of base plate  212  and secured therein via fourth cover plate  226  such that cylindrical objective lens  296  is positioned next to negative cylindrical lens  286  on an opposite side thereof relative to positive cylindrical lens  276 . 
     Each lens cradle  272 ,  282 ,  292  includes a finger  278 ,  288 ,  298  extending radially outwardly therefrom. Fingers  278 ,  288 ,  298  are configured to extend through slots (not explicitly shown) defined within base plate  212  adjacent chambers  221 ,  223 ,  225 , respectively, such that fingers  278 ,  288 ,  298  extend from base plate  212  on an underside thereof. 
     To assemble lens sub-assemblies  270 ,  280 ,  290  within clamp sub-assembly  210 , lenses  276 ,  286 ,  296  are engaged within pockets  274 ,  284 ,  294  of lens cradles  272 ,  282 ,  292 , respectively, and lens cradles  272 ,  282 ,  292  are positioned within chambers  221 ,  223 ,  225 , respectively. Cradles  272  and  292  define thicknesses that generally approximate the widths of chambers  221  and  225 , respectively, and/or include complementary features to maintain cradles  272  and  292  and, thus, lenses  276  and  296 , respectively, in fixed axial position within respective chambers  221  and  225  upon positioning therein. However, fingers  278  and  298  of lens cradles  272 ,  292  may be manipulated to rotate lens cradles  272 ,  292  and, thus, lenses  276  and  296 , respectively, relative to base plate  212 . Lens cradle  282 , on the other hand, defines a reduced thickness relative to the width of chamber  223  such that cradle  282  and, thus, lens  286 , may axially translate along barrel  218  upon corresponding manipulation of finger  288  of lens cradle  282 . Finger  288  may also be manipulated to rotate lens cradle  282  and, thus, lens  286 , relative to base plate  212 . The above-detailed configuration enabling rotational alignment of lenses  276 ,  286 ,  296  and axial positioning of lens  286  is advantageous as these have been found to be important alignments to ensure accurate performance of the flow cytometer. 
     During assembly, once collimation sub-assembly  230  is installed, lens sub-assembly  290  is then inserted into chamber  225 , rotationally adjusted using finger  298 , and secured using cover plate  226  and bolts  228  to fix lens sub-assembly  290  in position relative to base plate  212  under compression. Base plate  212  is configured such that lens sub-assembly  290  is installed at a distance from collimating lens  238  approximately equal to the sum of the focal lengths of the lens  296  and collimating lens  238 . Once lens sub-assembly  290  installed, as detailed above, a verification is conducted to ensure a beam waist 1/e 2  diameter of 6.7 μm to 9 μm, in a direction parallel to a direction along which the core stream flows through flow cell  340  (see  FIG.  2   ), has been achieved. 
     After the assembly and verification of lens sub-assembly  290 , lens sub-assembly  270  is inserted into chamber  221 , rotationally adjusted using finger  278 , and secured using cover plate  222  and bolts  228  to fix lens sub-assembly  270  in position relative to base plate  212  under compression. Positive cylindrical lens  276  of lens sub-assembly  270  is rotationally aligned such that its axis of dioptric power is perpendicular to that of cylindrical objective lens  296 , and this is verified by again confirming that the beam waist 1/e 2  diameter of 6.7 μm to 9 μm, in the parallel to core stream flow direction, is maintained. 
     Next, lens sub-assembly  280  is inserted into chamber  223 , rotationally and/or axially adjusted using finger  288 , and secured using cover plate  224  and bolts  228  and to fix lens sub-assembly  280  in position relative to base plate  212  under compression. Negative cylindrical lens  286  of lens sub-assembly  280  is rotationally aligned so that its axis of dioptric power is perpendicular to that of cylindrical objective lens  296  and parallel to that of positive cylindrical lens  276 , and this is verified by again confirming that the beam waist 1/e 2  diameter of 6.7 μm to 9 μm, in the parallel to core stream flow direction, is maintained. The axial spacing of negative cylindrical lens  286  is adjusted in order to achieve a beam 1/e 2  width of, in embodiments, 190 μm to 210 μm or, in embodiments, 200 μm, in a direction perpendicular to the direction the core stream flows through flow cell  340  (see  FIG.  2   ). 
     Suitable fixturing (not shown) for retaining the various components and facilitating manipulation of fingers  278 ,  288 ,  298  to enable adjustment during assembly may be utilized, as may any suitable test equipment to measure beam width during the above-noted verifications. Once fully assembled and verified as detailed above, laser optics assembly  200  provides a beam waist 1/e 2  diameter of 6.7 μm to 9 μm in the parallel direction and a 1/e 2  beam width of 190 μm to 210 μm (or 200 μm) in the perpendicular direction. 
     Referring to  FIGS.  12 - 17   ,  FIGS.  12 - 14    show the divergence range of different laser diodes relative to the fast (more divergent) axis of the laser diode  240 , which is set parallel to the core stream in flow cell  340  and, as stated previously, perpendicular to the bottom surface of the base plate  212 .  FIGS.  15 - 17    show the divergence range of different laser diodes relative to the slow (less divergent) axis of the laser diode  240 , which is set perpendicular to the core stream in flow cell  340  and parallel to the bottom surface of the base plate  212 . In one embodiment, laser diode  240  (e.g., a Ushio HL6363MG-A laser diode) will have a fast axis divergence greater than its slow axis divergence, but these two divergences are otherwise independent from one another. 
     The fast axis divergence of the laser diode  240  (parallel to the core stream in flow cell  340 ) governs the beam waist at the flow cell core stream in a well-aligned system. The most divergent beam from the laser diode (depicted in  FIG.  12   ) provides the minimum beam waist, 6.7 μm, at the core stream and the least divergent beam (depicted in  FIG.  14   ) laser diode provides the maximum beam waist, 9.0 μm, at the core stream. Again, in a well-aligned system, and regardless of the axial disposition of the negative cylindrical lens  286  within its adjustment range, the beam waist parallel to the core stream is maintained. 
     However, slight changes in the axial position of the negative cylindrical lens  286  allow adjustment of the 1/e 2  width of the laser diode beam (perpendicular to core stream flow) within flow cell  340  to a range of 190 μm to 210 μm or, in embodiments, 200 μm. These axial position changes of a few hundred microns are not perceptible in  FIGS.  15 - 17    but, in all three figures, the 1/e 2  laser beam width is 200 μm perpendicular to and at the core stream center line.  FIG.  15    depicts the most divergent slow axis laser beam, and  FIG.  17    the least divergent slow axis laser beam. In  FIG.  15   , negative cylindrical lens  286  is located farthest from laser diode  240 , while in  FIG.  17   , negative cylindrical lens  286  is located closest to laser diode  240 . Note also that the slow axis axial focus position varies with different slow axis divergences and negative cylindrical lens  286  placements; the slow axis focus is closest to laser diode  240  in  FIG.  15    and farthest from laser diode  240  in  FIG.  17   . 
     As detailed below, laser optics assembly  200 , having such a beam 1/e 2  diameter in the direction perpendicular to the flow of the core stream through flow cell  340  ( FIG.  2   ), is advantageously insensitive to radial core stream shifts (real or apparent) within a 15 μm radius and, thus, radial core stream shifts (real or apparent) within a 15 μm radius do not result in degradation of performance. 
     Turning back to  FIGS.  1 - 5   , the above-detailed assembly of laser optics assembly  200  not only facilitates assembly and alignment, but also provides a configuration wherein collimation sub-assembly  230  and lens sub-assemblies  270 - 290  are individually and independently secured to base plate  212 . This configuration of laser optics assembly  200  has shown the ability to withstand 10 G&#39;s of random-axis vibration for 30 seconds without more than a 5% change in a beam waist 1/e 2  diameter of the laser optics assembly  200 . More specifically, vibration testing was performed with a Qualmark OVTT™ 18 Omni-Axial Vibration Table Top System, available from ESPEC North America Inc. of Denver, Colo., USA. Vibration tests were carried out by securing laser optics assembly  200  onto the vibration table and setting the table to 10 G&#39;s of random-axis mechanical vibration for at least 30 seconds. Acceleration was verified with an Omega™ HHVB82 Accelerometer, available from Omega Engineering, Inc. of Norwalk, Conn., USA. 
     Referring to  FIGS.  1  and  2   , as noted above, flow cell assembly  300  is mounted on mounting platform  100 . Flow cell assembly  300 , more specifically, includes an input  310  coupled to a nozzle  320  defined by a housing  330  for delivering the sample and sheath fluid to nozzle  320 , a flow cell  340  connected downstream of nozzle  320  to receive the sample and sheath (not shown) fluid therefrom, and an output  350  disposed downstream of flow cell  340  to direct the sample and sheath fluid to a suitable collection reservoir after testing. Housing  330  of flow cell assembly  300  is seated within an aperture  120  defined through mounting platform  100  and is fixedly secured to mounting platform  100  using a plurality of bolts  110  to maintain a prescribed distance between flow cell  340  and cylindrical objective lens  296 , which is an important distance to control to ensure accuracy of the flow cytometer. 
     Housing  330  of flow cell assembly  300  and mounting platform  100  are configured to sufficiently maintain the prescribed distance between flow cell  340  and cylindrical objective lens  296  within a 30° C. range such as, for example, from 10° C. to 40° C., without performance degradation. This is accomplished by forming housing  330  of flow cell assembly  300  and mounting platform  100  from materials having different linear coefficients of thermal expansion, configured to maintain a prescribed axial distance between the objective lens  296  and the flow cell  340  across the 10° C. to 40° C. range. In embodiments, this is accomplished by forming housing  330 , which comes in direct contact with the sample, e.g., blood, and sheath fluid and, thus, must also be suitable for such purpose, from Eastman Tritan™ Copolyester MX811 (having a linear coefficient of thermal expansion of 8.0×10 −5 ), available from Eastman Chemical Company of Kingsport, Tenn., USA, and forming mounting platform  100  from aluminum (having a linear coefficient of thermal expansion of 2.38×10 −5 ), although other suitable material combinations having linear coefficients of thermal expansion that, in opposition, balance the response of the flow cytometer to temperature fluctuations in the 10° C. to 40° C. range are also contemplated. This balancing includes not only compensating for the linear coefficients of thermal expansion of some of the components of the flow cytometer, but also accounts for temperature-dependent changes in the refractive indices of the optical components of flow cytometer  10 . Similarly as above, “prescribed axial distance” is understood to encompass a range of distances so as to take into account, for example, temperature-dependent changes in the target axial distance between the objective lens  296  and the flow cell  340 . This range may include variations from the prescribed axial distance between the objective lens  296  and the flow cell  340  of no greater than 0.01% or, in embodiments, no greater than 0.005%. 
     With flow cell assembly  300  mounted on mounting platform  100 , the face of flow cell  340  is not oriented parallel to the planar face of cylindrical objective lens  296  but, rather, is offset an angle of 5° in order to ensure that any specular reflections from it do not couple back into the laser optics. Flow cell  340  is also coated with an anti-reflection coating for similar purposes. 
     Continuing with reference to  FIGS.  1  and  2   , sensor assembly  400  includes a forward scatter sub-assembly  410  and a side scatter sub-assembly  420 . Forward scatter sub-assembly  410  includes a board  412  and a sensor array  414  including an extinction sensor, a forward scatter low angle sensor, and a forward scatter high angle sensor. Side scatter sub-assembly  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 center capture angle of side scatter sub-assembly  420  is 78° from the laser beam direction, instead of a right angle (i.e., 90°), to increase the side-scattering signal. 
     Referring generally to  FIGS.  1 - 2   , the insensitivity of module  10  to radial core stream shifts (real or apparent) within a 15 μm radius, noted above, is described in more detail below. As is traditional, a Cartesian coordinate system is defined wherein the core stream flows in the positive y-axis direction and the laser beam flux points in the positive z-axis direction. By controlling the beam waist 1/e 2  diameter along the y-axis, and also the beam 1/e 2  diameter along the x-axis, it is possible to set elliptical areas within which the maximum intensity of the laser beam does not decrease more than a defined amount. As detailed below, module  10  is configured to remain insensitive to radial core stream shifts (real or apparent) within a 15 μm radius, thus maintaining performance (a decrease in intensity of equal to or less than 5%) despite such radial core stream shifts. 
     The laser beam from laser optics assembly  200  is aligned to the core stream flowing through flow cell  340  of flow cell assembly  300  while the scattered laser light, coming from particles flowing in it, are monitored and converted into electrical signals. The relative position of laser optics assembly  200  and flow cell  340  are adjusted in the x- and z-directions in order to maximize these signals. Prior to completion of this x- and z-axis alignment, the sensors of sensor assembly  400  are aligned horizontally to the laser beam; no further alignment in the y-direction is required. 
     The flow cytometer of the present disclosure makes measurements based on either the maximum scattering signal or the maximum area under the profile of the scattering signal and, thus, 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, the relative intensity is defined in equation (1): 
                 I   z       I     0   ,   z         =       (     1   +       (     z   ⁢     /     ⁢     z   R       )     2       )         -   1     ⁢     /     ⁢   2             
where I 0,z  is the intensity at z=0 (the location of the beam waist, aligned to the center of the core stream), and the z position and Rayleigh range z R  are defined in micrometers (μm). By definition, then, equation (2) is provided:
 
               z   R     =       πω     0   ,   y     2       4   ⁢   λ   ⁢           ⁢     M   2               
where the y-direction beam waist 1/e 2  diameter is ω 0,y , defined in μm, λ=0.64 μm for the nominal laser wavelength, and beam quality factor, M 2 =1.2.
 
     The x-direction beam 1/e 2  diameter being relatively large (e.g., 190 μm to 210 μm), minimizes the effect on the laser beam intensity as a function of distance along the z-axis. However, the x-direction beam diameter does affect how much beam intensity is available to be scattered, if the core stream shifts along the x-axis from its aligned position. In the x-direction, the relative intensity is defined in equation (3): 
                 I   x       I     0   ,   x         =     e       -   8     ⁢       (     x   ⁢     /     ⁢   ω   ⁢           ⁢   x     )     2               
where, similar to Equations (1) and (2), I 0,x  is the intensity at x=0 (again, the center of the beam diameter, aligned to the center of the core stream), and the x position and x-direction beam 1/e 2  diameter co are defined in μm.
 
     The product of Equations (1) and (3), for given beam waist 1/e 2  diameter ω 0,y  and beam 1/e 2  diameter ω x , can be solved for combinations of x and z positions that describe the outer limits of a beam intensity decrease of a considered value, such as 5%. For example, equation (4): 
     
       
         
           
             0.95 
             = 
             
               
                 
                   I 
                   x 
                 
                 * 
                 
                   I 
                   z 
                 
               
               
                 I 
                 0 
                 2 
               
             
           
         
       
     
     Since the beam diameters are defined along orthogonal axes that are equivalent to the coordinate axes, equation (5) holds true:
 
 I   0   =I   0,z   =I   0,x  
 
     According to the above, the beam diameters are selected to ensure that at a radial core stream shift of up to 15 μm, relative to the y-axis, the intensity at that shifted center is maintained within 5% of the original center, to which the system was aligned. These core stream shifts can be real (in the case that the core stream moves radially from its original center) or apparent (in the case that the focal point of the laser optics changes due to shifts in one or more components). 
     Taking into account the above, and also considering that the x-direction extent of the beam 1/e 2  diameter can be limited to mitigate reflections off of the internal edges of flow cell  340 , the 190 μm to 210 μm (or 200 μm) x-direction beam 1/e 2  diameter is selected. Taking into account the above and also considering that the y-direction extent of the beam waist 1/e 2  diameter can be limited to increase the ability to make relevant Time-of-Flight (TOF) measurements, as detailed below, the 6.7 μm to 9 μm y-direction beam waist 1/e 2  diameter is selected. 
     Another important consideration for both beam diameter components (x-axis and y-axis) is that a wider beam diameter spreads the laser power over a larger area. In fact, the beam intensity along a given axis is inversely proportional to its beam diameter along that same axis. Module  10  balances the above-detailed constraints by providing the 190 μm to 210 μm (or 200 μm) x-direction beam 1/e 2  diameter and the 6.7 μm to 9 μm y-direction beam waist 1/e 2  diameter. Thus, a large area within which the core stream may actually or apparently shift is achieved, while the contributions of stray light scattering off flow cell  340  side walls are mitigated. In addition, these balanced constraints allow for precise TOF measurements, as detailed below, and minimize the laser power requirements of module  10 . 
     With respect to TOF measurement, as a cell or particle flows through flow cell  340 , it first encounters increasing laser intensity, until the cell or particle is coincident with the maximum laser intensity, and then the particle encounters 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 laser beam intensity and the particle&#39;s cross-sectional, incremental volume. Thus, by considering how spherical particles of a range of diameters overlap with the laser beam, relative, scaled widths of the different particle overlaps can be compared. And, as long as the cell flow rates remain consistent, TOF will scale accordingly. 
     Based on the above, and utilizing full-width-at-half-maximum (FWHM) changes estimated as the maximum proportional change in the scattering intensity curve width, for a given particle or cell, it can be determined what y-axis beam widths will still allow that particle&#39;s or cell&#39;s diameter to be classified within ±1 μm of its actual diameter, to approximately a 95% confidence level. However, flow rate variability limits the ability to accurately determine a particle&#39;s or cell&#39;s diameter and, thus, must be taken into account. 
     Utilizing the 6.7 μm to 9 μm y-direction beam waist 1/e 2  diameter and controlling flow rate variability to within approximately 2% from the mean, as is provided by the presently-disclosed flow cytometer, enables TOF discrimination between particles or cells (of between 4 and 16 μm in diameter) having diameters differing by at least ±1 μm. Furthermore, periodic flow rate variability may be compensated, for example, using a pressure sensor to detect flow rate variability and, based thereupon, correcting for variations in pulsatile flow (from the pump module pumping the sample and sheath fluid through flow cell  340 ). 
     It is understood that reference to any specific numerical value herein encompasses a range of values to take into account material and manufacturing tolerances generally accepted in the art and/or margins of error of measurement equipment generally accepted in the art. 
     From the foregoing and with reference to the various figure drawings, those skilled in the art will appreciate that certain modifications can also be made to the present disclosure without departing from the scope of the same. While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.