Flow cytometer

Abstract

To improve the structure and operation of subassemblies of a flow cytometer.SOLUTION: A flow cytometer includes: a laser diode-based optical subsystem for impinging a beam of light upon particles passing through a viewing zone; a composite microscope objective for gathering and imaging light scattered from or fluorescence emitted by particles passing through the viewing zone; a fluidic subsystem for supplying a liquid sheath flow to the viewing zone; a peristaltic pump for injecting into the liquid sheath flow a liquid sample flow carrying particles that passes together with the liquid sheath flow through the viewing zone; a multimode optical fiber that receives scattered light and fluorescence from the viewing zone that the composite microscope objective gathers and images; and a wavelength division multiplexer for optically separating into color bands light received via the optical fiber.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Generally, the present disclosure relates to the technical field of flow cytometry, and more particularly, to the structure and operation of an improved flow cytometer having various individual sub-assemblies included therein.

Background Art

[0002] Flow cytometry is a biophysical technique used in cell counting, sorting, biomarker detection, and protein engineering. In flow cytometry, cells suspended in a liquid stream pass through an electronic detection device. Flow cytometry enables simultaneous multi-parameter analysis of the physical and / or chemical characteristics of thousands of cells per second at most.

[0003] Flow cytometry has various applications in the fields of molecular biology, pathology, immunology, plant biology, and marine biology. Also, flow cytometry has a wide range of applications in medicine (especially transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics, and sperm sorting for pre-selecting gender). In marine biology, the autofluorescence properties of photosynthetic plankton can be utilized by flow cytometry in characterizing abundance and community composition. In protein engineering, flow cytometry is used in both yeast display and bacterial display to identify protein variants displayed on the cell surface having desired properties. A common variant form of flow cytometry is to physically sort particles based on their properties, thereby purifying the population of interest.

[0004] The overall flow cytometry system includes the following main components: 1. A flow cell that carries and hydrodynamically aligns cells or particles with a liquid stream, usually called a sheath flow, liquid, or fluid, such that the cells or particles pass through a single file in the flow cell; 2. Detect cells or particles passing through the flow cell, usually, a. An impedance or conductivity measurement subsystem, or b. An optical illumination subsystem having a light detection subsystem A measurement subsystem connected to the flow cell, which is either one of them, and 3. A conversion subsystem for converting the output signal from the measurement subsystem into computer - processable data, and 4. A computer for analyzing the data generated by the conversion subsystem.

[0005] The optical illumination subsystem provides a collimated and then focused light beam, usually a laser beam of a single wavelength, that is incident on a hydrodynamically narrowed liquid stream passing through the flow cell. Thus, the flow cytometer system 1. One or more lamps, for example, mercury or xenon, 2. One or more high - power water - cooled lasers, for example, argon, krypton, or dye lasers, 3. One or more low - power air - cooled lasers, for example, argon (488 nm), helium - neon (red - 633 nm), helium - neon (green), and helium - cadmium (UV), and / or 4. One or more diode lasers (blue, green, red, violet) Having one or more light sources including.

[0006] The light detection subsystem includes one or more detectors that aim at the location where the narrowed liquid stream passes through the light beam. Such detectors are 1. A detector on the same line as the light beam (forward scatter, i.e., FSC), and 2. A detector perpendicular to it (side scatter, i.e., SSC), and 3. A fluorescence detector, and Including.

[0007] Each suspended particle passing through the beam scatters light, and a fluorescent material present within or attached to the particles excited by the incident light emits light at a wavelength longer than that of the incident light. By detecting and analyzing the change in brightness in the combination of scattered light and fluorescence at each detector (one for each peak of fluorescence emission), various types of information about the physical and chemical structure of each individual particle can be derived. FSC correlates with the volume of the cell. SSC is due to the scattered light of the internal components within the cell and depends on the internal complexity of the particle (i.e., the shape of the nucleus, the amount and type of cytoplasmic granules, or the roughness of the membrane). Some flow cytometers omit the fluorescence detector and detect only scattered light. Other flow cytometers form images of the fluorescence, scattered light, and transmitted light of each cell.

[0008] The conversion subsystem of a flow cytometer system, which may include one or more amplifiers that can be either linear or logarithmic, generally includes one or more analog-to-digital converters ("ADCs") for converting the output signal of the measurement subsystem into data that is then computer-processed.

[0009] Modern flow cytometers typically include four or fewer lasers and a number of fluorescence detectors. By increasing the number of lasers and detectors, cell labeling with several different antibodies becomes possible, and the target population can be more accurately identified by its phenotypic markers. Some instruments can further capture digital images of individual cells, enabling the analysis of the position of the fluorescence signal inside or on the surface of the cell.

[0010] Sample Illumination In most instruments, target particles such as blood cells or microspheres are transported into an observation zone inside a cuvette or a jet stream by sheath flow using hydrodynamic focusing and are irradiated there by a focused laser beam. This technique provides a means to accurately identify and count target particles without being hindered by background noise that occurs outside the recording time window (Practical Flow Cytometry, Howard M. Shapiro, Wiley (2003) ISBN 0471411256 (Non-Patent Document 1)). To increase detection sensitivity, the cross-section of the focused laser beam is typically elliptical with a short axis along the flow direction. To maintain threshold integrity, the laser profile needs to have a smooth or bell-shaped profile along the flow direction. One common method for generating such a beam is to elongate a nearly collimated circular Gaussian beam along the flow direction by a beam expander made from a prism or a cylindrical lens pair and then focus the beam by a spherical lens. Since the shape of the beam at the focus is the spatial Fourier transform of the beam in the far field, this generates a Gaussian-shaped elliptical spot with a short axis along the flow.

[0011] Conventional lasers were expensive, bulky, and of low power. More recently, laser diodes (“LDs”) have become available. Unlike conventional lasers, next-generation LDs are cost-effective, compact, and power-saving, and thus the future of next-generation compact biomedical devices is promising. An LD emits light with an elliptical cross-section having a major axis of an ellipse, often called the fast axis, perpendicular to the junction of the LD, and a minor axis of the ellipse, often called the slow axis, parallel to the junction of the LD. Unfortunately, the beam quality of typical LDs, especially along their fast axis, has many insufficient parts, thus hindering their wide adoption in flow cytometry applications.

[0012] Basically, the quality of the LD beam can be improved to a large extent by spatial filtering. When a small pinhole or a single-mode optical fiber is positioned at the focus of a lens so as to receive only the lowest-order spatial mode, the beam passing through the pinhole or the single-mode optical fiber has an almost perfect Gaussian shape. U.S. Patent No. 5,788,927 (Patent Document 1) discloses that such a beam can then be collimated, expanded in the direction of the flow through a cytometer, and finally focused into an elliptical Gaussian beam having a minor axis along the flow direction. However, due to the size of desktop instrumentation, the pinhole diameter is restricted to less than 5 microns. The core size of a visible-wavelength single-mode optical fiber also has a similar dimension. Attempts to manufacture such a highly accurate spatial filter and maintain its long-term stability not only increase the cost of the LD-based laser system but also reduce its reliability.

[0013] More recently, in an effort to reduce side lobes that can occur due to the edge effect of the limited numerical aperture of a collimating lens, U.S. Patent No. 6,713,019 (the “'019 Patent”) (Patent Document 2) discloses rotating the LD by 90° such that the slow axis is parallel to the flow direction. Subsequently, a beam diffusing portion such as a cylindrical concave lens is introduced to diffuse the collimated beam in a direction orthogonal to the flow, and then a beam spot forming portion such as a spherical focusing lens is introduced to form an elliptical spot within the particle observation zone of the cytometer. As described in detail in the '019 Patent (Patent Document 2), the laser beam after the spot forming portion is highly distorted. Specifically, the beam width in the observation zone in the direction orthogonal to the flow is equal to or wider than the width of the flow channel. This not only reduces the amount of laser energy incident on the particles and consequently also reduces the signal intensity, but also increases undesirable background scattering from the interface between the liquid and the flow cell. Instead of rotating the LD, U.S. Patent Nos. 7,385,682 (Patent Document 3) and 7,561,267 (Patent Document 4) disclose using an aspherical lens with a large numerical aperture for collimation of the LD. However, such a design cannot correct the interference fringe effect inherent in the beam profile of the LD. Accordingly, there is currently a need for a simple LD-based optical system for use in a flow cytometer that can reliably generate a focused elliptical beam having a generally Gaussian-like shape along the minor axis and a width along the major axis.

[0014] Observation zone Microscope objective lens Modern flow cytometers include a spatial filter, typically either a mechanical pinhole or a large-core optical fiber, placed at the imaging position of the objective lens to avoid unwanted background light entering the cytometer's detector. Since particles remain within the cytometer's observation zone for a few microseconds, it is necessary to use a microscope objective lens with a large numerical aperture to maximize the light collection efficiency. As disclosed in U.S. Patent No. 4,727,020 (Patent Document 5), it is also desirable to use an objective lens with a large field of view to support multiple spatially separated excitation laser beams within the flow cytometer. To achieve these goals, U.S. Patent Nos. 6,510,007 (Patent Document 6) and 7,110,192 (Patent Document 7) disclose a design for an objective lens that uses an improved apochromat having a substantially hemispherical lens gel-bonded or epoxy-bonded as the optical element closest to the sample, followed by multiple meniscus lenses. Such a microscope objective lens provides both a satisfactory numerical aperture and a field of view, but significantly degrades the image quality, thereby 1. Limiting the effective use of the spatial filter and 2. Having a low ability to distinguish background light. Furthermore, such refractive microscope objective lenses are bulky, costly to manufacture, and often exhibit significant chromatic aberration. To overcome these limitations, Patent Cooperation Treaty (「PCT」) Patent Application No. WO 01 / 27590 (Patent Document 8) discloses an alternative objective lens design based on a spherical concave mirror. This design provides a large numerical aperture and good image quality along the optical axis. However, due to its inferior off-axis characteristics, such a design was not suitable for flow cytometers having multiple spatially separated laser beams.

[0015] Sheath fluid supply The performance of a flow cytometer depends largely on a stable sheath fluid flow. Specifically, a flow cytometer having multiple spatially separated excitation laser beams or performing droplet sorting depends on a constant rate of sheath fluid flow for timing synchronization. As disclosed in U.S. Patent No. 5,245,318 (Patent Document 9), conventional flow cytometers 1. apply a constant air pressure to a sheath fluid reservoir to press the fluid through the flow cell, or 2. use a vacuum pump to suck the fluid from the sheath fluid reservoir through the flow cell to provide a stable sheath fluid flow by using an airtight fluid system.

[0016] These systems are bulky, costly to manufacture, and prone to failure. Recently, U.S. Patent No. 8,187,888 (Patent Document 10) discloses including a sheath fluid subsystem that pumps the sheath fluid from a sheath fluid reservoir into an observation zone and a sheath waste pump that pumps the sheath waste fluid from the observation zone into a waste fluid tank. The disclosed sheath fluid subsystem does not appear to have been used in flow cytometers where speed is critical, but this patent reports that the disclosed sheath fluid subsystem overcomes most of the deficiencies regarding the stability of conventional sheath fluid flow 1. a. one fluid capacitor between the sheath fluid pump and the flow cell, and b. another fluid capacitor between the flow cell and the waste pump, by placing them to attenuate pump pulsations, and 2. a pump controller whose operation responds to a pressure sensor that measures the differential pressure between the inlet and outlet of the flow cell to overcome.

[0017] The disclosed sheath fluid subsystem has other limitations. For example, a pressure sensor placed near the outlet of the flow cell can be a potential source of contamination.

[0018] Peristaltic pump Sample liquid supply A peristaltic pump is a positive displacement pump in which a set of linear or circular moving rollers continuously compress a flexible tube to propel the fluid passing through the tube. Peristaltic pumps are particularly widely used for pumping clean / sterile fluids or aggressive fluids by the pump to avoid cross-contamination of exposed pump parts.

[0019] Conventional peristaltic pumps exhibit pulsation due to a temporary increase in tube volume when the compressed tube expands to return to its original shape each time the roller rolls away from the tube near the pump outlet. Pulsation is not desirable for applications that require a smooth flow. Many attempts have been made to reduce pulsation. For example, U.S. Patent Nos. 3,726,613 (Patent Document 11) and 3,826,593 (Patent Document 12) introduce a cam-type presser that synchronously applies external pressure to the tube to compensate for tube expansion. In U.S. Patent No. 4,834,630 (Patent Document 13), a plurality of tubes attached to segmented rollers are joined together by a T-shaped connector at the pump inlet and outlet such that pulsation from individual tubes is reduced by averaging. U.S. Patent No. 7,645,127 (Patent Document 14) proposes arranging a pump tube with a slightly larger inner diameter near the inlet such that the decompression of the tube near the pump outlet is compensated by the compression of a larger volume of tube near the inlet. Each method either significantly increases the complexity of the peristaltic pump or fails to avoid the influence of pulsation.

[0020] Multicolor fluorescence detection In many multicolor fluorescence detection devices such as flow cytometers (Practical Flow Cytometry, Howard M. Shapiro, Wiley (2003) ISBN 0471411256 (Non-Patent Document 1)), the fluorescence emitted from the object of interest is 1. Condensed by a microscope objective lens, 2. Reimaged through a small pinhole or multimode optical fiber, 3. Then collimated and separated into multiple color bands, 4. Finally detected by a photodetector such as a photomultiplier tube (PMT), PIN photodiode, or avalanche photodiode (APD).

[0021] A photomultiplier tube (PMT) is essentially a special type of electron tube. This device from the "pre-semiconductor era" is bulky and expensive. Furthermore, the PMT had lower quantum efficiency and less reproducible spectral response than silicon-based semiconductor detectors, especially in the biologically important red in the near-infrared spectral region. Despite these drawbacks, the PMT has excellent noise characteristics. For example, the dark current of a typical 13 mm PMT (e.g., R9305, manufactured by Hamamatsu Corporation, Japan) was only 1 nA. In contrast, the dark current of an APD was more than 10 times higher, even when its active area was reduced to 1 / 20 of that of the PMT. As a result, the PMT was, in fact, the low-level photodetector in many commercially available fluorescence detection flow cytometers. The PMT was replaced by APD detectors only in some scientific applications where the event rate was low and the dark current could be distinguished by expensive photon counting techniques (see High-Throughput Flow Cytometric DNA Fragment Sizing, A.V. Orden, R.A. Keller, W.P. Ambrose, Anal. Chem., 2000, 72 (1), p 37-41 (Non-Patent Document 2)). More recently, Geiger-mode APD arrays have also been encouraged as alternatives to PMTs. (e.g., the multi-pixel photon counter manufactured by Hamamatsu Photonics, Japan and the solid-state photomultiplier tube manufactured by SensL Inc., Ireland). However, these detectors also had high dark currents and were non-linear at high event rates.

[0022] The only industry where APDs are widely accepted is optical communication. The active area of the APD is 1 mm 2When reduced to less than that, the corresponding dark current is known to drop to the same level as that of the PMT. In optical communication, the light is a laser beam from a single-mode optical fiber. Such a beam can be easily collimated and then focused to an area much smaller than 1 mm 2 . It should be noted that the color separation device used in the fluorescence detector described in U.S. Patent No. 6,683,314 (Patent Document 15) and its references has almost the same function and architecture as the wavelength division multiplexer (WDM) widely used in optical communication described in U.S. Patent Nos. 4,482,994 (Patent Document 16) and 5,786,915 (Patent Document 17). The basic reason for avoiding the use of a small-area APD in a fluorescence detection device lies in the well-known étendue conservation theorem. That is, the fluorescence emerging through a pinhole or a multimode optical fiber is a distributed light source having an étendue hundreds of times larger than that of a laser beam from a single-mode optical fiber. As a result, as shown in FIG. 26, the beam cannot be collimated over a long distance unless the beam diameter is significantly enlarged. Unfortunately, the larger the beam diameter, the more difficult it is to overcome the technical problems for focusing to a small spot. Since efficient color separation can be economically achieved only by a collimated optical beam, small-area APDs have not been considered suitable for multi-color fluorescence detection applications. There is a clear need for a technology that can collimate an optical beam with a large étendue over a long distance without significantly enlarging the beam diameter. Such a technology would enable the realization of devices such as WDMs for fluorescence detection with characteristics comparable to those of low-noise semiconductor detectors.

Prior Art Documents

Patent Documents

[0023]

Patent Document 1

Patent Document 2

Patent Document 3

[0024] [Non-Patent Document 1] Practical Flow Cytometry, Howard M. Shapiro, Wiley (2003) ISBN 0471411256 [Non-Patent Document 2] High-Throughput Flow Cytometric DNA Fragment Sizing, AV Orden, RA Keller, WP Ambrose, Anal. Chem., 2000, 72 (1), p 37-41 Summary of the Invention

[0025] Disclosure The present disclosure provides an improved flow cytometer that includes a variety of improved components.

[0026] An objective of the present disclosure is to provide a simple and reliable diode laser-based optical system capable of delivering a focused laser beam of elliptical cross section with a Gaussian-like intensity distribution along the minor axis and a width optimized for flow cytometry applications along the major axis.

[0027] An object of the present disclosure is an imaging quality microscope objective that is easy to manufacture, has a long working distance, a large numerical aperture, a wide field of view, and minimal chromatic aberration.

[0028] The objective of the present disclosure is a simple fluidics system for flow cytometers that is not only robust, compact, and easy to manufacture, but also capable of supporting speed-critical applications, such as in instruments with multiple spatially separated excitation laser beams or in droplet sorters.

[0029] The objective of the present disclosure is a simple design for a peristaltic pump that can provide highly desirable, pulse-free liquid flow.

[0030] It is an object of the present disclosure to provide a peristaltic pump with minimal pulsation.

[0031] It is an object of the present disclosure to provide a peristaltic pump that is simple to manufacture and operate.

[0032] An object of the present disclosure is to provide an apparatus capable of collimating an optical beam from a distributed light source over a long distance without significantly expanding the beam diameter. Another object of the present disclosure is to provide a WDM system using the above apparatus for separating an optical beam into a plurality of color bands. Furthermore, an object of the present disclosure is to provide such a WDM system that is compatible with a low-noise semiconductor detector. In addition, due to the diversity of fluorescence probes, an object of the present disclosure is to provide a reconfigurable such WDM system.

[0033] Disclosed herein is a flow cytometer including the following: 1. An LD-based optical subsystem for incident an optical beam on particles passing through an observation zone; 2. A compound microscope objective lens for collecting and imaging light scattered from particles passing through the observation zone or fluorescence emitted by the particles; 3. A fluid subsystem for supplying a sheath fluid flow to the observation zone; 4. A peristaltic pump for injecting a sample fluid flow carrying particles passing through the observation zone together with the sheath fluid flow into the sheath fluid flow; 5. A multimode optical fiber for receiving scattered light and fluorescence from the observation zone that the compound microscope objective lens collects and images; 6. A wavelength division multiplexer for optically separating the light received via the optical fiber into color bands.

[0034] Generally, the LD-based optical subsystem according to the present disclosure for irradiating particles passing through the observation zone of a flow cytometer 1. A laser diode whose slow axis is oriented parallel to the flow direction; 2. A collimating lens for converting the diverging beam from the LD into a collimated elliptical beam having a major axis orthogonal to the flow; 3. A focusing lens system for reducing the laser beam in the observation zone to an optimal width in a direction orthogonal to the flow; 4. Finally, a high-magnification cylindrical focusing element arranged close to the observation zone, with the axis of the cylindrical focusing element orthogonal to the flow direction, and comprising.

[0035] The high-magnification cylindrical focusing element, while maintaining the lateral beam profile, displaces the far-field profile of the LD along its slow axis to its Fourier conjugate in the observation zone along the flow direction, whereby the laser beam profile in the observation zone is optimized for flow cytometry applications.

[0036] The compound microscope objective according to the present disclosure generally 1. a concave spherical mirror, and 2. a transparent aberration compensation plate, and the observation zone of the flow cytometer is arranged between the mirror and the plate. The scattered light and fluorescence emitted from the particles within the observation zone are collected by the mirror and reflected backward toward the compensation plate. The optical aberration caused by the mirror is significantly reduced after the light passes through the compensation plate. In one aspect of the present disclosure, the observation zone is arranged inside a flow cell provided by a rectangular glass cuvette having a small rectangular flow channel through which the particle-carrying liquid flows. The concave mirror is made of an optically transparent material such as glass or optically quality plastic, in a plano-convex shape having a highly reflective coating on the convex surface for internal reflection. The plane of the mirror is either gel-bonded or joined to one side of the cuvette. The planar aspherical compensation plate is made of a transparent material such as glass or optically quality plastic, with the plane gel-bonded or joined to the opposite side of the cuvette. Also, the plano-convex mirror and the aspherical compensation plate can be integrally formed with the cuvette. In yet another aspect of the present disclosure, the observation zone is within a jet flow, and the concave mirror and the compensation plate are both independent of the observation zone, and the mirror is preferably a concave surface mirror.

[0037] A fluid system according to the present disclosure generally includes a sheath fluid reservoir in which a liquid pump draws the sheath fluid. The sheath fluid then flows from the liquid pump to the inlet of a T-junction. One outlet arm of the T-junction is connected to a bypass, returning a portion of the pumped sheath fluid to the sheath fluid reservoir, and the returned sheath fluid flows into the air in the sheath fluid reservoir. The second outlet arm of the T-junction is connected to a sheath path, which includes a particle filter and then a flow cell following a reservoir capsule. Next, the sheath fluid exiting the flow cell flows to a waste tank. The fluid resistance along the bypass is designed to be lower than the fluid resistance along the sheath path. This allows only a small portion of the sheath fluid to pass through the flow cell. Note that a typical sheath flow rate in flow cytometry applications is in the tens of milliliters per minute. Thus, this bypass enables the use of a higher flow rate liquid pump that is not only less expensive and more reliable but also operates at a higher pulsation frequency that makes damping easier. Since the outlet of the bypass path is in communication with the air, this also functions as a large fluid capacitor to significantly reduce the pulsation of the sheath fluid flowing along the sheath path. During operation, the inlet portion of the filter cartridge is filled with air. Thus, the filter cartridge also functions as a fluid capacitor to further reduce the pulsation of the sheath fluid in the flow cell to a negligible level. Due to the large fluid resistance in the flow cell, the air trapped near the inlet of the filter cartridge is compressed. When the liquid pump stops, the compressed air in the filter cartridge pushed back towards the sheath fluid reservoir is stored in the reservoir capsule. The size of the capsule is selected such that the trapped air does not reach the T-junction.

[0038] Generally, a peristaltic pump according to the present disclosure includes a plurality of rollers disposed on an outer periphery of a rotor that moves the rollers circularly within an arcuate curved track of a housing, and compressible tabs against which these rollers compress against the track. In one aspect of the present disclosure, the track of the housing of the peristaltic pump has one recess, whereby each time one of the rollers moves through the recess, the compressible tube is continuously decompressed to full expansion and then compressed to full closure. The position and shape of the recess are maintained such that the total liquid volume within the compressible tube from the recess to the pump outlet is substantially invariant. The effect of tube expansion as the roller moves past the pump outlet is compensated by tube compression as another different roller upstream of the pump outlet moves into the compression portion of the recess. In another aspect of the present disclosure, the track of the pump housing includes a plurality of recesses, and a plurality of rollers are provided upstream of the pump outlet to continuously modify tube compression at a plurality of portions along the compressible tube. The position and shape of the plurality of recesses are designed such that the modification of tube compression at these portions substantially compensates for the effect of tube expansion near the pump outlet. In yet another aspect of the present disclosure, the compressible tube remains fully closed below the rollers except for the inlet and outlet portions. A variable speed motor is used to drive the pump. As the roller reaches the outlet portion, the rotation of the motor is programmably accelerated to compensate for tube expansion.

[0039] A wavelength division multiplexer (WDM) according to the present disclosure generally includes at least two optical elements. The first optical element collimates an optical beam received from a diffuse light source, such as light from a pinhole or from a multimode optical fiber. The first optical element magnifies the diffuse light source, defined, for example, by the core of the pinhole or multimode optical fiber, into an image having a size similar to the effective cross-section of the first optical element, thereby generating a collimated optical beam between the first optical element and its image. The second optical element is positioned near the image and relays the first optical element with unit magnification downstream in the optical path. In this way, the second optical element effectively doubles the collimated optical path length. Additional optical elements in the same 1:1 image relay configuration can also be included in the present disclosure and can further extend the collimated optical path. The cascaded unit magnification image relay architecture of the present disclosure provides a greatly extended collimated optical path length without greatly magnifying the beam. As a result, the well-established WDM technology in the optical communication industry can be easily adapted for fluorescence detection. Specifically, a plurality of color bands present in the optical beam are separated using dichroic filters arranged along the optical path, and the separated light is tightly focused into a small spot compatible with a low-noise semiconductor photodetector.

[0040] In one aspect of the WDM, the first optical element is a lens and the second element is a concave mirror. However, it will also be apparent to those skilled in the art that other types of refractive and / or reflective optical components can be used to achieve the same design goals. Similar to its counterpart in optical communication, the optical path in the WDM of the present disclosure can be folded using dichroic filters. In one aspect of the present disclosure, the optical path is folded into a zigzag configuration. Preferably, to facilitate reliable reconfiguration of the flow cytometer, each dichroic filter is joined to a mechanical holder having a reference plane that is optically parallel to the reflective surface of the filter. As a result, all WDM filters can be accurately positioned along the optical path by referencing the filter holder with respect to a common optical plane.

[0041] In another aspect of the disclosure, the collimated beam passing through the dichroic filter is further split into a plurality of color bands using a secondary dichroic filter. The dichroic filter can be inserted at any location along the elongated collimated beam path obtained by the relay imaging of the disclosure, and thus, for example, the star configuration described in U.S. Patent No. 6,683,314, the branching configuration described in U.S. Patent No. 4,727,020, and other types of WDM optical configurations widely implemented in the optical communication industry. It will be apparent to those skilled in the art that a tightly focused beam can be transmitted to a photodetector using various optical configurations. Instead of the concave mirror, the WDM can be replaced with a curved dichroic filter to further increase the number of color bands selected by the WDM.

[0042] [Invention 1001] A flow cytometer (40) comprising the following: (a) A laser diode ("LD") - based optical subsystem (50) for directing a light beam into an observation zone of a flow cytometer (40) through which a sample fluid carrying particles flows, wherein the sample fluid is hydrodynamically constricted within the observation zone by a sheath fluid flow also passing through the observation zone, and the optical subsystem (50) comprises i. An LD for emitting a diverging light beam from an end face of the LD, the diverging light beam having an elliptical cross - sectional profile having both a major axis and a minor axis; ii. A collimating lens for converting the diverging light beam emitted from the LD into a collimated elliptical light beam, the minor axis of the collimated elliptical light beam being oriented parallel to the direction in which particles pass through the observation zone; iii. A beam compression optical element for reducing the size of the elliptical light beam in the observation zone, whereby the width of the major axis of the elliptical light beam oriented perpendicular to the direction in which particles pass through the observation zone is shorter than the width of the sheath fluid flow. v. A cylindrical focusing element positioned adjacent to the observation zone, with the axis of the cylindrical focusing element oriented perpendicular to the direction in which particles pass through the observation zone, whereby (1) the minor axis of the light beam is focused in the observation zone, and (2) the magnitude of the major axis of the elliptical light beam in the observation zone remains essentially unchanged, a cylindrical focusing element, and an optical subsystem (50) including the same, (b) A compound microscope objective lens (60) for imaging light scattered from particles present in the observation zone and fluorescence emitted by the particles, the compound microscope objective lens (60) comprising i. A concave mirror onto which scattered light and fluorescence are incident, and ii. An aberration correction plate made of an optically transparent material, the aberration correction plate comprising (1) the aberration correction plate having A. The thinnest, and B. A negative optical power, a first zone of the aberration correction plate outside the intermediate zone of the aberration correction plate, and (2) a second zone of the aberration correction plate inside the intermediate zone having a positive optical power, an aspherical lens having the same, with light reflected from the compound microscope objective lens (60) passing through the aberration correction plate, an aberration correction plate, and a compound microscope objective lens (60) including the same, with the observation zone of the flow cytometer (40) disposed between the concave mirror and the aberration correction plate, (c) A fluid subsystem (70) for supplying a non-pulsatile sheath fluid flow to the observation zone, comprising 1. A liquid pump for supplying liquid drawn from a reservoir, and ii. A T-connector having at least one inlet and two outlets, wherein (1) the inlet of the T-connector receives liquid from the liquid pump, (2) A first portion of the liquid received by the inlet flows through a bypass conduit to a first outlet of the outlets and returns to the reservoir, and (3) A second portion of the liquid received by the inlet flows through a particle filter to the observation zone of the flow cytometer (40) via a second outlet of the outlets, A T-shaped connector, A fluid subsystem (70) including (d) A peristaltic pump 80 for supplying a sample liquid carrying particles, wherein the sample liquid is hydrodynamically constricted in the observation zone by a sheath liquid flow, and the peristaltic pump 80 has i. A pump housing having an arcuate curved track formed therein extending between a pump inlet and a pump outlet, ii. A plurality of rollers attached to a rotor, the rollers having a substantially equal angular spacing between each directly adjacent pair of rollers, the rotor being rotatable with the rollers attached to the rotor inside the pump housing, a plurality of rollers, ii. A compressible tube sandwiched between the roller and the arcuate curved track of the pump housing, the arcuate curved track having (1) An outlet portion, when the roller rolls through the outlet portion, the compressible tube adjacent to the roller continuously expands from a fully closed state at the start of the outlet portion to a fully open state at the pump outlet where the contact between the roller and the compressible tube is interrupted, an outlet portion, (2) At least one pump portion along the arcuate curved track between the pump inlet and the pump outlet, the compressible tube being compressed to a fully closed state by at least one of the rollers, at least one pump portion, A compressible tube including A peristaltic pump 80 including (e) A wavelength division multiplexer (WDM(90)) that separates the light beam initially emitted from the observation zone and imaged into an optical fiber by a compound microscope objective lens (60) for transmission to the wavelength division multiplexer 90 (''WDM(90)'') into a plurality of color bands, i. A collimating optical element that magnifies to produce an image substantially the same size as the effective size of the collimating optical element; ii. At least one dichroic filter disposed between the collimating optical element and the image, the at least one dichroic filter separating the collimated light beam into two characteristic color branches; iii. A focusing optical element disposed within one of the branches, the focusing optical element focusing the light beam within the branch into a spot having a diameter of less than 1.0 mm; iv. An image relay optical element disposed near the image generated by the collimating optical element in the other branch, the image relay optical element producing an image of the collimating optical element at substantially unit magnification; The WDM(90) comprising. [Invention 1002] The flow cytometer (40) of Invention 1001, wherein the cuvette has a rectangular shape and the observation zone of the flow cytometer (40) is disposed within a flow path having a rectangular cross-section disposed within the cuvette. [Invention 1003] The flow cytometer (40) of Invention 1001, wherein the cuvette has a tubular cross-section and the observation zone of the flow cytometer (40) is disposed within a flow path having a circular cross-section disposed within the cuvette. [Invention 1004] The flow cytometer (40) of Invention 1001, wherein the sample liquid and the sheath liquid flow form a jet flow within which the observation zone of the flow cytometer (40) is disposed. [Invention 1005] The flow cytometer (40) of the present invention 1002, wherein the cylindrical focusing element is in optical contact with the incident surface of the rectangular cuvette. [The present invention 1006] The flow cytometer (40) of the present invention 1002, wherein the cylindrical focusing element is separated from the rectangular cuvette. [The present invention 1007] The flow cytometer (40) of the present invention 1003, wherein the cylindrical focusing element is separated from the tubular cuvette. [The present invention 1008] The flow cytometer (40) of the present invention 1004, wherein the cylindrical focusing element is separated from the jet stream. [The present invention 1009] The flow cytometer (40) of the present invention 1001, further comprising a polarization adjustment element through which the collimated elliptical light beam passes. [The present invention 1010] The flow cytometer (40) of the present invention 1001, wherein the optical image of the observation zone is formed outside the compound microscope objective lens (60). [The present invention 1011] The flow cytometer (40) of the present invention 1010, wherein the observation zone is arranged in a flow channel contained within a rectangular cuvette made of an optically transparent material. [The present invention 1012] The flow cytometer (40) of the present invention 1011, wherein the concave mirror is a plano-concave rear mirror made of an optically transparent material. [The present invention 1013] The flow cytometer (40) of the present invention 1012, wherein the plane of the plano-concave rear mirror is optically coupled to the plane of the cuvette. [The present invention 1014] The flow cytometer (40) of the present invention 1013, wherein an optical adhesive material realizes the optical coupling. [The present invention 1015] The flow cytometer (40) of the present invention 1013, wherein a refractive index matching gel realizes the optical coupling. [The present invention 1016] The flow cytometer (40) of the present invention 1013, wherein the refractive index matching fluid realizes the optical coupling. [The present invention 1017] The flow cytometer (40) of the present invention 1013, wherein the optical contact joint realizes the optical coupling. [The present invention 1018] The flow cytometer (40) of the present invention 1013, wherein the plano-concave rear mirror is integrally formed with the cuvette means. [The present invention 1019] The flow cytometer (40) of the present invention 1011, wherein the aberration correction plate is a planar aspherical lens. [The present invention 1020] The flow cytometer (40) of the present invention 1019, wherein the plane of the aberration correction plate is optically coupled to the plane of the cuvette on the opposite side of the plano-concave rear mirror. [The present invention 1021] The flow cytometer (40) of the present invention 1020, wherein the refractive index matching gel realizes the optical coupling. [The present invention 1022] The flow cytometer (40) of the present invention 1020, wherein the refractive index matching fluid realizes the optical coupling. [The present invention 1023] The flow cytometer (40) of the present invention 1020, wherein the optical contact joint realizes the optical coupling. [The present invention 1024] The flow cytometer (40) of the present invention 1020, wherein the planar aspherical lens is integrally formed with the cuvette. [The present invention 1025] The flow cytometer (40) of the present invention 1019, wherein the aberration correction plate is separated from the cuvette. [The present invention 1026] The flow cytometer (40) of the present invention 1010, wherein the observation zone is inside the jet flow. [The present invention 1027] The flow cytometer (40) of the present invention 1025, wherein the concave mirror is a surface mirror. [The present invention 1028] The flow cytometer (40) of the present invention 1010, wherein the observation zone is arranged on the surface of a flat transparent substrate. [The present invention 1029] The flow cytometer (40) of the present invention 1028, wherein the concave mirror is a plano-concave rear mirror made of an optically transparent material. [The present invention 1030] The flow cytometer (40) of the present invention 1029, wherein the plane of the plano-concave rear mirror is optically coupled to the flat transparent substrate. [The present invention 1031] The flow cytometer (40) of the present invention 1030, wherein an optical adhesive material realizes the optical coupling. [The present invention 1032] The flow cytometer (40) of the present invention 1030, wherein a refractive index matching gel realizes the optical coupling. [The present invention 1033] The flow cytometer (40) of the present invention 1030, wherein a refractive index matching fluid realizes the optical coupling. [The present invention 1034] The flow cytometer (40) of the present invention 1030, wherein an optical contact joint realizes the optical coupling. [The present invention 1035] The flow cytometer (40) of the present invention 1029, wherein the plano-concave rear mirror is integrally formed with the flat transparent substrate. [The present invention 1036] The flow cytometer (40) of the present invention 1028, wherein the aberration correction plate is separated from the flat transparent substrate. [The present invention 1037] The flow cytometer (40) of the present invention 1001, wherein the particle filter has an inlet for receiving liquid from the T-shaped connector, and the inlet of the particle filter is arranged such that air is confined within the particle filter at the inlet. [The present invention 1038] The flow cytometer (40) of the present invention 1037, wherein air cannot enter the bypass conduit when the liquid pump stops. [The present invention 1039] The flow cytometer (40) of the present invention 1038 further includes a small capsule disposed between the second outlet of the outlets of the T-shaped connector and the particle filter to store the air discharged from the particle filter when the liquid pump stops. [The present invention 1040] The flow cytometer (40) of the present invention 1038 further includes a tube of a certain length disposed between the second outlet of the outlets of the T-shaped connector and the particle filter to store the air discharged from the particle filter when the liquid pump stops. [The present invention 1041] The flow cytometer (40) of the present invention 1038 further includes an adjustment valve disposed in the bypass conduit between the first outlet and the reservoir to limit the liquid flow between the first outlet of the outlets of the T-shaped connector and the reservoir. [The present invention 1042] The flow cytometer (40) of the present invention 1038 further includes an adjustment valve disposed between the second outlet and the observation zone to limit the liquid flow between the second outlet of the outlets of the T-shaped connector and the observation zone. [The present invention 1043] The throughput of the liquid pump of the flow cytometer (40) of the present invention 1038 is adjustable. [The present invention 1044] The arcuate curved track of the pump housing includes at least two pump portions, the arcuate curved track further includes at least one recess portion disposed between the pump portions along the arcuate curved track, and when one of the rollers rolls through the recess portion, the compressible tube in the recess portion is depressurized to full expansion and then compressed to a fully closed state, in the flow cytometer (40) of the present invention 1001. [The present invention 1045] A flow cytometer (40) including a plurality of recessed portions along the arcuate curved track upstream of the pump outlet, wherein an angular interval between a compression portion of the recessed portion adjacent to the pump outlet and an outlet portion of the arcuate curved track is substantially the same as the angular interval between each directly adjacent pair of rollers, the flow cytometer (40) of the present invention 1044. [The present invention 1046] The compression portion of the recessed portion adjacent to the pump outlet has a shape that complements the shape of the outlet portion of the arcuate curved track, and when one of the rollers continuously rolls away from the outlet portion of the arcuate curved track, the total fluid volume inside the portion of the compressible tube extending from the recessed portion to the pump outlet is maintained to be substantially invariant, the flow cytometer (40) of the present invention 1044. [The present invention 1047] The flow cytometer (40) of the present invention 1044 having a plurality of recessed portions respectively scattered between directly adjacent pairs of a plurality of pump portions. [The present invention 1048] (a) The angular interval between adjacent pairs of recessed portions, (b) The angular interval between the outlet portion of the arcuate curved track and the recessed portion adjacent to the outlet portion, both of which are substantially the same as the angular interval between each directly adjacent pair of rollers, the flow cytometer (40) of the present invention 1046. [The present invention 1049] The shapes of the plurality of recessed portions of the arcuate curved track complement the shape of the outlet portion of the arcuate curved track, and when one of the rollers continuously rolls away from the outlet portion of the arcuate curved track, the fluid volume in the portions of the compressible tube in the plurality of recessed portions and the outlet portion is maintained to be substantially invariant, the flow cytometer (40) of the present invention 1047. [The present invention 1050] The flow cytometer (40) of the present invention 1001, wherein the speed of the rotor is programmably controlled so as to vary substantially inversely to the rate of change of the fluid volume in the compressible tube due to the compression change of the compressible tube near the outlet portion of the arcuate curved track. [The present invention 1051] The flow cytometer (40) of the present invention 1001, wherein at least one additional dichroic filter is disposed between the image relay optical element and the image generated by the image relay optical element, and the dichroic filter generates two branches of the light beam having a characteristic color. [The present invention 1052] The flow cytometer (40) of the present invention 1051, wherein another focusing optical element is disposed within one of the branches, and the light beam within the branch is focused into a spot having a diameter of less than 1.0 mm. [The present invention 1053] The flow cytometer (40) of the present invention 1052, wherein a continuous combination of the image relay optical element, the dichroic filter, and the focusing optical element is cascaded to generate additional focused spots having a diameter of less than 1.0 mm for a plurality of color bands of the light beam. [The present invention 1054] The flow cytometer (40) of the present invention 1052, wherein the dichroic filter is assembled using a template including two optically flat glass plates joined in optical contact, and the dichroic filter is joined to a filter holder using the template, whereby the coated filter surface of the dichroic filter is recessed with respect to a reference surface of the filter holder and is optically parallel to the reference surface. [The present invention 1055] The flow cytometer (40) of the present invention 1054, wherein the reference surface of the filter holder is supported by an optically flat surface of a reference block included within a WDM (90), thereby providing consistent optical alignment when installing the dichroic filter within the WDM (90). [Invention 1056] An LD-based optical subsystem (50) for injecting a light beam into an observation zone in which particles are present, (a) an LD for emitting a divergent light beam from an end face of the LD, the divergent light beam having an elliptical cross-sectional profile having both a major axis and a minor axis; (b) a collimating lens for converting the divergent light beam emitted from the LD into a collimated elliptical light beam, the minor axis of the collimated elliptical light beam being oriented parallel to the direction in which particles pass through the observation zone; (c) a beam compression optical element for reducing the size of the elliptical light beam in the observation zone, whereby the width of the major axis of the elliptical light beam oriented perpendicular to the direction in which particles pass through the observation zone is shorter than the width of the sheath liquid flow; (d) a cylindrical focusing element positioned adjacent to the observation zone, the axis of the cylindrical focusing element being oriented perpendicular to the direction in which particles pass through the observation zone, whereby i. the minor axis of the light beam is focused in the observation zone and ii. the size of the major axis of the elliptical light beam in the observation zone remains essentially unchanged, a cylindrical focusing element; comprising an optical subsystem (50). [Invention 1057] An optical subsystem (50) further comprising a cuvette having a rectangular cross-section, the observation zone being disposed within a flow path having a rectangular cross-section disposed within the cuvette, the optical subsystem (50) of Invention 1056. [Invention 1058] An optical subsystem (50) further comprising a cuvette having a tubular cross-section, the observation zone being disposed within a flow path having a circular cross-section disposed within the cuvette, the optical subsystem (50) of Invention 1056. [Invention 1059] The optical subsystem (50) of the present invention 1056, wherein a sample liquid flow and a sheath liquid flow form a jet flow inside which the observation zone is disposed. [The present invention 1060] The optical subsystem (50) of the present invention 1057, wherein the cylindrical focusing element is in optical contact with the incident surface of the rectangular cuvette. [The present invention 1061] The optical subsystem (50) of the present invention 1057, wherein the cylindrical focusing element is separated from the rectangular cuvette. [The present invention 1062] The optical subsystem (50) of the present invention 1058, wherein the cylindrical focusing element is separated from the tubular cuvette. [The present invention 1063] The optical subsystem (50) of the present invention 1059, wherein the cylindrical focusing element is separated from the jet flow. [The present invention 1064] The optical subsystem (50) of the present invention 1056, further comprising a polarization adjustment element through which the collimated elliptical light beam passes. [The present invention 1065] A method for transmitting an elliptical light beam using an LD-based optical subsystem (50), wherein the light beam has a smooth profile at the focus of the minor axis of the light beam disposed in an observation zone through which a sample liquid flows, and the sample liquid is hydrodynamically constricted within the observation zone by a sheath liquid flow that also passes through the observation zone, the method comprising: (a) providing an LD that emits a divergent light beam from an end face of the LD, the divergent light beam having an elliptical cross-sectional profile having both a major axis and a minor axis; (b) incidenting the divergent light beam emitted by the LD on a collimating lens for converting the divergent light beam emitted by the LD into a collimated elliptical light beam, the minor axis of the collimated elliptical light beam being oriented parallel to the direction in which the sample liquid passes through the observation zone; (c) After passing through the collimating lens, the collimated elliptical light beam is incident on a beam compression optical element for reducing the size of the elliptical light beam in the observation zone, whereby the width of the major axis of the elliptical light beam directed perpendicular to the direction in which the sample liquid passes through the observation zone becomes shorter than the width of the sheath liquid flow; (d) After passing through the beam compression optical element, the light beam is incident on a cylindrical focusing element positioned adjacent to the observation zone, the axis of the cylindrical focusing element being directed perpendicular to the direction in which the sample liquid passes through the observation zone, whereby i. the minor axis of the light beam is focused in the observation zone and ii. the size of the major axis of the elliptical light beam in the observation zone remains essentially unchanged; The method comprising; A method comprising. [Invention 1066] The method of Invention 1065, wherein the observation zone is disposed in a flow path having a rectangular cross-section disposed within a cuvette. [Invention 1067] The method of Invention 1065, wherein the observation zone is disposed in a flow path having a circular cross-section disposed within a cuvette. [Invention 1068] The method of Invention 1065, wherein the observation zone is disposed within a jet flow. [Invention 1069] The method of Invention 1066, further comprising the step of establishing an optical contact between the cylindrical focusing element and the entrance surface of the cuvette. [Invention 1070] The method of Invention 1066, further comprising the step of establishing a gap between the cylindrical focusing element and the cuvette. [Invention 1071] The method of Invention 1067, further comprising the step of establishing a gap between the cylindrical focusing element and the cuvette. [Invention 1072] The method of the present invention 1068, further comprising the step of establishing a gap between the cylindrical focusing element and the jet flow. [The present invention 1073] The method of the present invention 1065, further comprising the step of inserting a polarization adjusting element between the collimating lens and the beam compression optical element, whereby the collimated elliptical light beam passes through the polarization adjusting element. [The present invention 1074] A compound microscope objective lens (60) adapted to image light scattered from particles present in an observation zone and fluorescence emitted by the particles, the compound microscope objective lens (60) comprising: (a) A concave mirror onto which scattered light and fluorescence are incident; (b) An aberration correction plate made of an optically transparent material, the aberration correction plate comprising: i. The aberration correction plate, (1) being the thinnest and (2) having a negative optical power, a first zone of the aberration correction plate outside the intermediate zone of the aberration correction plate; ii. A second zone of the aberration correction plate inside the intermediate zone, having a positive optical power; and an aspherical lens having the above, and light reflected from the compound microscope objective lens (60) passes through the aberration correction plate; an aberration correction plate, and the compound microscope objective lens (60) includes the above, and the observation zone is disposed between the concave mirror and the aberration correction plate. [The present invention 1075] The compound microscope objective lens (60) of the present invention 1074, wherein an optical image of the observation zone is formed outside the compound microscope objective lens (60). [The present invention 1076] The compound microscope objective lens (60) of the present invention 1075, wherein the observation zone is disposed in a flow channel included in a rectangular cuvette made of an optically transparent material. [The present invention 1077] The compound microscope objective lens (60) of the present invention 1076, wherein the concave mirror is a plano-concave rear mirror made of an optically transparent material. [The present invention 1078] The compound microscope objective lens (60) of the present invention 1077, wherein the plane of the plano-concave mirror is optically coupled to the plane of the cuvette. [The present invention 1079] The compound microscope objective lens (60) of the present invention 1078, wherein an optical adhesive material realizes the optical coupling. [The present invention 1080] The compound microscope objective lens (60) of the present invention 1078, wherein a refractive index matching gel realizes the optical coupling. [The present invention 1081] The compound microscope objective lens (60) of the present invention 1078, wherein a refractive index matching fluid realizes the optical coupling. [The present invention 1082] The compound microscope objective lens (60) of the present invention 1078, wherein an optical contact joint realizes the optical coupling. [The present invention 1083] The compound microscope objective lens (60) of the present invention 1078, wherein the plano-concave mirror is integrally formed with the cuvette means. [The present invention 1084] The compound microscope objective lens (60) of the present invention 1076, wherein the aberration correction plate is a plano-aspherical lens. [The present invention 1085] The compound microscope objective lens (60) of the present invention 1084, wherein the plane of the aberration correction plate is optically coupled to the plane of the cuvette on the opposite side of the plano-concave mirror. [The present invention 1086] The compound microscope objective lens (60) of the present invention 1085, wherein a refractive index matching gel realizes the optical coupling. [The present invention 1087] The compound microscope objective lens (60) of the present invention 1085, wherein a refractive index matching fluid realizes the optical coupling. [The present invention 1088] The compound microscope objective lens (60) of the present invention 1085, wherein an optical contact joint realizes the optical coupling. [The present invention 1089] The compound microscope objective lens (60) of the present invention 1085, wherein the plano-aspherical lens is integrally formed with the cuvette. [The present invention 1090] The compound microscope objective lens (60) of the present invention 1084, wherein the aberration correction plate is separated from the cuvette. [The present invention 1091] The compound microscope objective lens (60) of the present invention 1075, wherein the observation zone is inside the jet flow. [The present invention 1092] The compound microscope objective lens (60) of the present invention 1090, wherein the concave mirror is a surface mirror. [The present invention 1093] The compound microscope objective lens (60) of the present invention 1075, wherein the observation zone is arranged on the surface of a flat transparent substrate. [The present invention 1094] The compound microscope objective lens (60) of the present invention 1093, wherein the concave mirror is a plano-concave rear surface mirror made of an optically transparent material. [The present invention 1095] The compound microscope objective lens (60) of the present invention 1094, wherein the plane of the plano-concave rear surface mirror is optically coupled to the flat transparent substrate. [The present invention 1096] The compound microscope objective lens (60) of the present invention 1095, wherein an optical adhesive material realizes the optical coupling. [The present invention 1097] The compound microscope objective lens (60) of the present invention 1095, wherein a refractive index matching gel realizes the optical coupling. [The present invention 1098] The compound microscope objective lens (60) of the present invention 1095, wherein a refractive index matching fluid realizes the optical coupling. [The present invention 1099] The compound microscope objective lens (60) of the present invention 1095, wherein an optical contact bonding realizes the optical coupling. [The present invention 1100] The compound microscope objective lens (60) of the present invention 1094, wherein the plano-concave rear surface mirror is integrally formed with the flat transparent substrate. [The present invention 1101] The compound microscope objective lens (60) of the present invention 1093, wherein the aberration correction plate is separated from the flat transparent substrate. [The present invention 1102] A method for characterizing microscopic species using a microscope objective lens device comprising the following: (a) A concave mirror, and (b) An aberration correction plate made of an optically transparent material, wherein the thickness of the plate means is thinnest, and which has a negative optical power, a zone of the plate means outside the intermediate zone, and a zone of the plate means inside the intermediate zone having a positive optical power, the aberration correction plate being an aspherical lens, and (c) An observation zone disposed between the concave mirror and the aberration correction plate. [The present invention 1103] The method of the present invention 1102, wherein an optical image of the observation zone is formed outside the device. [The present invention 1104] The method of the present invention 1103, wherein the observation zone is disposed within a flow channel contained within rectangular cuvette means made of an optically transparent material. [The present invention 1105] The method of the present invention 1104, wherein the concave mirror is a plano-concave rear mirror made of an optically transparent material. [The present invention 1106] The method of the present invention 1105, wherein the plane of the plano-concave rear mirror means is optically coupled to the plane of the cuvette means. [The present invention 1107] The method of the present invention 1106, wherein an optical adhesive material realizes the optical coupling. [The present invention 1108] The method of the present invention 1106, wherein a refractive index matching gel realizes the optical coupling. [The present invention 1109] The method of the present invention 1106, wherein a refractive index matching fluid realizes the optical coupling. [The present invention 1110] The method of the present invention 1106, wherein an optical contact joint realizes the optical coupling. [The present invention 1111] The method of the present invention 1106, wherein the plano-concave rear mirror is integrally formed with the cuvette. [The present invention 1112] The method of the present invention 1104, wherein the aberration correction plate is a plano-aspherical lens. [The present invention 1113] The method of the present invention 1112, wherein the plane of the aberration correction plate is optically coupled to the plane of the cuvette means on the opposite side of the concave mirror. [The present invention 1114] The method of the present invention 1113, wherein the refractive index matching gel realizes the optical coupling. [The present invention 1115] The method of the present invention 1113, wherein the refractive index matching fluid realizes the optical coupling. [The present invention 1116] The method of the present invention 1113, wherein the optical contact bonding realizes the optical coupling. [The present invention 1117] The method of the present invention 1113, wherein the plano-aspherical lens is integrally formed with the cuvette. [The present invention 1118] The method of the present invention 1112, wherein the aberration correction plate is separated from the cuvette. [The present invention 1119] The method of the present invention 1102, wherein the observation zone is inside the jet flow. [The present invention 1120] The method of the present invention 1119, wherein the concave mirror is a surface mirror. [The present invention 1121] The method of the present invention 1102, wherein the observation zone is arranged on the surface of a flat transparent substrate. [The present invention 1122] The method of the present invention 1121, wherein the concave mirror is a plano-concave rear mirror made of an optically transparent material. [The present invention 1123] The method of the present invention 1122, wherein the plane of the plano-concave rear mirror means is optically coupled to the flat transparent substrate. [The present invention 1124] The method of the present invention 1123, wherein the optical adhesive material realizes the optical coupling. [The present invention 11 The method of the present invention 1123, in which an index matching gel realizes the optical coupling. [The present invention 1126] The method of the present invention 1123, in which an index matching fluid realizes the optical coupling. [The present invention 1127] The method of the present invention 1123, in which an optical contact joint realizes the optical coupling. [The present invention 1128] The method of the present invention 1122, in which the plano-concave back mirror is integrally formed with the flat transparent substrate. [The present invention 1129] The method of the present invention 1128, in which the aberration correction plate is separated from the flat transparent substrate. [The present invention 1130] A fluid subsystem (70) for supplying a non-pulsating liquid flow to an outlet of the fluid subsystem (70), comprising: (a) a liquid pump for supplying liquid drawn from a reservoir; (b) a T-shaped connector having at least one inlet and two outlets, wherein: i. the inlet of the T-shaped connector receives liquid from the liquid pump; ii. a first portion of the liquid received by the inlet flows through a bypass conduit to a first outlet of the outlets and returns to the reservoir; and iii. a second portion of the liquid received by the inlet flows through a particle filter to a second outlet of the outlets and to the outlet of the fluid subsystem (70); the T-shaped connector; and the fluid subsystem (70). [The present invention 1131] The fluid subsystem (70) of the present invention 1130, wherein the particle filter has an inlet for receiving liquid from the T-shaped connector, and the inlet of the particle filter is arranged such that air is trapped within the particle filter at the inlet. [The present invention 1132] The fluid subsystem (70) of the present invention 1131, wherein air cannot enter the bypass conduit when the liquid pump stops. [The present invention 1133] The fluid subsystem (70) of the present invention 1132, further comprising a small capsule disposed between a second outlet of the outlets of the T-shaped connector and the particle filter for storing air discharged from the particle filter when the liquid pump stops. [The present invention 1134] The fluid subsystem (70) of the present invention 1132, further comprising a tube of a certain length disposed between a second outlet of the outlets of the T-shaped connector and the particle filter for storing air discharged from the particle filter when the liquid pump stops. [The present invention 1135] The fluid subsystem (70) of the present invention 1132, further comprising a regulating valve disposed in the bypass conduit between the first outlet and the reservoir to restrict liquid flow between the first outlet of the T-shaped connector and the reservoir. [The present invention 1136] The fluid subsystem (70) of the present invention 1132, further comprising a regulating valve disposed between the second outlet of the outlets of the T-shaped connector and the outlet of the fluid subsystem (70) to restrict liquid flow therebetween. [The present invention 1137] The fluid subsystem (70) of the present invention 1132, wherein the throughput of the liquid pump is adjustable. [The present invention 1138] A method for supplying a liquid flow without pulsation to the outlet of the fluid subsystem (70), comprising: (a) a liquid pump for supplying liquid drawn from a reservoir; (b) a T-shaped connector having at least one inlet and two outlets, wherein: i. the inlet of the T-shaped connector receives liquid from the liquid pump; ii. A first portion of the liquid received by the inlet flows through a bypass conduit to a first outlet of the outlets and returns to the reservoir, and iii. A second portion of the liquid received by the inlet flows through a particle filter to a second outlet of the outlets and to the outlet of the fluid subsystem (70), a T-shaped connector, and A method comprising. [Invention 1139] The method of Invention 1138, wherein during normal operation, an amount of air is trapped near the inlet of the filter cartridge means. [Invention 1140] The reservoir means holds a sufficient amount of liquid so that a portion of the tube between the T-shaped means and the reservoir means is still filled with liquid when the pump means stops, preventing the trapped air from leaking into the bypass means. The method of Invention 1139. [Invention 1141] The method of Invention 1140, wherein the reservoir means is a capsule. [Invention 1142] The method of Invention 1140, wherein the reservoir means is a portion of a tube. [Invention 1143] The method of Invention 1140, wherein adjustable flow restrictor means is disposed within the bypass path. [Invention 1144] The method of Invention 1140, wherein adjustable flow restrictor means is disposed within the sheath path. [Invention 1145] The method of Invention 1140, wherein the throughput of the sheath pump is adjustable. [Invention 1146] (a) A pump housing having an arcuate curved track formed therein that extends between a pump inlet and a pump outlet, (b) A plurality of rollers attached to the rotor, wherein the rollers have substantially equal angular intervals between each pair of directly adjacent rollers, and the rotor is rotatable together with the rollers attached to the rotor inside the pump housing, the plurality of rollers; (c) A compressible tube sandwiched between the roller and the arcuate curved track of the pump housing, wherein the arcuate curved track is i. An outlet portion, when the roller rolls through the outlet portion, the compressible tube adjacent to the roller continuously expands from a completely closed state at the beginning of the outlet portion to a completely open state at the pump outlet where the contact between the roller and the compressible tube is interrupted, the outlet portion; ii. At least one pump portion along the arcuate curved track between the pump inlet and the pump outlet, wherein the compressible tube is compressed to a completely closed state by at least one of the rollers, at least one pump portion; including a compressible tube; including a peristaltic pump 80. [Invention 1147] The arcuate curved track of the pump housing includes at least two pump portions, the arcuate curved track further includes at least one recessed portion disposed between the pump portions along the arcuate curved track, and when one of the rollers rolls through the recessed portion, the compressible tube in the recessed portion is decompressed to a fully expanded state and then compressed to a completely closed state, the peristaltic pump 80 of Invention 1146. [Invention 1148] A peristaltic pump 80 including a plurality of recessed portions along the arcuate curved track upstream of the pump outlet, wherein the angular interval between the compression portion of the recessed portion adjacent to the pump outlet and the outlet portion of the arcuate curved track is substantially the same as the angular interval between each pair of directly adjacent rollers, the peristaltic pump 80 of Invention 1147. [Invention 1149] The compression part of the recessed part adjacent to the pump outlet has a shape that complements the shape of the outlet part of the arcuate curved track, and when one of the rollers continuously rolls away from the outlet part of the arcuate curved track, the total fluid volume inside the part of the compressible tube extending from the recessed part to the pump outlet is maintained to be substantially unchanged, the peristaltic pump 80 of the present invention 1147. [The present invention 1150] The peristaltic pump 80 of the present invention 1147, which has a plurality of recessed parts scattered between pairs directly adjacent to a plurality of pump parts. [The present invention 1151] (a) The angular interval between pairs of adjacent recessed parts, (b) The angular interval between the outlet part of the arcuate curved track and the recessed part adjacent to the outlet part, Both of which are substantially the same as the angular interval between each pair of directly adjacent rollers, the peristaltic pump 80 of the present invention 1149. [The present invention 1152] The shapes of the plurality of recessed parts of the arcuate curved track complement the shape of the outlet part of the arcuate curved track, and when one of the rollers continuously rolls away from the outlet part of the arcuate curved track, the fluid volume in the parts of the compressible tube at the plurality of recessed parts and the outlet part is maintained to be substantially unchanged, the peristaltic pump 80 of the present invention 1150. [The present invention 1153] The speed of the rotor is programmably controlled so as to vary substantially inversely to the rate of change of the fluid volume in the compressible tube due to the compression change of the compressible tube near the outlet part of the arcuate curved track, the peristaltic pump 80 of the present invention 1146. [The present invention 1154] A method for delivering a liquid using a peristaltic pump 80 including the following: (a) A pump housing having an arcuate curved track, (b) A plurality of rollers attached to rotor means rotatable inside the pump housing, (c) the plurality of rollers spaced apart from each other at substantially equal angular intervals; (d) a compressible tube sandwiched between the roller and the arcuate curved track of the pump housing; (e) the arcuate curved track of the pump housing including an outlet portion, wherein when one of the rollers rolls away from the compressible tube at the pump outlet, the compressible tube is continuously decompressed to full expansion, the arcuate curved track; (f) at least one pump portion along the arcuate curved track of the pump housing between the pump inlet and the pump outlet, wherein the compressible tube is compressed to full closure by one of the rollers, at least one pump portion. [Invention 1155] The arcuate curved track of the pump housing includes at least two pump portions, the arcuate curved track further includes at least one recessed portion disposed between the pump portions along the arcuate curved track, and when one of the rollers rolls through the recessed portion, the compressible tube in the recessed portion is decompressed to full expansion and then compressed to a fully closed state, the method of Invention 1154. [Invention 1156] The peristaltic pump 80 includes a plurality of recessed portions along the arcuate curved track upstream of the pump outlet, and the angular interval between the compression portion of the recessed portion adjacent to the pump outlet and the outlet portion of the arcuate curved track is substantially the same as the angular interval between each directly adjacent pair of rollers, the method of Invention 1155. [Invention 1157] The compression portion of the recessed portion adjacent to the pump outlet has a shape that complements the shape of the outlet portion of the arcuate curved track, and when one of the rollers continuously rolls away from the outlet portion of the arcuate curved track, the total fluid volume inside the portion of the compressible tube extending from the recessed portion to the pump outlet is maintained to be substantially invariant, the method of Invention 1155. [Invention 1158] The method of the present invention 1155 having a plurality of recessed portions scattered between pairs directly adjacent to a plurality of pump portions. [The present invention 1159] (a) The angular interval between pairs of adjacent recessed portions, and (b) The angular interval between the outlet portion of the arcuate curved track and the recessed portion adjacent to the outlet portion, and both are substantially the same as the angular interval between each pair of directly adjacent rollers, the method of the present invention 1157. [The present invention 1160] The shape of the plurality of recessed portions of the arcuate curved track complements the shape of the outlet portion of the arcuate curved track, and when one of the rollers rolls continuously away from the outlet portion of the arcuate curved track, the fluid volume in the plurality of recessed portions and the portion of the compressible tube at the outlet portion is maintained to be substantially invariant, the method of the present invention 1158. [The present invention 1161] The speed of the rotor of the peristaltic pump 80 is programmably controlled so as to vary substantially inversely to the rate of change of the fluid volume in the compressible tube due to the compression change of the compressible tube near the outlet portion of the arcuate curved track, the method of the present invention 1154. [The present invention 1162] A WDM (90) for separating an emitted light beam into a plurality of color bands, (a) A collimating optical element that magnifies to generate an image having substantially the same size as the effective size of the collimating optical element, and (b) At least one dichroic filter disposed between the collimating optical element and the image, the at least one dichroic filter separating the collimated light beam into two branch paths of characteristic colors, and (c) A focusing optical element disposed in one of the branch paths, the light beam in the branch path being focused by the focusing optical element into a spot having a diameter of less than 1.0 mm, the focusing optical element (d) An image relay optical element disposed near an image generated by the collimating optical element in a branch of the other party, the image relay optical element generating an image of the collimating optical element substantially at the same magnification, and A WDM (90) including. [Invention 1163] The WDM (90) of Invention 1162, wherein at least one additional dichroic filter is disposed between the image relay optical element and the image generated by the image relay optical element, and the dichroic filter generates two branches of the optical beam having characteristic colors. [Invention 1164] The WDM (90) of Invention 1163, wherein another focusing optical element is disposed in one of the branches, and the optical beam in the branch is focused into a spot having a diameter of less than 1.0 mm. [Invention 1165] The WDM (90) of Invention 1164, wherein a continuous combination of the image relay optical element, the dichroic filter, and the focusing optical element is cascaded to generate additional focused spots having a diameter of less than 1.0 mm for a plurality of color bands of the optical beam. [Invention 1166] The WDM (90) of Invention 1164, wherein the dichroic filter is assembled using a template including two optically flat glass plates joined together in optical contact, and the dichroic filter is joined to a filter holder using the template, whereby the coated filter surface of the dichroic filter is recessed with respect to a reference surface of the filter holder and is optically parallel to the reference surface. [Invention 1167] The WDM (90) of Invention 1166, wherein the reference surface of the filter holder is supported by an optically flat surface of a reference block included in the WDM (90), thereby providing consistent optical alignment when installing the dichroic filter in the WDM (90). [Invention 1168] A method for separating an emitted optical beam into color bands using a WDM (90) comprising: (a) a collimating optical element that magnifies to produce an image substantially the same size as the effective size of the collimating optical element; (b) at least one dichroic filter disposed between the collimating optical element and the image, the at least one dichroic filter separating the collimated optical beam into two characteristic color branches; (c) a focusing optical element disposed within one of the branches, the focusing optical element focusing the optical beam within the branch into a spot having a diameter of less than 1.0 mm; (d) an image relay optical element disposed near the image produced by the collimating optical element in the other branch, the image relay optical element producing a substantially same magnification image of the collimating optical element's image. [Invention 1169] The method of Invention 1168, wherein at least one additional dichroic filter is disposed between the image relay optical element and the image produced by the image relay optical element, the dichroic filter producing two branches of the optical beam having characteristic colors. [Invention 1170] The method of Invention 1169, wherein another focusing optical element is disposed within one of the branches to focus the optical beam within the branch into a spot having a diameter of less than 1.0 mm. [Invention 1171] The method of Invention 1170, wherein a continuous combination of the image relay optical element, dichroic filter, and focusing optical element is cascaded to produce additional focused spots having a diameter of less than 1.0 mm for a plurality of color bands of the optical beam. [Invention 1172] The method of the present invention 1170, wherein the dichroic filter is assembled using a template including two optically flat glass plates joined together by optical contact, and the dichroic filter is joined to a filter holder using the template, whereby the coated filter surface of the dichroic filter is recessed with respect to the reference surface of the filter holder and is optically parallel to the reference surface. [The present invention 1173] The method of the present invention 1172, wherein the reference surface of the filter holder is supported by the optically flat surface of a reference block included in a WDM (90), thereby providing consistent optical alignment when installing the dichroic filter within the WDM (90). These features and other features, objects, and advantages will be clearly understood by those skilled in the art from the various drawings and the following detailed description of the preferred embodiments of the present disclosure.

Brief Description of the Drawings

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[0044] BEST MODE FOR CARRYING OUT THE INVENTION Flow Cytometer FIG. 1 shows a flow cytometer according to the present disclosure identified by general reference numeral 40. The flow cytometer 40 includes 1. an LD-based optical subsystem 50, 2. a compound microscope objective lens 60, 3. a fluid subsystem 70 for supplying a sheath fluid flow, 4. a peristaltic pump 80 for injecting a sample fluid flow containing particles to be analyzed into the sheath fluid flow supplied by the fluid subsystem 70, wherein the sample fluid flow hydrodynamically constricted by the sheath fluid flow passes through an observation zone, and the compound microscope objective lens 60 collects and images light scattered by the particles and / or fluorescence emitted by the particles in the observation zone, the peristaltic pump 80; 5. an optical fiber 852 that receives light scattered by particles and / or fluorescence emitted by the particles in the observation zone, which is collected and imaged by the compound microscope objective lens 60; 6. a wavelength division multiplexer 90 ( "WDM90") for optically processing the scattered light and / or fluorescence received from the optical fiber 852, and includes.

[0045] Optical subsystem 50 As shown in more detail in FIG. 2, the optical subsystem 50 includes an LD 501 that emits a diverging light beam from the end face of the LD. As visually shown in FIGS. 2 and 2A, the diverging light beam has an elliptical cross-sectional profile having both a major axis, also known as the fast axis, and a minor axis, also known as the slow axis. The diverging light beam emitted from the LD 501 is incident on the collimating lens 502, and the collimating lens 502 converts the diverging light beam emitted by the LD 501 into a collimated light beam having an elliptical cross-section. Although not necessarily essential, the optical subsystem 50 includes an optional mirror 503 positioned to direct the collimated elliptical light beam toward the compound microscope objective lens 60. The plano-convex lens 504 positioned near the compound microscope objective lens 60 reduces the major axis of the elliptical light beam such that the sample fluid and the sheath fluid surrounding it are directed perpendicular to the direction in which they flow through the observation zone within the compound microscope objective lens 60. In the observation zone, the width of the elliptical light beam is 1. The width perpendicular to the direction in which the sample fluid flow passes through the observation zone is preferably slightly shorter than the width of the sheath fluid flow, while 2. It is still wide enough so that particles within the sample fluid flow pass through a substantially flat portion of the elliptical light beam at the beam maximum intensity.

[0046] According to the present disclosure, it will be apparent to those skilled in the art that the plano-convex lens 504 can be replaced with other types of optical elements such as an achromatic lens, or a combination of a spherical lens, a cylindrical lens, and / or a prism pair. Alternatively, the mirror 503 and the lens 504 can also be replaced with a concave mirror. In the case of highly polarization-sensitive applications of the flow cytometer 40, any polarization adjustment element such as a half-wave plate can also be disposed within the collimated portion of the light beam extending from the collimating lens 502 to the lens 504. Finally, before passing through the observation zone, the light beam passes through a high-magnification cylindrical lens 505 positioned adjacent to the observation zone. As shown in FIG. 1, the axis of the cylindrical lens 505 is directed perpendicular to the direction in which the sample fluid flow passes through the observation zone, and the focal length of the cylindrical lens 505 causes a dense focusing of the short axis of the light beam in the observation zone.

[0047] The advantages of the optical subsystem 50 when compared to a conventional LD-based optical subsystem are more clearly shown in FIGS. 2 and 2A. Most commercially available laser diodes suitable for use in a flow cytometer emit a light beam from the end face of the laser diode. As shown in FIG. 2, the gain portion 509 of such an LD chip 510 is highly confined in the transverse direction indicated by arrow 511. Thus, in order to achieve high output, often the manufacturer of the LD sacrifices the beam quality along the transverse direction or the fast axis direction, which is particularly the direction parallel to arrow 511. FIG. 2A shows the characteristics of the light emitted from the LD, and a plurality of interference fringes 512 resulting from gain confinement can be clearly seen in the far field in the short axis direction of the emitted light beam. The interference fringes 512 seen in the figure of FIG. 2A contain only a minute amount of the total energy of the light beam, and thus have little effect on the conventional M 2 It should be noted that it has little effect on the characteristic evaluation. However, as will be described in more detail below, the interference fringes 512 have an adverse effect on the performance of a conventional flow cytometer. Alternatively, the gain confinement along the slow axis direction of the edge-emitting LD, which is perpendicular to arrow 511, is much looser. As a result, as shown in FIG. 2A, the far field beam profile becomes smoother along the slow axis of the LD's light beam.

[0048] Figure 3A shows a conventional LD-based optical subsystem for a flow cytometer. These elements shown in Figure 3A, which are common to the optical subsystem 50 shown in Figure 1, are given the same reference numerals distinguished by a prime (') symbol. As shown in Figure 3A, the conventional optical subsystem aligns the fast axis of the LD 501 parallel to the direction in which the sample fluid flow passes through the observation zone. In its simplest configuration, the elliptical beam profile of the LD 501' is directly redirected into the observation zone by the spherical focusing lens 504'. In an attempt to achieve an optimal aspect ratio for the focused light beam, various different conventional LD-based optical subsystems include beam shaping optical elements in addition to those shown in Figure 3A.

[0049] The adverse effect of the interference fringes 512 along the fast axis of the LD 501' for the conventional optical subsystem configuration is clearly evident in the time profile of the light scattering shown in Figure 3B. Since the scattered light intensity or fluorescence intensity is directly proportional to the local laser output incident on the particles, any fine structure in the profile of the light beam along the direction in which the sample fluid flow passes through the observation zone will appear in the time profile of the signal generated by the flow cytometer. Such a structure in the time profile cannot be distinguished from the signal generated by small particles, thus causing the flow cytometer to malfunction and misidentify the particles. Furthermore, the interference fringes 512 also cause inaccurate measurement of other cytometry parameters such as the area and width of the pulsation shown in Figure 3B.

[0050] Figures 4A and 4B show yet another prior art optical subsystem for LD-based flow cytometry applications disclosed in the '019 patent shown above. These elements shown in Figures 4A and 4B are common to the optical subsystem 50 shown in either Figure 1 or 3A and are given the same reference numerals distinguished by double prime (") symbols. As shown in Figures 4A and 4B, by orienting the slow axis of the LD 501" parallel to the direction in which the sample fluid flow passes through the observation zone, the optical subsystem shown in Figures 4A and 4B effectively overcomes the problems caused by the interference fringes 512 as described above. Unfortunately, the beam diffusing element 513" disposed in front of the spherical focusing lens 504" in Figures 4A and 4B for diffusing the light beam perpendicular to the direction in which the sample fluid flow passes through the observation zone generates an optical beam with a large amount of spherical aberration near the observation zone. Specifically, by focusing this aberrated optical beam in the observation zone in the direction in which the sample fluid flow passes through the observation zone, the width of the optical beam perpendicular to the direction in which the sample fluid flow passes through the observation zone increases, and thus the width of the beam becomes the same as or even wider than the sheath flow. As a result, the optical subsystem shown in Figures 4A and 4b not only reduces the amount of energy of the light incident on the particles flowing through the observation zone, but also increases the unwanted scattered light from the interface between the sheath fluid flow and the adjacent portion of the compound microscope objective lens 60.

[0051] Figure 5 emphasizes the main differences between the optical subsystem disclosed in the '019 patent and the optical subsystem 50 shown in FIG. 1. As shown in FIG. 4, instead of arranging the off-plane beam diffusing element 513″ in front of the spherical beam focusing lens 504, the high-magnification cylindrical lens 505 shown in FIGS. 5A and 5B as a cylindrical plano-convex lens is arranged along the optical beam behind the spherical beam focusing lens 504 and is preferably juxtaposed with the compound microscope objective lens 60. As shown in FIGS. 5A and 5B, the cylindrical lens 505 focuses the minor axis of the optical beam within the observation zone, while the major axis of the optical beam remains essentially unchanged. Thus, the optical subsystems 50 shown in FIGS. 1, 5A, and 5B 1. establish a tightly focused minor axis spanning the combined sample fluid flow and sheath fluid flow, 2. a smooth minor axis profile in the direction of the combined sample fluid flow and sheath fluid flow that is the Fourier conjugate of the far-field beam profile along the slow axis of LD501, resulting in an elliptical optical beam profile having in the observation zone.

[0052] On the other hand, as shown in FIG. 5B, the off-plane beam width is not affected by the cylindrical lens 505. FIG. 5C shows the measured temporal profile of the light scattered from the microparticles using the optical subsystem 50 shown in FIGS. 1, 5A, and 5B. The LD501 used in making the measurements presented in FIG. 5C is the same as that used to create the measured temporal profile of the light scattered from the microparticles, represented in FIG. 3B. As shown in FIG. 5C, the side lobes caused by the interference fringes 512 along the fast axis of LD501 no longer significantly affect the performance of the flow cytometer 40.

[0053] FIG. 6 shows yet another alternative diode laser-based optical subsystem according to the present disclosure adapted for use in a flow cytometer. These elements shown in FIGS. 6 and 6A, which are common to the optical subsystem 50 shown in FIGS. 1, 5A, and 5B, are given the same reference numerals distinguished by triple prime (''') symbols. The optical subsystem 50''' shown in FIGS. 6A and 6B is substantially the same as that shown in FIGS. 1, 5A, and 5B, except that an observation zone is created within a free-flow jet stream 519 that includes both a sample flow and a sheath flow emitted from nozzle 518, so that the observation zone is created without using a compound microscope objective lens 60. Thus, in the case of the configuration of the optical subsystem 50''' shown in FIGS. 6A and 6B, the high-magnification cylindrical lens 505 is remote from the observation zone disposed within the jet stream 519.

[0054] In exemplary embodiments of the present disclosure shown in FIGS. 1, 5A, 5B, 6A, and 6B, the short axis of LD501, i.e., the slow axis, is oriented perpendicular to the direction in which the sample liquid flow passes through the observation zone. However, it will be apparent to those skilled in the art that other optical configurations can be used to orient the long axis of LD501, i.e., the fast axis, perpendicular to the direction in which the sample liquid flow passes through the observation zone. FIG. 7 shows an example of another configuration of such an optical element. These elements shown in FIG. 7, which are common to the optical subsystem 50 shown in FIGS. 1, 5A, 5B, 6A, and 6B, are given the same reference numerals distinguished by quadruple prime ('''') symbols. As shown, the slow axis of LD501'''' is oriented in the z direction. The light beam emitted from LD501'''' is then rotated into the y-direction plane by a pair of 90° reflecting mirrors 523a and 523b. In FIG. 7, the normal to mirror 523a that changes the direction of the first elliptical light beam is oriented at 45° with respect to the x-axis within the x-y plane, and the normal to mirror 523b that changes the direction of the second elliptical light beam is oriented at 45° with respect to the z-axis within the y-z plane.

[0055] Compound microscope objective lens 60 FIG. 8 shows the compound microscope objective lens 60 shown in FIGS. 1, 5A, 5B and 7 according to one aspect of the present disclosure. As shown in FIG. 8, the compound microscope objective lens 60 images an observation zone disposed inside the prism-shaped glass cuvette 603 in the small flow channel 604. Preferably, the small flow channel 604 has a rectangular cross-sectional shape through which a combined sample liquid flow and a sheath liquid flow carrying particles pass. The plano-concave mirror 601 included in the compound microscope objective lens 60 is made of an optically transparent material, such as glass or optically quality plastic, preferably having the same refractive index as the glass cuvette 603. To minimize optical losses, the mirror 601 includes a flat front surface optically coupled to the abutting plane of the prism-shaped cuvette 603. For the optical coupling of the mirror 601 to the cuvette 603, a refractive index matching gel, an optical adhesive, or a direct optical bond may be used. Alternatively, the mirror 601 may be integrally formed with the cuvette 603.

[0056] In addition, the compound microscope objective lens 60 also includes a planar aspheric correction plate 602 made of an optically transparent material, such as glass or optically quality plastic, preferably having the same refractive index as the glass cuvette 603. To reduce optical losses, the plane of the correction plate 602 is optically coupled to the abutting plane of the prism-shaped cuvette 603 on the surface of the prism-shaped cuvette 603 that is directly opposite the back mirror 601. For the optical coupling of the correction plate 602 to the cuvette 603, a refractive index matching gel, an optical adhesive, or direct optical bonding may be used. The aspheric surface of the correction plate 602 farthest from the correction plate 602 can have an anti-reflection coating to reduce the loss of light transmission, but such a coating is not an essential requirement for the compound microscope objective lens 60 according to the present disclosure. The aspheric shape of the correction plate 602 is similar to that of a classical Schmidt camera (Schmidt, B., Mitt. Hamburg Sternwart 7 (36) 1932). As is known to those skilled in the art, the correction plate of a Schmidt camera includes a circular intermediate zone where the correction plate does not deflect the light rays of the light passing through the plate. When used in the compound microscope objective lens 60, outside the intermediate zone of the correction plate 602 with the thinnest plate thickness, the correction plate 602 has a negative optical power, but inside the intermediate zone, the correction plate 602 has a positive optical power. The exact shape of the aspheric correction plate 602 can be easily obtained by those skilled in the art using any commercially available optical ray tracing tool. In the flow cytometer 40, it should be noted that the light beam generated by the optical subsystem 50 shown in FIGS. 1, 5A, 5B, and 7 enters the cuvette 603 perpendicular to the flow channel 604 through one of the two surfaces of the cuvette 603 that does not abut either the back mirror 601 or the correction plate 602.

[0057] FIG. 9A shows the results of ray tracing for the embodiment of the compound microscope objective lens 60 shown in FIG. 8. As shown in FIG. 9A, the scattered and fluorescent emissions from three spatially separated positions in the flow channel 604 near the center of the cuvette 603 1. First, propagate towards the back mirror 601 and be internally reflected by the back mirror 601, and 2. Next, first pass through the cuvette 603, 3. Then, pass through the aspherical correction plate 602, 4. Finally, form three different images near the image plane 605.

[0058] Note that the light rays crossing the compound microscope objective lens 60 shown in FIG. 9A are almost optically uniform, and the light emitted near the center of the cuvette 603 crosses the correction plate 602 at almost normal incidence. Thus, the compound microscope objective lens 60 causes little chromatic dispersion in the light emitted near the center of the cuvette 603.

[0059] Furthermore, it is well known in the astrophysics world that a Schmidt camera provides an unparalleled combination of a bright f-number and an optical performance that is almost diffraction-limited over a wide field of view. The main drawback of a conventional Schmidt camera is that the image plane is inside the instrument. In the case of the compound microscope objective lens 60, the light near the center of the cuvette 603 propagates in the opposite direction to that of a conventional Schmidt camera, and thus, the image plane is outside the compound microscope objective lens 60. This allows the present disclosure to fully utilize only the advantages of the optical performance of a Schmidt camera without being subject to the constraints of a conventional Schmidt camera. FIGS. 9B1 to 9B3 show spot diagrams near the image plane 605 for three emission positions within the observation zone in the flow channel 604 that are spaced 150 microns apart from each other. The diameter of all the images shown in FIGS. 9B1 to 9B3 is less than 35 microns.

[0060] The light emitted from the observation zone within the flow channel 604 of the compound microscope objective lens 60 shown in FIGS. 8 and 9A that traverses the aspherical correction plate 602 is subject to a small amount of chromatic aberration. FIG. 10 shows an alternative embodiment for the compound microscope objective lens 60 shown in FIGS. 1, 5A, 5B, and 7. These elements shown in FIG. 10, which are common to the compound microscope objective lens 60 shown in FIGS. 8 and 9A, are assigned the same reference numerals distinguished by a prime (') symbol. The shapes of the rear mirror 601' and the aberration correction plate 602' shown in FIG. 10 are slightly modified to produce a collimated infinite focus image of the light emitting position near the observation zone within the flow channel 604'. In FIG. 10, the compound microscope objective lens 60' also includes a color correction doublet 609 inserted between the correction plate 602' and the image plane 605'. In addition to focusing the light emitted from the correction plate 602' onto the image plane 605', the doublet 609 also serves to further reduce the residual chromatic aberration caused by the aspherical correction plate 602'.

[0061] The flat surface of the correction plate 602 does not necessarily have to be optically coupled to the cuvette 603. FIG. 11 shows a compound microscope objective lens 60 according to an alternative aspect of the present disclosure. These elements shown in FIG. 11, which are common to the compound microscope objective lens 60 shown in FIGS. 8 and 9A, are given the same reference numerals distinguished by a double prime (″) symbol. FIG. 11 shows an aberration correction plate 602″ optically separated from the cuvette 603″. Although not essential for the operation of the compound microscope objective lens 60″, the exposed surfaces of both sides of the aberration correction plate 602″ and the cuvette 603″ can have an anti-reflection coating to improve light transmission efficiency. It will be appreciated that the aberration correction plate 602″ shown in FIG. 11 is held by a mechanical support (not shown in FIG. 11) in the fixed relationship of the combined rear mirror 601 and the cuvette 603. Similar to the compound microscope objective lenses 60 and 60′ shown in FIGS. 9A and 10 respectively, the compound microscope objective lens 60″ having a separate correction plate 602″ can be configured to provide either a finite focal length image or an infinite focus system that is now focused on the finite distance image plane by adding a color correction doublet 609.

[0062] FIG. 12 shows a compound microscope objective lens 60 according to yet another alternative embodiment of the present disclosure. These elements shown in FIG. 12, which are common to the compound microscope objective lens 60 shown in FIGS. 8, 9A and 11, are given the same reference numerals distinguished by triple prime (''') symbols. The compound microscope objective lens 60''' shown in FIG. 12 is adapted to collect scattered and fluorescent emissions from cells or other microscopic particles carried in the jet stream 519 emitted by the nozzle 518. The compound microscope objective lens 60''' consists of a concave spherical surface mirror 610 and an aberration correction plate 612. The surface mirror 610 can be made of glass or other types of hard materials having a highly reflective coating on the concave surface 611, or can be made of metal having a polished concave surface 611. Similar to the correction plate 602, the planar aspherical correction plate 612 is made from a thin sheet of a transparent material such as glass or optical quality plastic. The aspherical surface is formed on either surface of the correction plate 612. Preferably, both surfaces of the correction plate 612 are coated with an anti-reflection coating to reduce light transmission loss, although such a coating is not an essential requirement for the correction plate 612 according to the present disclosure. It will be understood that the surface mirror 610 and the correction plate 612 are held in a fixed relationship to each other by a mechanical support (not shown in FIG. 12). Scattered light and fluorescence emitted from cells or other types of microscopic particles within the observation zone inside the jet stream 519 are reflected by the concave surface 611 of the surface mirror 610. Aberrations due to reflection from the concave surface 611 are corrected by the correction plate 612 after the light has traversed the correction plate 612. The compound microscope objective lens 60''' is configured to provide either a finite focus image similar to that shown in FIG. 9A, or a collimated infinite focus image focused at a finite distance from the compound microscope objective lens 60''' by a chromatic aberration corrected compound lens similar to the compound lens 609 shown in FIG. 10.

[0063] Figure 13 shows the adaptation of a compound microscope objective lens 60 for imaging a test piece fixed to the surface of a transparent substrate such as a slide glass. These elements shown in Figure 13, which are common to the compound microscope objective lens 60 shown in Figures 8, 9A, and 11, are given the same reference numerals distinguished by a quadruple prime (''') symbol. The compound microscope objective lens 60'''' shown in Figure 13 includes a plano-concave rear mirror 617 made of a transparent material such as glass or optically quality plastic, which are two optical elements, and an aberration correction plate 618. As shown in Figure 13, the test piece to be imaged is fixed to the front surface 615 of a transparent, usually slide glass 616. The slide 616 is optically coupled to the plane of the rear mirror 617, preferably using a thin layer of refractive index matching fluid. The scattered light and fluorescence emitted by the test piece 1. First propagate through the slide 616 and the rear mirror 617, 2. Are internally reflected by the rear mirror 617 and pass through the slide 616, 3. Then pass through the correction plate 618, 4. And finally form an image at the image plane located above the correction plate 618.

[0064] Fluid subsystem 70 Figure 15 shows a fluid subsystem 70 according to the present disclosure, including a sheath fluid reservoir 702 and a liquid pump 701 that draws sheath fluid from the sheath fluid reservoir 702. The liquid pump 701 may be a diaphragm pump, or a peristaltic pump, or a piston pump, or any kind of continuous fluid pump. The outlet of the liquid pump 701 is connected to the inlet of a T-connector 703 that receives sheath fluid from the liquid pump 701. The T-connector 703 has two outlets, and the first outlet is connected to a bypass conduit 710 to return a portion of the sheath fluid received by the T-connector 703 from the liquid pump 701 to the sheath fluid reservoir 702. Returning a portion of the sheath fluid received by the T-connector 703 from the liquid pump 701 to the sheath fluid reservoir 702 is advantageous for the following two reasons: 1. As shown in FIG. 1, the bypass conduit 710 remains open to the surrounding atmosphere, which effectively attenuates the pulsations, thereby significantly reducing the pulsations characteristic of the operation of the liquid pump 701. 2. Returning a portion of the sheath fluid received from the liquid pump 701 by the T-connector 703 to the sheath fluid reservoir 702 also effectively reduces the throughput of the liquid pump 701, thereby enabling the use of a relatively high-flow, low-cost pump in the flow cytometer 40.

[0065] Let the flow resistance of the bypass conduit 710 be represented by "r", and the flow resistance of the path from the T-connector 703 to the flow channel 604 of the cuvette 603 be represented by "R". Therefore, the output resistance to the sheath pump is TIFF2025111685000002.tif11128.

[0066] Since R >> r, the behavior of the liquid pump 701 is dominated by the resistance of the bypass conduit 710 whose hydrodynamic characteristics are temperature-insensitive. Therefore, the configuration of the fluid subsystem 70 shown in FIG. 15 also provides a simple mechanism for realizing a sheath fluid flow that is temperature-insensitive to the flow channel 604.

[0067] As shown in FIG. 15, the second outlet of the T-connector 703 is preferably connected to the flow channel 604 extending through the cuvette 603 via a small reservoir capsule 704 first and then via a filter cartridge 705. As shown in FIG. 16, a tube 704' about 4 feet long replaces the small reservoir capsule 704. In the initial stage of the fluid subsystem 70, some air is trapped within the filter cartridge 705 near its inlet, which is disposed above the outlet of the filter cartridge 705 as shown in FIG. 15. The air trapped within the filter cartridge 705 acts as an additional fluid capacitor and can effectively reduce the pulsation in the sheath fluid discharged into the flow channel 604 to a very low level. Due to the large fluid resistance in the flow channel 604, the air trapped within the filter cartridge 705 becomes compressed. When the liquid pump 701 stops, the air trapped within the filter cartridge 705 is pushed back towards the T-connector 703 similar to an exhaust capacitor. Even without the small reservoir capsule 704, some air discharged from the filter cartridge 705 reaches the bypass conduit 710 due to its low fluid resistance, and when the liquid pump 701 operates again, the air is pushed out of the fluid subsystem 70. Without an additional air supply, such a scenario is repeated until most of the air is purged from the fluid subsystem 70 and the filter cartridge 705 loses its effectiveness as a pulsation damper. Thus, the purpose of the small reservoir capsule 704 or the tube 704' is to provide a reservoir for isolating the filter cartridge 705 from the bypass conduit 710, thereby ensuring that the air trapped inside the filter cartridge 705 remains within the fluid subsystem 70 regardless of the repeated start-stop operations of the liquid pump 701.

[0068] The pulsation damping effect of the air trapped near the inlet of the filter cartridge 705 is clearly visible in the histograms shown in FIGS. 16A and 16B. FIG. 16A shows the particle flight time measured in the flow channel 604 when an air pocket is trapped near the inlet of the filter cartridge 705. FIG. 16B shows the particle flight time measured in the flow channel 604 when the trapped air is purged from the fluid subsystem 70. The results shown in the histograms of FIGS. 16A and 16B are obtained using two knife-edge shaped laser beams focused near the center of the flow channel 604 and separated by an interval of approximately 200 μm. The horizontal axes of FIGS. 16A and 16B are the flight times taken for one particle to travel from one laser beam to the other, measured by recording the peak arrival time of the light scattered from the particles at an angle of 90° from the excitation beam. In both cases, the average flight time for the particles to cross the two laser beams is the same. As shown in FIG. 16A, when the filter cartridge 705 holds some air, all particles take approximately the same amount of time to cross the two laser beams. As shown in FIG. 16B, when the filter cartridge 705 does not hold air, not only does the distribution of the flight times broaden, but it also becomes bimodal. In other words, some particles take less time, while other particles take longer than the average time to cross the two laser beams. This is a phenomenon that can be easily attributed to the pulsation of the sheath fluid velocity in the flow channel 604.

[0069] In the aspects of the present disclosure described so far, the fluid resistance along the bypass conduit 710 and between the T-connector 703 and the flow channel 604 is not adjustable. As will be apparent to those skilled in the art, flow restrictors such as fixed restrictors or regulating valves 711, 711' and 712, 712' can be advantageously inserted within the bypass conduit 710 and between the T-connector 703 and the flow channel 604 to adjust the flow rate through the flow channel 604. Alternatively, the velocity of the sheath fluid flowing through the flow channel 604 can also be adjusted using a liquid pump 701 driven by a variable speed brushless DC motor.

[0070] Peristaltic pump 80 A peristaltic pump 80 according to one aspect of the present disclosure is shown in FIG. 17. The pump includes a housing 809 having an arcuate curved track 808, three rollers 810, 811, and 812 attached to a rotor 816 rotatable within the housing 809, and a compressible tube 807 sandwiched between the compressible tube 807 of the housing 809 and the rollers �10, 811, and 812. As schematically shown in FIGS. 18A-18D, the rollers 810, 811, and 812 of the peristaltic pump 80 are spaced from each other at substantially equal angular distances, separation distances, or intervals around the rotor 816. For clarity, in the following description, it is assumed that the rotor 816 rotates counterclockwise, but it should be understood that this description equally applies to peristaltic pumps having a rotor that rotates clockwise. The compressible tube 807 of the housing 809 can be divided into the following several parts: 1. An open portion between point 801 and point 806 that is not compressed, and 2. A pump inlet portion between point 801 and point 802 that is continuously compressed to a completely closed state when the roller rolls over this portion, and 3. Two pump portions between point 802 and point 803 and between point 804 and point 805 that are completely closed by the roller, and 4. A recessed portion between point 803 and point 804 that continuously expands from a completely closed state to a completely open state when the roller rolls through the enlarged portion of the recessed portion from point 803 to point 813, and 5. Then, when the roller rolls through the compressed portion of the recessed portion from point 813 to point 804, it is continuously compressed to a completely closed state, and 6. An outlet portion between point 805 and point 806 that continuously expands from a completely closed state to a completely open state when the roller rolls through this portion.

[0071] In other words, when the roller rolls counterclockwise above from the inlet point 801 to the outlet point 806, the internal gap is 1. Continuously decreasing from the fully open state at point 801 to the fully closed state at point 802, and remaining closed until point 803; 2. Then, continuously expanding again to the fully open state at point 813; 3. Then, continuously decreasing to the fully closed state at point 804 and remaining closed until the roller reaches point 805; 4. Finally, continuously expanding again to the fully open state at point 806.

[0072] The size of the internal gap is schematically shown in FIGS. 18A - 18D as the distance between the dashed circle and the solid compressible tube 807. As shown in FIGS. 18A - 18D, in the peristaltic pump 80 according to this aspect, the angular distances, separation distances, or intervals between point 801 and 803, between point 802 and 813, between point 813 and 805, and between point 804 and 866 are consistent with the angles between adjacent rollers. Thus, as shown from FIGS. 18A to 18B, when the roller 810 rolls through the pump portion from point 804 to point 805, its interaction completely determines the fluid flow rate of the peristaltic pump 80. As shown in FIG. 18C, when the roller 810 reaches the outlet portion between point 805 and point 806, the area below the roller 810 begins to continuously expand and the gap begins to increase. On the other hand, the roller 811 reaches the compression portion of the recessed part and begins to continuously compress. In the peristaltic pump 80, the shape of the compression portion of the recessed part between point 813 and point 804 along the compressible tube 807 is such that the volume of the liquid extruded by the compression under the roller 811 within the compression portion of the recessed part between point 813 and point 804 substantially fills the volume generated by the expansion under the roller 810 within the outlet portion between point 5 and point 6. During this period, the compressibility is partially open below both rollers 810 and 811 and completely closed below the roller 12. Thus, the pumping action is mainly realized by the roller 12. Specifically, by design, since the total volume of the liquid within the portion between point 13 and point 6 remains substantially constant during this period, the flow rate of the peristaltic pump 80 in the state shown in FIG. 18C remains substantially the same as the flow rate in the states shown in FIGS. 18A and 18B. When the roller 810 passes point 806, the roller 811 reaches the pump portion between point 804 and point 805. Note that since there is no physical difference between the rollers 810, 811, and 812, the flow rate of the peristaltic pump 80 remains substantially constant throughout the process.

[0073] The mechanism of the peristaltic pump without pulsation of the present disclosure will be more clearly understood when viewed along the circular coordinates following the movement of the roller. Referring to FIG. 19, the volume of the fluid inside the compressible tube 819 from the outlet to the nearest roller 820 closing the compressible tube 819, that is, the amount of fluid represented by the hatched area 818 shown in FIG. 19, is represented as V. Clearly, V depends on the angular position θ of the roller 20 and the amount of tube compression δ applied by all other downstream rollers. TIFF2025111685000003.tif6128

[0074] Accordingly, the flow rate F of the peristaltic pump is related to the time derivative of Vc by the following equation. TIFF2025111685000004.tif9128

[0075] Here, R is the rotational speed of the rotor, and the subscript is used to identify a plurality of downstream rollers. The first term on the right side of Equation (2) represents the contribution from the roller closing the tube. Therefore, the partial derivative TIFF2025111685000005.tif8128 does not depend on θ. The summation term represents the contribution from all other downstream rollers that partially compress the compressible tube 819. Here, let ΔS be the cross-sectional area change due to the compression of the compressible tube 819, and L be the length of the tube whose cross-sectional shape is affected by the compression of the tube. Therefore, it is clear to those skilled in the art that L is proportional to the tube compression δ, and ΔS is proportional to δ squared. Accordingly, the volume ΔV of the fluid loss caused by the compression of the tube by the roller is represented by the following Equation (3). 2 It is clear to those skilled in the art that this is the case. Accordingly, the volume ΔV of the fluid loss caused by the compression of the tube by the roller is represented by the following Equation (3). In TIFF2025111685000006.tif7128, D is the inner diameter of the compressible tube, and G is the minimum gap shown in FIGS. 19, 19A, and 19B, which is also indicated by the distance between the dashed circle and the solid compressible tube 807 of the housing 809 in FIGS. 18A - 18D. Referring now to FIGS. 20A and 20B, in the circular coordinate system, FIG. 20A corresponds to the state of the pump shown in FIGS. 18A and 18B. During this period, since there is no roller downstream of the roller 810', the summation term in Equation (2) disappears. FIG. 20B corresponds to the state of the pump shown in FIG. 18C. It is blocked by the roller 12' and partially compressed by the rollers 810' and 811'. However, the volume changes caused by the two rollers 810' and 811' substantially cancel each other out. As a result, the summation term in Equation (2) also disappears. Therefore, the flow rate of the peristaltic pump 80 remains substantially constant regardless of the position of the rollers.

[0076] The shape of the compressible tube 807 that satisfies the above requirements can be easily derived from Equation (3). Referring to FIG. 18C, the gap G of the arcuate compressible tube 807 in the compression part of the recess between the point 813 and the point 804 13,4 and the gap G of the arcuate compressible tube 807 in the outlet part between the point 805 and the point 806 5,6 are given by the following equation: In TIFF2025111685000007.tif8128, as shown in FIG. 21, the total fluid volume of these two parts remains substantially constant. In the peristaltic pump 80, the shape of the pump housing 809 is symmetric about its center line, whereby the inlet half of the pump housing 809 is a mirror image of the outlet half of the housing 809, as shown in FIG. 17. Therefore, the peristaltic pump 80 can operate in both counterclockwise and clockwise rotations with little pulsation. However, it will be understood that symmetry is not required to realize a peristaltic pump without pulsation according to the present disclosure. For example, the gap G of the arcuate compressible tube 807 in the portion between the point 13 and the point 3 13,3and the gap G of the arcuate compressible tube 807 in the portion between point 2 and point 1 2,1 is given by the following equation (5): As long as it conforms to TIFF2025111685000008.tif8128, the peristaltic pump according to the present disclosure shows almost no pulsation when the rotor 816 rotates clockwise.

[0077] FIG. 22 shows an alternative embodiment of the peristaltic pump according to the present disclosure. These elements shown in FIG. 22 that are common to the peristaltic pump 80 shown in FIG. 17 are given the same reference numeral distinguished by a prime (') symbol. The peristaltic pump 80' includes a compressible tube 807' having two recesses 820 and 821, and four rollers 822, 823, 824, and 825. In the embodiment shown in FIG. 22, the volume loss of the fluid due to the tube expansion near the pump outlet is compensated by the combined effect of the compression of the compressible tube by the rollers 822 and 823 near the two recesses 820 and 821.

[0078] FIG. 23 shows yet another alternative embodiment of the peristaltic pump according to the present disclosure. These elements shown in FIG. 23 that are common to the peristaltic pump 80 shown in FIG. 17 and the peristaltic pump 80' shown in FIG. 22 are given the same reference numeral distinguished by a double prime (") symbol. The peristaltic pump 80" includes six rollers and an arcuate compressible tube 807" having two recesses 818" and 819". In the peristaltic pump 80", the volume loss of the fluid due to the tube expansion near the pump outlet is compensated by the action of the roller immediately upstream of one of the recesses 818" or 819" near the pump outlet.

[0079] Pulsations resulting from the expansion of the compressed compliant tube near the outlet of the peristaltic pump can also be overcome by a peristaltic pump having a programmable rotor speed. FIGS. 24A-24C are diagrams showing relevant aspects of an alternative mechanism for minimizing the pulsations of a peristaltic pump according to the present disclosure for a three-roller peristaltic pump. As shown in FIG. 24B, the track 828 is substantially circular between the pump inlet and the pump outlet portion. Thereby, as indicated by the distance between the dashed circle 829 and the solid curve of the track 828, the compliant tube is completely closed by any one of the three rollers 826, 827, and 829 of the pump between the pump inlet and the pump outlet. FIG. 24A schematically shows the roller positions in a polar coordinate system for the peristaltic pump shown in FIG. 24B. Since there is only one roller downstream of the roller closing the tube, Equation (2) is simplified. TIFF2025111685000009.tif8128

[0080] Here, the tube compression δ(θ) is explicitly expressed as a function of the roller position θ. The terms within the parentheses represent the rate of change of the fluid volume with respect to the roller position. The first term is the contribution from the roller that closes the tube, i.e., roller 827 in FIG. 24A, and the second term is the contribution from the roller at the outlet portion. It should be noted that by definition, when the rate of change of volume is negative and there is no roller at the outlet portion, the second term within the parentheses disappears. The dotted curve in FIG. 24C is a representative plot of the negative rate of change of volume with respect to the roller position. The bulge along the curve due to tube expansion when the roller rolls away from the tube near the pump outlet causes pulsation in a conventional peristaltic pump having a constant rotor speed. However, in the case of the peristaltic pumps shown in FIGS. 24A to 24C, the rotor speed R shown by the dashed curve in FIG. 24C is set to vary in synchronization with the rotor position and be inversely proportional to the rate of change of the fluid volume. As a result, as shown by the solid line in the upper part of FIG. 24C, the flow rate of the pump, which is the product of the rotor speed and the rate of change of the fluid volume, remains constant. It should be noted that the terms within the parentheses in Equation (6) are uniquely determined by the mechanical structure of the pump. Therefore, the rotor speed profile can be easily created from the shape of the track 828 according to Equation (3). For those skilled in the art, there are numerous ways to implement a programmable rotor, for example, using a stepping motor or a DC servo motor.

[0081] WDM device 90 FIG. 25 shows an optical ray trace for an exemplary six-port wavelength division multiplexer of the present disclosure using a zigzag configuration. As shown in FIG. 25, the fluorescence passing through the pinhole or emitted from the end face of a multimode optical fiber such as the optical fiber 852 shown in FIG. 1 forms a dispersive object or a dispersive light source, i.e., the optical input of the WDM 90, at position 901. The size of the object is defined by the diameter of the pinhole or the core diameter of the multimode optical fiber. It should be noted that the practical size of the pinhole or the core diameter of the multimode optical fiber is measured in millimeters, in contrast to the diameter of a single-mode optical fiber measured in micrometers. Thus, the étendue of the fluorescence light source, defined as the product of the beam size and its divergence angle, is hundreds of times larger than its counterpart in optical communications. According to the étendue conservation theorem (Julio Chaves, Introduction to Nonimaging Optics, CRC Press, 2008 [ISBN 978-1420054293]), like the light from a flash light, the light from such a dispersive light source can only be collimated for a very limited distance, especially when the diameter of the collimated portion needs to be small.

[0082] As shown in FIG. 25, a collimating optical element, in this case, the achromatic lens 902, captures the light from the light source 901 and projects an enlarged image of the object near the last focusing lens 905. The size of the image near 905 is kept approximately the same as the effective size of the collimating optical element 902. Thus, the optical beam propagating between the lens 902 and the lens 905 is effectively collimated. As shown in FIG. 25, as long as the magnification is kept small, for example, less than about 10, a simple single lens 905 can be used to easily focus the collimated optical beam into a spot smaller than the spot of the optical beam received by the WDM 90 at position 901. Since the optical beam can be focused into such a small size, it becomes possible to place a small-area semiconductor detector at the focus 906 of the focusing lens 905 for efficient optical detection.

[0083] A dichroic filter 903 oriented at an inclined angle is inserted into the optical path approximately midway between the collimating optical element 902 and the lens 905. The dichroic filter 903 passes the target color band and reflects the remaining colors of the light beam for further processing within the WDM90. An optional bandpass filter 904 is inserted after the dichroic filter 903 to further improve the color separation performance of the WDM90.

[0084] The light reflected from the dichroic filter 903 is incident on a second optical element 907, preferably a concave mirror. The concave mirror 907 has a radius of curvature approximately equal to the distance between the collimating optical element 902 and the image near the focusing lens 905. Thus, the concave mirror 907 forms a second image of the collimating lens 902 near the second focusing lens 908. The light beam between the concave mirror 907 and the second image in the lens 908 has substantially the same diameter as the light beam between the collimating lens 902 and the first image near the focusing lens 905. Thus, the relay imaging concave mirror 907 can effectively double the collimated beam path without expanding the beam diameter. Here too, the expanded and collimated beam can be easily focused into a spot smaller than the spot of the light source at 901. Thereafter, a second dichroic filter 909 is inserted approximately midway between the relay imaging concave mirror 790 and the second image near the focusing lens 908. The second dichroic filter 909 passes another color band within the light beam received by the WDM90 at position 901 and reflects the remainder of the incident light beam for further processing.

[0085] As shown in FIG. 25, additional relay collimating optical elements 910, 911, 912, 913 and dichroic filters 914, 915, 916, 917 are similarly cascaded to generate a plurality of images in the vicinity of focusing lenses 918, 919, 920, and 921, and each of these images corresponds to a specific color band of the light received by the WDM90 at position 1. As shown in FIG. 25, due to the 1:1 image relay architecture of the present disclosure, all the light spots generated by the focusing lenses 906, 908, 918, 919, 920 and 921 are smaller than the light source of the light beam, and thus can be easily captured by a small-area APD.

[0086] FIG. 25 shows a 6-port wavelength division multiplexer for an optical beam from a distributed light source, but it will be readily understood by those skilled in the art that a WDM having a different number of ports can be easily constructed in accordance with the present disclosure. Also, although the WDM90 preferably uses achromatism as the first collimating optical element, it will be apparent to those skilled in the art that a single lens can also be used since all the images generated in front of the focusing lenses 906, 908, 918, 919, 920 and 921 are substantially monochromatic. Instead of using a concave mirror to relay the light beam reflected from the dichroic filter, it is also possible to use a refractive optical system as a relay element to extend the path of the collimated light beam. However, an obvious advantage of the zigzag architecture used in the WDM90 is that an array detector can be used, whereby a more compact WDM suitable for portable devices can be obtained.

[0087] FIG. 26 shows an optical ray trace for a prior art collimating device. The method shown in FIG. 26 is widely used in conventional multi-color fluorescence instruments, such as those disclosed in U.S. Patent No. 6,683,314. As shown in FIG. 26, the light beam diverges rapidly beyond the image 924 created by the collimating optical element 923. As a result, the only option for constructing a multi-color device is to insert a dichroic filter between the collimating element 923 and its image 924.

[0088] Due to the constraints of étendue conservation, the diameter of the collimated beam must be increased significantly to accommodate the multiple dichroic filters within this section. The beam expansion poses a significant challenge in refocusing the collimated beam into a small spot suitable for a small area semiconductor detector. To overcome these difficulties, some instrument manufacturers have chosen to use Mt for fluorescence detection only, for example, in mainstream flow cytometers manufactured by Becton-Dickinson, Becman Coulter, and Partec, and in the MegaBACE series of DNA sequencers by GE Amersham. Other instruments, such as the Luminex multiplex beam analyzer, select specific color bands containing known bright fluorescence and use a large area APD to detect the light within the selected color bands.

[0089] FIG. 27 is a perspective view of an alternative embodiment for a 6-port WDM90 using a combination of a zigzag configuration and a branching configuration. The design is a modified form of the zigzag configuration shown in FIG. 25. In the alternative embodiment shown in FIG. 27, the bandpass filter 904 of FIG. 25 is replaced by a dichroic filter 904'. The filter 904' is positioned to pass one color and reflect the other color at 90°. The optical path lengths of the light beams passing through the dichroic filter 904' and the light beams reflected from 904' are substantially the same, whereby one arm is focused by the lens 905 and the other arm is focused by the lens 905' into small spots that are adapted to the small area semiconductor detectors arranged at the focusing positions 906 and 906'. As shown in FIG. 25, the remaining colors of the light reflected by the dichroic filter 903 are relayed and imaged by the concave mirror 907, and the configuration including the optical elements 903, 904', 905 and 905' is further cascaded twice to form a 6-port WDM.

[0090] FIG. 28 shows a perspective view of an alternative embodiment for an 8-port WDM90. By replacing the relay imaging concave mirrors 907 and 910 in FIG. 27 with concave dichroic filters 907' and 910', the WDM shown in FIG. 28 provides two more color bands compared to the WDMs shown in FIGS. 25 and 27.

[0091] Numerous fluorescent probes for use in flow cytometry have been developed over the years. More recently, multiple fluorescent proteins have also become important tools in biomedical research. To accommodate different types of fluorescent probes, various techniques have been developed to enable user selection of dichroic filters suitable for a user's specific needs. An important issue regarding interchangeable dichroic filters is to avoid the coated filter surface coming into direct contact with the rigid flow cytometer reference frame. Repeated direct contact between the coated filter surface and any rigid reference frame can damage the interchangeable dichroic filter. Currently, most conventional solutions to this problem use precisely machined mechanical spacers to hold the interchangeable dichroic filter in place. An example of such a solution is disclosed in U.S. Patent No. 6,683,314. However, such a solution is less reliable when the active area of the detector is less than 1.0 mm 2 in size.

[0092] Figures 29A and 29B show the fabrication of the interchangeable dichroic filter assembly 934 shown in Figure 29C, which is suitable for small area detectors. The assembly of the interchangeable dichroic filter assembly 934 starts from Figure 29A, which shows the construction of a reference template for its fabrication. The reference template is a stepped structure made from two optically parallel glass plates 925 and 926. By joining the two glass plates 925 and 926 in optical contact with each other, it is ensured that the surface 929 of glass plate 925 is optically parallel to the surface 930 of glass plate 926. Thereafter, the front surface 932 of the interchangeable dichroic filter 927 is pressed against the surface 929 of the template. The filter holder 928, which loosely fits the dichroic filter 927, includes a reference surface 931 and a filter slot 933. During the assembly of the interchangeable dichroic filter, the filter slot 933 is partially filled with an epoxy adhesive, and the reference surface 931 of the filter holder 928 is pressed against the surface 930 of the template, while the filter holder 928 slides towards the dichroic filter 927. While the epoxy adhesive is setting, while pressure is applied to the dichroic filter 927 and the filter holder 928, a portion of the dichroic filter 927 remains fixed within the filter slot 933. It will be apparent to those skilled in the art that the epoxy adhesive may be UV curable or thermosetting, or may be made by blending the components of an A / B mixture. Figure 29C shows the E934~ fabricated as shown in Figures 29A and 29B and described above. By the assembly process shown in Figures 29A and 29B and described above, it is ensured that the front surface 932 of the interchangeable dichroic filter assembly 934 is optically parallel to the reference surface 931 and is recessed from the reference surface 931 at an interval accurately determined by the thickness of the glass plate 925.

[0093] Figures 30A and 30B illustrate one aspect of the present disclosure in which the above-described exchangeable dichroic filter assembly 934 is used in the WDM 90 to optically process an optical beam from a distributed light source. A notable feature of the WDM 90 is the glass reference block 935 having an optically flat surface. As will be apparent to those skilled in the art, the glass reference block 935 can be made of other materials. As shown in FIG. 30B, when installing the dichroic filter 927, the reference surface 931 of the exchangeable dichroic filter assembly 934 slides with respect to the plane of the glass reference block 935 and is held in contact therewith by the spring-suspended screw 936. Thereby, the coated front surface 932 of the exchangeable dichroic filter assembly 934 is maintained optically parallel to the optical plane and accurately positioned. On the other hand, the retraction of the front surface 932 with respect to the reference surface 931 protects the reference surface 931 from physical contact with any object during filter replacement.

[0094] It will be apparent to those skilled in the art that numerous changes and modifications are possible to the described aspects of the exchangeable dichroic filter assembly 934. For example, an alternative aspect of the present disclosure is a pedestal assembled using first and second circular optical planes. When assembling the exchangeable dichroic filter assembly 934, the reference surface of the filter holder abuts against the first optical plane, and the coated surface of the dichroic filter abuts against the second optical plane. Thereafter, by epoxy bonding, the coated surface of the dichroic filter is held optically parallel to the reference surface of the filter holder and is recessed at a distance accurately determined by the thickness of the second optical plane.

[0095] Industrial Applicability While one aspect of the present disclosure of an LD-based optical system for flow cytometry applications has been described in detail and some similarly advantageous aspects of a stream-based flow cytometry device have also been described, it will be apparent to those skilled in the art that numerous changes and modifications of the described aspects are possible without departing from the principles and concepts of the present disclosure as set forth in the appended claims, taking into account the above teachings.

[0096] While one aspect of the present disclosure of a wavelength division multiplexer for separating a light beam from a distributed light source into a plurality of color bands has been described in detail and several other similarly advantageous aspects have also been described, it will be apparent to those skilled in the art that numerous changes and modifications of the described aspects are possible without departing from the principles and concepts of the present disclosure as set forth in the appended claims, taking into account the above teachings.

[0097] The present invention has been described with respect to the presently preferred aspects, but it is to be understood that such disclosure is solely for illustrative purposes and is not to be construed as limiting the present disclosure. Thus, various changes, modifications, and / or alternative uses of the present disclosure will surely be presented to those skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the present disclosure. Accordingly, the appended claims are intended to be construed to cover all changes, modifications, or alternative uses that fall within the true spirit and scope of the present disclosure.

Claims

1. A compound optical objective lens for a particle analyzer, comprising: a concave mirror configured to collect light from an observation zone configured such that particles are observed, and to reflect the collected light to form an image; and a correction lens disposed to receive the light reflected from the concave mirror, the correction lens including an aspherical surface having an intermediate zone and configured to reduce spherical aberration in the reflected light caused by the concave mirror. The compound optical objective lens according to claim 1, further comprising a correction lens.

2. The compound optical objective lens according to claim 1, wherein the intermediate zone is a circular intermediate zone.

3. The compound optical objective lens according to claim 2, wherein the circular intermediate zone is configured not to deflect light rays passing through the correction lens.

4. wherein the image is an image of the observation zone, and the correction lens is configured to reduce the spherical aberration caused by the concave mirror based on the reflected light passing through the correction lens. The compound optical objective lens according to claim 1.

5. wherein the concave mirror includes a plano-concave mirror including a spherical mirror surface, the correction lens includes a plano-aspherical lens, and the observation zone is positioned between the concave mirror and the correction lens. The compound optical objective lens according to claim 1.

6. The compound optical objective lens according to claim 1, wherein the correction lens includes one aspherical surface.

7. The compound optical objective lens according to claim 1, wherein the concave mirror is configured to collect light emitted from a plurality of spatially separated positions in the observation zone, and to reflect the collected light through the correction lens to form a corresponding plurality of images with respect to an image plane.

8. The compound optical objective lens according to claim 1, wherein the intermediate zone is located between a first zone and a second zone.

9. wherein the first zone has a negative refractive power, and the second zone has a positive refractive power. The compound optical objective lens according to claim 8.

10. wherein the first zone is disposed outside the intermediate zone, or the second zone is disposed inside the intermediate zone, or the thickness of the correction lens is thinnest in the first zone, or the thickness of the correction lens in the second zone is greater than the thickness of the correction lens in the intermediate zone, or any combination thereof. The compound optical objective lens according to claim 8.

11. A particle analyzer comprising: The compound optical objective lens according to claim 1; and A detector configured to measure the brightness of light corrected by the correction lens.

12. The particle analyzer according to claim 11, wherein the detector is configured to receive light corrected by the correction lens.

13. The particle analyzer according to claim 11, further comprising an optical fiber disposed in an optical path between the correction lens and the detector.

14. The optical fiber is Located near the image plane of the concave mirror, Configured to transmit the corrected light to a wavelength division multiplexer in order to optically separate the light corrected by the correction lens into at least two color bands, or A combination thereof, The particle analyzer according to claim 13.

15. A flow cytometer comprising the particle analyzer according to claim 11.

Images