Biological sample staining module and biological analysis system and method

Abstract

A bioimaging analyzer includes a staining module configured to produce stained cells. The staining module includes: one or more chambers configured to receive a biological sample and a staining composition; and a heater configured to heat the biological sample and the staining composition. The analyzer further comprises: an illumination module configured to illuminate the stained cells; and an imaging module configured to capture an image of the stained cell. A method of staining a biological sample, the method comprising depositing a staining composition into one or more chambers, and preheating the staining composition in the one or more chambers. The method further includes depositing the biological sample into the one or more chambers, and heating the biological sample and the staining composition in the one or more chambers.

Description

[0001] priority

[0002] This application claims the benefit of U.S. patent application No. 63 / 391,536, filed on July 22, 2022, entitled “Lighting Module for Biological Analysis,” U.S. patent application No. 63 / 391,545, filed on July 22, 2022, entitled “Biological Sample Staining Module,” and U.S. patent application No. 63 / 391,549, filed on July 22, 2022, entitled “Flowcell Holder,” the disclosures of which are incorporated herein by reference. Background Art

[0003] Various types of tests related to patient diagnosis and treatment can be performed by analyzing patient samples. This can include analyzing the patient's microorganism or "microbe" and analyzing samples to determine chemistry, antigens, antibodies, blood cell counts, particulate matter (e.g., sediment) and other factors that may affect the patient's health. Microorganisms are microscopic living organisms such as bacteria, fungi or viruses, which can be unicellular or multicellular. When analyzing microorganisms, biological samples containing the patient's microorganism can be taken from the patient's infection, body fluids or abscesses, and can be placed in a test panel or array, combined with various reagents, incubated and analyzed to assist the patient's treatment. Patient chemistry, immunoassays, blood cell counts, particulate matter and other characteristics can be similarly performed. For these different analyses, automated biochemical analyzers or biological testing systems have been developed to meet the following needs of health care facilities and other institutions: to facilitate the analysis of patient samples and to improve the accuracy and reliability of the results compared to the analysis using manual operations, and to help determine the effectiveness of various antimicrobial agents.

[0004] Blood cell analysis is one of the most commonly performed medical tests for providing an overview of a patient's health status. A blood sample can be extracted from a patient and stored in a test tube containing an anticoagulant to prevent blood clotting. A whole blood sample typically contains three main categories of blood cells, including red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Each class can be further divided into subclasses of members. For example, the five main types or subclasses of white blood cells (WBC) have different shapes and functions. White blood cells can include neutrophils, lymphocytes, monocytes, eosinophils, and basophils. There are also subclasses of red blood cell types. The appearance of the particles in the sample may vary depending on pathological conditions, cell maturity, and other reasons. Red blood cell subclasses can include reticulocytes and nucleated red blood cells.

[0005] Traditional blood analysis methods involve using indirect analysis methods (e.g., impedance, light scattering, fluorescence intensity distribution) to collect information about blood cells, such as cell counts and cell types. However, due to these indirect methods, these techniques may have limitations in the quality of the information obtained about the cells.

[0006] Newer analytical techniques can utilize imaging as part of the analytical process. However, obtaining images of sufficient quality to determine the correct cell count and cell type can be challenging. Therefore, there is a need to ensure appropriate image quality in order to obtain sufficient cellular information to be useful in blood and biological analysis. Summary of the invention

[0007] Described herein are apparatus, systems, and methods for classifying objects, such as cells, in images captured by an analyzer, such as a bioassay system that captures images of blood cells from a blood sample.

[0008] In some embodiments, a biological imaging analyzer is described. The analyzer includes a staining module configured to produce stained cells. The staining module includes: one or more chambers configured to receive a biological sample and a staining composition; and a heater configured to heat the biological sample and the staining composition. The analyzer also includes: an illumination module configured to illuminate the stained cells; and an imaging module configured to capture images of the stained cells.

[0009] In some embodiments, the analyzer also includes a flow cell configured to flow the stained cells therethrough, the flow cell also including an imaging region in which images are captured. The imaging module may include a camera. The one or more chambers may also be configured to receive a lysis composition. Additionally or alternatively, the staining composition may also include a lysis compound. The stained cells may be leukocytes having a stained nuclear region.

[0010] In some embodiments, the heater comprises an induction coil. The one or more chambers may comprise two or more chambers, and the heater may be coupled to all of the two or more chambers. The heater may comprise a mounting bracket coupled to all of the two or more chambers and a heating element mounted to the mounting bracket.

[0011] In some embodiments, a method for staining a biological sample using a bio-imaging analyzer is described. The method includes depositing a staining composition into one or more chambers and preheating the staining composition in one or more chambers. The method also includes depositing the biological sample into one or more chambers, and heating the biological sample and the staining composition in one or more chambers. The method may also include mixing the biological sample and the staining composition in one or more chambers. Additionally or alternatively, the staining composition may also include a cleavage compound.

[0012] Although multiple examples are described herein, other examples of the described subject matter will become apparent to those skilled in the art from the following detailed description and accompanying drawings, which show and describe illustrative examples of the disclosed subject matter. As will be appreciated, the disclosed subject matter is capable of modification in various aspects, all without departing from the spirit and scope of the described subject matter. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] While the specification concludes with claims particularly pointing out and distinctly claiming the invention, it is believed that the invention will be better understood from the following description of certain examples taken in conjunction with the accompanying drawings, in which like reference numerals represent like elements, and in which:

[0014] Figure 1 is a schematic diagram, partially in cross-section and not to scale, illustrating various operational aspects of an exemplary flow cell and high optical resolution imaging device for sample image analysis using digital image processing.

[0015] Figure 2 A slide-based visual inspection system is illustrated in which aspects of the disclosed technology may be used.

[0016] Figure 3 A perspective view of an exemplary illumination module in combination with another exemplary flow cell and a high optical resolution imaging device for sample image analysis using digital image processing is illustrated.

[0017] Figure 4 Picture shows Figure 3A perspective view of a lighting module of FIG. 1 , wherein a portion of the housing is omitted to illustrate a light emitter, a focusing lens, a dichroic element, and a collimating lens of the lighting module.

[0018] Figure 5 Picture shows Figure 3 FIG. 1 is a top plan view of a lighting module showing light traveling from each of the light emitters to a collimating lens.

[0019] Fig. 6A A perspective view of an exemplary staining module for mixing a stain with a sample to form a sample mixture and for incubating the sample mixture is illustrated, showing a ferromagnetic sheet laminated to a housing of the staining module.

[0020] Figure 6B Picture shows Fig. 6A A perspective view of a dyeing module showing the ferromagnetic sheet being wrapped to the housing using tape.

[0021] Figure 6C Picture shows Fig. 6A A stereoscopic view of a dyeing module showing the winding of the heating coil of the dyeing module around the housing.

[0022] Fig. 7A A side view of an exemplary multi-chamber staining module for mixing a stain with a sample to form a sample mixture and for incubating the sample mixture is illustrated.

[0023] Figure 7B Picture shows Fig. 7A A top plan view of the multi-chamber staining module.

[0024] Figure 8 Illustration of a process that can be used to stain a sample.

[0025] Fig. 9 A perspective view of an exemplary flow cell holder in combination with another exemplary flow cell and a high optical resolution imaging device for sample image analysis using digital image processing is illustrated.

[0026] Fig.10 Picture shows Fig. 9 Side view of the flow cell holder, flow cell, and high optical resolution imaging device.

[0027] Fig.11 Picture shows Fig. 9 A stereoscopic view of a flow cell holder and a flow cell.

[0028] Fig.12 Picture shows Fig. 9 A top plan view of the flow cell holder and flow cell.

[0029] Fig.13 Picture shows Fig. 9 Cross-sectional view of the flow cell holder and the subassembly of the flow cell.

[0030] Fig.14 Picture shows Fig.13 Exploded cross-sectional view of a subassembly.

[0031] Fig.15 Picture shows Fig.13 A perspective view of the adapter plate of the subassembly.

[0032] Fig.16 Picture shows Fig.13 Exploded perspective views of alternative arrangements of subassemblies.

[0033] Fig.17 Picture shows Fig. 9 Side view of an alternative arrangement of a flow cell holder, flow cell and high optical resolution imaging device.

[0034] Fig.18 A perspective view of another exemplary lighting module is illustrated.

[0035] Fig.19 Picture shows Fig.18 A perspective view of an alternative arrangement of lighting modules.

[0036] Fig. 20 A schematic diagram of another exemplary lighting module is illustrated.

[0037] Fig.21 Picture shows Fig. 20 A partial perspective view of an alternative arrangement of lighting modules.

[0038] Fig. 22 Picture shows Fig. 20 Schematic diagram of another alternative arrangement of lighting modules.

[0039] Fig.23 A perspective view of an exemplary heating module is illustrated.

[0040] Fig.24 Picture shows Fig.23 Exploded perspective view of an alternative arrangement of a heating module.

[0041] Fig.25 A perspective view of an exemplary chamber is shown.

[0042] Fig.26A The diagram shows the Fig.25 Schematic diagram of the first step of an exemplary backflow mixing process in a chamber.

[0043] Fig.26B The diagram shows the Fig.25 Schematic diagram of the second step of the chamber backflow mixing process.

[0044] Fig.27A A schematic diagram of an exemplary AC heater is illustrated, showing the first step of an exemplary heating process.

[0045] Fig.27B Picture shows Fig.27A Schematic diagram of an AC heater showing the second step of the heating process.

[0046] Fig.28 Picture shows Fig.27A Schematic diagram of an alternative arrangement of an AC heater.

[0047] Fig.29 The diagram shows the FIG. 27A to FIG. 28 Schematic diagram of an exemplary voltage-controlled amplitude amplifier that supplies voltage to an AC heater.

[0048] Fig.30 The diagram shows the FIG. 27A to FIG. 28 Schematic diagram of an exemplary voltage controlled oscillator and bandpass filter supplying voltage to an AC heater.

[0049] Fig.31 The diagram shows the integration in a microcontroller / DSP for FIG. 27A to FIG. 28 Schematic diagram of an exemplary control loop for an AC heater supply voltage.

[0050] The drawings are not intended to be limiting in any way, and it is contemplated that various embodiments of the invention may be performed in a variety of other ways, including those not necessarily depicted in the drawings. The accompanying drawings, which are incorporated in and form a part of the specification, illustrate several aspects of the invention and, together with the description, serve to explain the principles of the invention; however, it should be understood that the invention is not limited to the precise arrangements shown. DETAILED DESCRIPTION

[0051] The present disclosure relates to devices, systems, compositions and methods for analyzing samples containing particles. In one embodiment, the present invention relates to an automatic particle imaging system, which includes an analyzer, which can be, for example, a visual analyzer. In some embodiments, the visual analyzer can also include a processor for facilitating automatic analysis of images.

[0052] According to some aspects of the present disclosure, a system for obtaining an image of a sample including particles suspended in a liquid including a visual analyzer can be provided. Such a system can be useful, for example, in characterizing particles in biological fluids, such as detecting and quantifying red blood cells, reticulocytes, nucleated red blood cells, platelets, and white blood cells, including white blood cell differential counting, classification and subclassification, and analysis. Other similar uses are also contemplated, such as characterizing blood cells from other fluids.

[0053] Although other types of body fluid samples can be used, the identification and / or classification of blood cells in blood samples is an exemplary application to which this subject is particularly suitable. For example, aspects of the disclosed technology can be used to analyze non-blood body fluid samples including blood cells (e.g., white blood cells and / or red blood cells), such as serum, bone marrow, lavage fluid, effusion, exudate, cerebrospinal fluid, pleural fluid, peritoneal fluid, and amniotic fluid. The sample can also be a solid tissue sample, such as a biopsy sample that has been processed to produce a cell suspension. The sample can also be a suspension obtained from a processed excreta sample. The sample can also be a laboratory or production line sample including particles, such as a cell culture sample. The term sample can be used to refer to a sample obtained from a patient or a laboratory, or any fragment, portion, or aliquot thereof. In some processes, the sample can be diluted, divided into several parts, or stained.

[0054] In some respects, present, image and analyze sample in an automated manner. In the case of a blood sample, the sample can be diluted substantially with a suitable diluent or saline solution, which reduces the degree to which the visual field of some cells in an undiluted or less diluted sample may be blocked by other cells. Cells can be processed with a reagent that enhances the contrast of some cell aspects, for example, using a permeabilizing agent to make the cell membrane permeable, and using a histological stain to adhere to such as particles and cell nuclei and reveal features. In some cases, it may be necessary to dye an aliquot of the sample for counting and characterizing the particles comprising reticulocytes, nucleated red blood cells and platelets, and for leukocyte classification, characterization and analysis. In other cases, the sample comprising red blood cells can be diluted before being introduced into a circulation cell and / or being imaged in a circulation cell or otherwise.

[0055] As previously described, imaging cells can provide enhanced benefits for characterization and provide an advance over traditional non-imaging techniques. However, capturing images with sufficient quality and resolution to achieve such characterization can be difficult. Aspects of the present invention include the use of flow imaging, i.e., using a sample driven through a flow cell having an imaging area. It is challenging to capture a "still" image of a sample cell within a biological sample as the sample moves through an analysis area of ​​a flow cell. For example, as the sample moves through the analysis area of ​​a flow cell, the sample may move at a speed of about 20 cm / s, making it difficult to capture a clear still image of the sample cell.

[0056] In some embodiments presented herein, and as will be explained in further detail herein, a biological sample can be driven through a flow cell of a biological testing system for analysis. For example, such a testing system can include a microscope imaging system that can be equipped with an imaging device, such as a high-speed, high-resolution camera, configured to image a biological sample (e.g., blood) as the biological sample passes through an analysis region of the flow cell. Such a microscope imaging system can include an optical element, known as an "objective lens," that collects light from the biological sample to form a magnified image of the sample, which can then be focused, for example, via a tube lens, onto an imaging sensor of a camera at an image formation plane.

[0057] Ⅰ. System Overview

[0058] Turning now to the accompanying drawings, Figure 1 An exemplary flow cell 22 for conveying a sample fluid through an observation region 23 of a high optical resolution imaging device 24 is schematically shown, and the high optical resolution imaging device 24 is configured to image microscopic particles in a sample flow stream 32 using digital image processing. The flow cell 22 is coupled to a source 25 of sample fluid, which may have undergone processing, such as contact with a particle contrast agent composition and heating. The flow cell 22 is also coupled to one or more sources 27 of particles and / or intracellular organelle alignment liquid (PIOAL), such as a clear glycerol solution having a viscosity greater than that of the sample fluid.

[0059] The sample fluid is injected through the flat opening at the distal end 28 of the sample feed tube 29 and is injected into the interior of the circulation cell 22 at a point where the PIOAL flow has been substantially established, resulting in a stable and symmetrical laminar flow of the PIOAL above and below (or on opposite sides thereof) the ribbon-like sample flow. The sample flow and the PIOAL flow can be supplied by a precision metering pump that moves the PIOAL along the substantially narrowed flow path together with the injected sample fluid. The PIOAL encapsulates and compresses the sample fluid in the region 21 where the flow path is narrowed. Thus, the reduction in the thickness of the flow path at the region 21 can contribute to the geometric focusing of the sample flow 32. The sample fluid ribbon 32 is encapsulated and carried with the PIOAL downstream of the narrowed region 21, passing in front of or otherwise through the observation region 23 of the high optical resolution imaging device 24 where images are collected, for example, using a CCD 48. In this manner, flow imaging is performed, wherein images from the flowing sample stream and the cellular material contained therein are collected. The processor 18 may receive pixel data as input from the CCD 48. The sample fluid band flows with the PIOAL to the outlet 33.

[0060] As shown here, the narrowed region 21 can have a proximal flow path portion 21a having a proximal thickness PT and a distal flow path portion 21b having a distal thickness DT, such that the distal thickness DT is less than the proximal thickness PT. Thus, a sample fluid can be injected through the distal end 28 of the sample tube 29 at a location distal to the proximal portion 21a and proximal to the distal portion 21b. Thus, when the PIOAL flow is compressed by the region 21, the sample fluid can enter the PIOAL sheath, wherein the sample fluid injection tube has a distal outlet port through which the sample fluid is injected into the flowing sheath fluid, the distal outlet port being defined by a reduced portion of the flow path dimension of the flow cell.

[0061] The digital high optical resolution imaging device 24 having an objective lens 46 is oriented along an optical axis that intersects the strip sample stream 32. The relative distance between the objective lens 46 and the flow cell 22 can be changed by operation of a motor drive 54 for resolving and collecting a focused digitized image on the optical sensor array. Figure 1 Additional information on the construction and operation of the exemplary flow cell shown in is provided in the following patents: U.S. Patent No. 9,322,752, entitled “Flow cell Systems and Methods for Particle Analysis in Blood Samples,” published on April 26, 2016, the entire disclosure of which is incorporated herein by reference; and / or U.S. Patent No. 9,470,618, entitled “Sheath Fluid Systems and Methods for Particle Analysis in Blood Samples,” published on October 18, 2016, the entire disclosure of which is incorporated herein by reference.

[0062] Aspects of the disclosed technology may also be applied to applications other than Figure 1 The flow cell system is shown in the background outside. For example, Figure 2 2 illustrates a slide-based visual inspection system 200 in which aspects of the disclosed technology may be used. Figure 2 In the system shown in FIG. 1 , a slide 202 including a sample, such as a blood sample, is placed in a slide holder 204. Figure 2 As shown in , the slide holder 204 may be adapted to hold a plurality of slides, or only one slide. An image capture device 206 including an optical system 208 and an image sensor 210 is adapted to capture image data depicting a sample in the slide 202 .

[0063] The image data captured by the image capture device 206 may be transmitted to an image processing device 212. The image processing device 212 may be an external device, such as a personal computer, connected to the image capture device 206. Alternatively, the image processing device 212 may be incorporated into the image capture device 206. The image processing device 212 may include a processor 214 associated with a memory 216, the processor 214 being configured to determine the changes required to determine the difference between the actual focus and the correct focus of the image capture device 206. When the difference is determined, instructions may be transmitted to the steering motor system 218. The steering motor system 218 may change the distance z between the slide 202 and the optical system 208 based on the instructions from the image processing device 212. Descriptions of methods that can be used for focusing using this type of setup are provided in the following patents: U.S. Patent No. 9,857,361, published on January 2, 2018, entitled “Flow cell, Sheath Fluid, and Autofocus Systems and Methods for Particle Analysis in Urine Samples,” the entire disclosure of which is incorporated herein by reference; and / or U.S. Patent No. 10,705,008, published on July 7, 2020, entitled “Autofocus Systems and Methods for Particle Analysis in Blood Samples,” the entire disclosure of which is incorporated herein by reference.

[0064] II. Example of lighting module

[0065] In the context of imaging, including the flow imaging concepts discussed for biological imaging, proper illumination is important in order to enable proper visualization of biological material (e.g., blood cells). Illumination is an important criterion for image capture devices (e.g., cameras) to capture clear and well-illuminated images of samples - for example, so that algorithms can correctly identify cell types.

[0066] In such Figure 1 or Figure 2 In the system shown in Figure 3 The lighting module (also referred to as a lighting system or lighting device) 300 shown in FIG. 1 can be used to illuminate a device such as Figure 1 A camera or a high optical resolution imaging device 24 Figure 2For example, the illumination module 300 may be combined with an image sensor 210 to replace Figure 1 The light source 42 is shown in FIG. Figure 3 An illumination module 300 is shown in combination with an exemplary flow cell 302 that can be imaged and a high optical resolution imaging device 304. Figure 1 The high optical resolution imaging device 304 can be constructed and operated like the flow cell 22 shown in FIG. Figure 1 As shown, the illumination module 300 is positioned on the opposite side of the flow cell 302 from the high optical resolution imaging device 304 for illuminating the analysis region, such as the observation region (also referred to as the image capture region or the imaging region), of the flow cell 302 when the sample moves through the analysis region, so as to facilitate capturing an image of the sample by the high optical resolution imaging device 304. The cells of the sample may be stained by a staining module, such as any one of the staining modules 400, 500 described below, before moving through the flow cell 302.

[0067] In the example shown, the lighting module 300 includes a housing 310, a plurality of light emitters 312a, 312b, 312c, a plurality of focusing lenses 314a, 314b, 314c, a plurality of dichroic elements 316a, 316b, 316c, and a collimating lens 318. The light emitters 312a, 312b, 312c can each be any suitable light source, including, for example, an arc lamp, a light emitting diode (LED), or any other suitable light emitter for providing pulsed or continuous illumination. In some embodiments, the light emitters 312a, 312b, 312c can each be configured to emit light of a different color than the other light emitters 312a, 312b, 312c. For example, the first light emitter 312a may include a red LED configured to emit red light having a wavelength between about 600 nanometers and about 650 nanometers, such as about 620 nanometers; the second light emitter 312b may include a green LED configured to emit green light having a wavelength between about 470 nanometers and about 600 nanometers, such as about 525 nanometers; and / or the third light emitter 312c may include a blue LED configured to emit blue light having a wavelength between about 400 nanometers and about 470 nanometers, such as about 450 nanometers.

[0068] The light emitters 312a, 312b, 312c are each mounted to one side of the housing 310 in a row extending substantially parallel to the optical axis of the high optical resolution imaging device 304, so that light emitted by each light emitter 312a, 312b, 312c can initially be projected into the interior of the housing 310 in a direction substantially perpendicular to the optical axis of the high optical resolution imaging device 304. As shown, each focusing lens 314a, 314b, 314c is mounted within the housing 310 and axially aligned with a corresponding one of the light emitters 312a, 312b, 312c for focusing the light emitted by the corresponding light emitter 312a, 312b, 312c.

[0069] Each dichroic element 316a, 316b, 316c is mounted within the housing 310 in line with a corresponding one of the light emitters 312a, 312b, 312c for reflecting and / or filtering light emitted from one or more of the light emitters 312a, 312b, 312c (and focused by the corresponding focusing lens 314a, 314b, 314c). In this regard, each dichroic element 316a, 316b, 316c of the present example includes a corresponding reflective side portion 320a, 320b, 320c and a corresponding filtering side portion 322a, 322b, 322c. Each dichroic element 316a, 316b, 316c is oriented obliquely relative to the optical axis of the high optical resolution imaging device 304 and relative to the light received from the corresponding focusing lens 314a, 314b, 314c. For example, each dichroic element 316a, 316b, 316c can be oriented at an angle of about 45 degrees relative to the optical axis of the high optical resolution imaging device 304. More specifically, each dichroic element 316a, 316b, 316c is oriented so that the corresponding reflective side portion 320a, 320b, 320c generally faces both the corresponding focusing lens 314a, 314b, 314c and the high optical resolution imaging device 304, while the corresponding filtering side portion 322a, 322b, 322c generally faces away from both the corresponding focusing lens 314a, 314b, 314c and the high optical resolution imaging device 304.

[0070] In this way, the reflective side 320a, 320b, 320c of each dichroic element 316a, 316b, 316c can be configured to reflect light emitted from the corresponding light emitter 312a, 312b, 312c (and focused by the corresponding focusing lens 314a, 314b, 314c) and traveling approximately perpendicular to the optical axis of the high optical resolution imaging device 304, so that the reflected light travels approximately parallel to the optical axis of the high optical resolution imaging device 304. For example, the reflective side portion 320a of the first dichroic element 316a can be configured to reflect light emitted from the first light emitter 312a (and focused by the first focusing lens 314a) so that the reflected light travels approximately parallel to the optical axis of the high optical resolution imaging device 304; the reflective side portion 320b of the second dichroic element 316b can be configured to reflect light emitted from the second light emitter 312b (and focused by the second focusing lens 314b) so that the reflected light travels approximately parallel to the optical axis of the high optical resolution imaging device 304; and / or the reflective side portion 320c of the third dichroic element 316c can be configured to reflect light emitted from the third light emitter 312c (and focused by the third focusing lens 314c) so that the reflected light travels approximately parallel to the optical axis of the high optical resolution imaging device 304.

[0071] In addition, the filtering side portions 322a, 322b, 322c of at least some of the dichroic elements 316a, 316b, 316c can be configured to filter light received from one or more of the other dichroic elements 316a, 316b, 316c. For example, the filtering side portion 322b of the second dichroic element 316b can be configured to filter light reflected from the first dichroic element 316a; and / or the filtering side portion 322c of the third dichroic element 316c can be configured to filter light reflected from the second dichroic element 316b, and / or filter light reflected from the first dichroic element 316a (and filtered by the second dichroic element 316b). In this regard, the filtering side portion 322a, 322b, 322c of each dichroic element 316a, 316b, 316c can be configured to inhibit light having a wavelength below a corresponding predetermined threshold from passing therethrough. For example, the filter side portion 322b of the second dichroic element 316b can be configured to inhibit light having a wavelength below a predetermined threshold of about 596 nanometers from passing therethrough; and / or the filter side portion 322c of the third dichroic element 316c can be configured to inhibit light having a wavelength below a predetermined threshold of about 484 nanometers from passing therethrough. In some cases, the filter side portion 322b of the second dichroic element 316b and the filter side portion 322c of the third dichroic element 316c can be configured to allow about 95% of light having a wavelength above the corresponding predetermined threshold to pass therethrough, and / or inhibit about 99% of light having a wavelength below the corresponding predetermined threshold from passing therethrough.

[0072] Thus, light emitted by the first light emitter 312a may be focused by the first focusing lens 314a, reflected by the reflective side portion 320a of the first dichroic element 316a, filtered by the filtering side portion 322b of the second dichroic element 316b, and filtered by the filtering side portion 322c of the third dichroic element 316c; light emitted by the second light emitter 312b may be focused by the second focusing lens 314b, reflected by the reflective side portion 320b of the second dichroic element 316b, and filtered by the filtering side portion 322c of the third dichroic element 316c; and / or light emitted by the third light emitter 312c may be focused by the third focusing lens 314c and reflected by the reflective side portion 320c of the third dichroic element 316c. In this way, light emitted by light emitters 312a, 312b, 312c can be tuned via dichroic elements 316a, 316b, 316c to improve the whiteness of the light before being converged together by collimating lens 318 into a single collimated white light beam, which can then be transmitted out of housing 310 toward flow cell 302.

[0073] It will be understood that where the first light emitter 312a comprises a red LED, the red light emitted by the first light emitter 312a may be substantially unaffected by the filtering side portion 322b of the second dichroic element 316b and the filtering side portion 322c of the third dichroic element 316c due to the relatively higher wavelength of the red light, which may be greater than the threshold of either filtering side portion 322b, 322c.

[0074] The collimated white light beam formed by the collimating lens 318 can be transmitted toward the circulation pool 302 via a light pipe (also referred to as an illumination column or light guide) such as a hexagonal light pipe. The light pipe can be configured to collect the collimated white light beam, randomize the collimated white light beam, and / or converge the collimated white light beam onto the circulation pool 302 (e.g., at the observation area of ​​the circulation pool 302). The light pipe can be positioned relative to the collimating lens 318 so that the collimated light beam converges to a point (e.g., phase) at the entrance of the light pipe. The light pipe can be mounted to a circulation pool holder (not shown) that holds the circulation pool 302 to securely fix the outlet of the light pipe relative to the circulation pool 302, such as the circulation pool holder 700 described below. In this way, since the light pipe and the circulation pool 302 are operatively connected by the circulation pool holder 700, the distance between the light pipe and the circulation pool 302 is fixed.

[0075] In some embodiments, the light emitters 312a, 312b, 312c can be configured to provide pulsed illumination in a distribution in a synchronized manner with each other (e.g., simultaneously) so as to capture a still image of a sample cell moving through the circulation pool 302. For example, the objective lens of the high optical resolution imaging device 304 can be turned on; then, the light emitters 312a, 312b, 312c can simultaneously emit light pulses; then, an image of the sample cell can be captured by the high optical resolution imaging device 304; then, the objective lens of the high optical resolution imaging device 304 can be turned off. This process can be repeated for any suitable number of iterations. In some cases, the duration of each pulse can be between about 1 microsecond and about 3 microseconds, such as about 2 microseconds. The pulse frequency can depend on the camera frame acquisition frequency, which can be between about 220 frames per second and about 300 frames per second. For example, 220 frames per second can correspond to one picture about every 4.5 milliseconds. The objective lens can be opened for about 100 microseconds, which can be sufficient to capture one image. In some embodiments, a higher frame rate may be used, such as with a reduced field of view. Increased speed may be used depending on the specific application, the type of camera used, etc. It will be appreciated that an increase in speed may provide more data in less time (e.g., more images of more sample cells), while a smaller field of view may remove visual landmarks (e.g., "black bars") that may be used for focusing. It will also be appreciated that the pixel rate may contribute to the amount of data provided. For example, increasing the pixel rate may compensate for a decrease in the frame rate to provide the same amount of data.

[0076] In some embodiments, the light emitters 312a, 312b, 312c can be configured to sequentially emit light pulses to operate in a diagnostic mode. For example, a time delay can be provided between each flash to capture three separate images of a particular sample cell at three different moments along the path of the sample cell. The speed of the sample cell can then be calculated using the pixel representation of the distance to determine whether the sample cell is accelerating or decelerating, and / or to determine whether the flow is too fast to obtain reliable data. The diagnostic mode can be selectively entered and exited. For example, after operating in the diagnostic mode, the light emitters 312a, 312b, 312c can be configured to simultaneously emit light pulses in the main operating mode as described above.

[0077] III. Example of the staining module

[0078] In some embodiments, the flow imaging system combines a stain and an associated staining module to enhance the visualization of biological materials (e.g., blood cells). Staining can be used, for example, to stain the internal cell region of a leukocyte to visualize the internal nuclear structure, thereby helping to identify the cell type (e.g., a subset of leukocyte types, e.g., identified as neutrophils, lymphocytes, monocytes, eosinophils, or basophils). In some examples, a stain is applied to the outer surface of the cell to enhance the visualization of the cell (e.g., an external stain for red blood cells or platelets).

[0079] A. Example of a single chamber staining module

[0080] In such Figure 1 or Figure 2 In the system shown in FIG. 6A to FIG. 6C The staining module (also referred to as staining device) 400 shown in FIG. 4 can be used to stain cells in a sample mixture by, for example, Figure 1 A camera or a high optical resolution imaging device 24 Figure 2 The sample is mixed with a stain and the sample mixture is heated and incubated before imaging by the image sensor 210. For example, the staining module 400 may be combined with Figure 1 The location of the source 25 shown in FIG. 1 or in combination with Figure 1between the source 25 and the sample feed tube 29 shown in FIG. 2 to facilitate mixing the sample with the stain and incubating the sample mixture before capturing an image of the sample by the high optical resolution imaging device 24. The stain may include any suitable composition. For example, the stain can be composed according to any one or more of the teachings of: U.S. Patent No. 9,279,750, published on March 8, 2016, entitled “Method and Composition for Staining and Sample Processing,” the entire disclosure of which is incorporated herein by reference; and / or U.S. Patent No. 9,322,753, published on April 26, 2016, entitled “Method and Composition for Staining and Processing a Urine Sample,” the entire disclosure of which is incorporated herein by reference; and / or U.S. Publication No. 2021 / 0108994, published on April 15, 2021, entitled “Method and Composition for Staining and Sample Processing,” the entire disclosure of which is incorporated herein by reference.

[0081] In the illustrated embodiment, the dyeing module 400 includes a housing 410, a pair of ferromagnetic sheets 412, and a heater in the form of a heating coil 414 ( Figure 6C In various embodiments, the heating coil 414 may include a resistive coil, or alternatively, an induction coil. Fig. 6A As best shown in FIG. 4 , the housing 410 includes a plurality of (e.g., four) side walls 420 that together define an interior chamber 422 (also referred to as a sample reservoir) for receiving a stain and a sample, mixing the stain and the sample to form a sample mixture, and incubating the sample mixture. The housing 410 also includes a top wall 424 and a port 426 extending through the top wall 424 to the interior chamber 422. The port 426 can allow a stain dispenser (not shown) to deliver a stain to the interior chamber 422, and / or can allow a sample dispenser (not shown) to deliver a sample to the interior chamber 422 for addition to the stain.

[0082] In some embodiments, the housing 410 can include a metallic material having a relatively high thermal conductivity, such as aluminum, to promote uniform heating of the housing 410 and similarly promote uniform heating of the contents of the interior chamber 422. In the illustrated embodiment, the side walls 420 of the housing 410 are laminated with corresponding ferromagnetic sheets 412 to improve the efficiency of heating (e.g., resistive heating or alternatively inductive heating) performed by the dyeing module 400 (e.g., due to the relatively low ferromagnetic properties of aluminum). More specifically, each ferromagnetic sheet 412 is secured to an outer surface of a corresponding pair of side walls 420. It will be appreciated that any suitable number of ferromagnetic sheets 412 may be used to laminate the side walls 420. In the illustrated embodiment, a thermally conductive compound 430 is deposited on the outer surface of the side walls 420 for adhering the ferromagnetic sheets 412 to the side walls 420. As shown in FIG. Figure 6B As best shown in FIG. 4 , tape 432 is wrapped tightly around ferromagnetic sheet 412 to securely engage the inner surface of ferromagnetic sheet 412 with the outer surface of sidewall 420 .

[0083] like Figure 6C As best shown in , the heating coil 414 includes a wire 440 wound around the side wall 420 of the housing 410 (and around the ferromagnetic sheet 412). The wire 440 may include a metal material with a relatively high electrical conductivity, such as copper. The wire 440 may have any suitable cross-sectional area and / or thickness, and may be wound to define any suitable number of turns for the heating coil 414. In one embodiment, the heating coil 414 acts as an inductor or induction coil and is operatively coupled to a power unit 450 that may be configured to drive the heating coil 414 to a frequency at which the heating coil 414 behaves as a resonant circuit that generates an alternating current under excitation, thereby generating an alternating magnetic field at or near the heating coil 414. The field may generate an electromagnetic field (EMF) on the outer surface of the side wall 420, which in turn may cause an alternating current. The current combined with the resistivity of the housing 410 may generate power dissipation and heat the outer surface of the side wall 420. This heat can be transferred to the contents of chamber 422, such as staining agents and / or samples. It will be appreciated that this induction heating can be performed using relatively low input power, and / or can achieve uniform heating of the contents of chamber 422, and thereby improve staining and / or lysis performance. In this regard, maximum power can be delivered with a resonant frequency excitation circuit, and power delivery can be effectively regulated with an increased frequency excitation circuit. Alternative embodiments can use a resistive heater / resistive heating coil for heating coil 414.

[0084] In some embodiments, a temperature sensor, such as a thermistor (not shown), can be configured to continuously sense the temperature of the contents of chamber 422. The temperature sensor can be configured to send a feedback signal indicating the sensed temperature to a controller (not shown), which in turn can be configured to send a control signal to power unit 450 for selectively driving heating coil 414. In this way, the controller can stop heating the contents of chamber 422 when a threshold temperature is reached. In one example, the controller utilizes a heating control algorithm, and the feedback signal is incorporated into an element of the algorithm or computer-driven instructions provided to power unit 450 and / or heating coil 414 to optimally regulate the temperature. In some embodiments, the controller can be configured to send a control signal to a maintenance heater (not shown) for maintaining the contents of chamber 422 at a threshold temperature.

[0085] In one embodiment, multiple dyeing modules are contemplated, each utilizing FIG. 6A to FIG. 6C The structure (i.e., multiple structural elements 400) is provided. In this way, multiple samples can be stained, incubated, or otherwise prepared at the same time. In one example, each staining module has its own unique heating element. In one example, the staining module has: multiple chambers 422, each chamber can accommodate a sample; and a common heating structure connected to the entire module (e.g., a single housing 410 with multiple chambers 422 and a common heating coil 414 surrounding the housing 410).

[0086] B. Example of a multi-chamber staining module

[0087] In such Figure 1 or Figure 2 In the system shown in Fig. 7A and Figure 7B The multi-chamber staining module (also referred to as a staining device) 500 shown in FIG. 1 can be used to stain cells in a sample mixture by, for example, Figure 1 A camera or a high optical resolution imaging device 24 Figure 2 The sample is mixed with a stain and the sample mixture is incubated by induction heating before imaging by the image sensor 210. For example, the staining module 500 may be combined with Figure 1 The location of the source 25 shown in FIG. 1 or in combination with Figure 1between the source 25 and the sample feed tube 29 shown in FIG. 2 to facilitate mixing the sample with the stain and incubating the sample mixture before capturing an image of the sample by the high optical resolution imaging device 24. The stain may include any suitable composition. For example, the stain can be composed according to any one or more of the teachings of: U.S. Patent No. 9,279,750, published on March 8, 2016, entitled “Method and Composition for Staining and Sample Processing,” the entire disclosure of which is incorporated herein by reference; and / or U.S. Patent No. 9,322,753, published on April 26, 2016, entitled “Method and Composition for Staining and Processing a Urine Sample,” the entire disclosure of which is incorporated herein by reference; and / or U.S. Publication No. 2021 / 0108994, published on April 15, 2021, entitled “Method and Composition for Staining and Sample Processing,” the entire disclosure of which is incorporated herein by reference. Note that these stains generally describe stains containing a lysing agent that lyses red blood cells, a permeabilizing agent that permeates white blood cells, a staining element that stains the internal contents of white blood cells, and a repairing element that fixes white blood cells so that the stain does not escape.

[0088] In the illustrated embodiment, the staining module 500 includes a housing 510, a support (also referred to as a sleeve) 512, and a heater in the form of a heating coil 514. The housing 510 includes a plurality of internal chambers 522a, 522b, 522c, 522d (also referred to as sample reservoirs) for receiving a stain and a sample, mixing the stain and the sample to form a sample mixture, and incubating the sample mixture. The housing 510 also includes a top wall 524 and a plurality of ports 526a, 526b, 526c, 526d extending through the top wall 524 to the corresponding internal chambers 522a, 522b, 522c, 522d. Ports 526a, 526b, 526c, 526d can allow a dye dispenser (not shown) to deliver dye to the corresponding internal chambers 522a, 522b, 522c, 522d, and / or can allow a sample dispenser (not shown) to deliver a sample to the corresponding internal chambers 522a, 522b, 522c, 522d for addition to the dye. Although four interior chambers 522a, 522b, 522c, 522d and corresponding ports 526a, 526b, 526c, 526d are shown, it will be understood that any suitable number of interior chambers 522a, 522b, 522c, 522d and corresponding ports 526a, 526b, 526c, 526d may be provided, such as two, three, or more than four interior chambers 522a, 522b, 522c, 522d and corresponding ports 526a, 526b, 526c, 526d. In some versions, the first interior chamber 522a and the second interior chamber 522b may be configured to function as white blood cell (WBC) chambers 522a, 522b, while the third interior chamber 522c may be configured to function as a red blood cell (RBC) chamber 522c.

[0089] In some embodiments, the housing 510 may include a metallic material having a relatively high thermal conductivity, such as aluminum, to promote uniform heating of the housing 510 and likewise the contents of the interior chambers 522a, 522b, 522c, 522d.

[0090] In the illustrated embodiment, a bracket or sleeve 512 is positioned around the housing 510. The bracket 512 includes an inner bore 530 that is sized and configured to receive at least a portion of the housing 510. In some embodiments, the inner bore 530 can be sized and configured to slidably receive a portion of the housing 510 so that the portion of the housing 510 can be selectively inserted into and / or removed from the inner bore 530. The bracket 512 also includes an upper lip 532 and a lower lip 534 that define a recessed area 536 therebetween. The recessed area 536 is sized and configured to accommodate at least a portion of the heating coil 514.

[0091] The heating coil 514 includes a wire 540 wound around the bracket 512 on the recessed area 536. The wire 540 may include a metal material with relatively high electrical conductivity, such as copper. The wire 540 may have any suitable cross-sectional area and / or thickness, and may be wound to define any suitable number of turns for the heating coil 514. The heating coil 514 is operatively connected to a power unit 550, which may be configured to drive the heating coil 514 to a frequency at which the heating coil 514 behaves as a resonant circuit, which generates alternating current under excitation, thereby generating an alternating magnetic field at or near the heating coil 514, in this way, the heating coil 514 may be used as an inductor and configured as an induction coil or an induction heating coil. The field may generate an electromagnetic field (EMF) on the outer surface of the housing 510, which in turn may cause alternating current. The current may generate power dissipation and heat the outer surface of the housing 510 in combination with the resistivity of the housing 510. This heat can be transferred to the contents of one or more chambers 522a, 522b, 522c, 522d, such as dyes and / or samples. It will be appreciated that this induction heating can be performed using relatively low input power, and / or can achieve uniform heating of the contents of one or more chambers 522a, 522b, 522c, 522d, thereby improving staining and / or lysis performance. In this regard, the maximum power can be delivered with the resonant frequency excitation circuit, and the power delivery can be effectively regulated with the increased frequency excitation circuit.

[0092] In some embodiments, the bracket or sleeve 512 is made of a conductive material (e.g., a metallic material such as aluminum) to enhance heat transfer to the housing and the inner surface that receives the blood sample and stain. In some embodiments, the bracket or sleeve 512 is made of a ferromagnetic material. In some embodiments, the bracket or sleeve 512 is not used, and the heater / heating coil 514 is directly mounted to the outer surface of the housing 510.

[0093] In some embodiments, a temperature sensor, such as a thermistor (not shown), can be configured to continuously sense the temperature of the contents of one or more chambers 522a, 522b, 522c, 522d. The temperature sensor can be configured to send a feedback signal indicative of the sensed temperature to a controller (not shown), which in turn can be configured to send a control signal to the power unit 550 for selectively driving the heating coil 514. In this way, the controller can stop heating the contents of the chamber 522 when a threshold temperature is reached. In some embodiments, the controller can be configured to send a control signal to a maintenance heater (not shown) for maintaining the contents of one or more chambers 522a, 522b, 522c, 522d at a threshold temperature.

[0094] C. Example of sample preparation process

[0095] In such Figure 1 or Figure 2 In the system shown in Figure 8 The procedure shown in can be used to generate Figure 1 A camera or a high optical resolution imaging device 24 Figure 2 Sample preparation is performed prior to imaging with the image sensor 210. Initially, Figure 8 In the process, at step 601, a dye can be delivered to a chamber, such as FIG. 6A to FIG. 6C Chamber 422 of the staining module 400 shown in FIG. FIG. 7A to FIG. 7B One or two WBC chambers 522a, 522b of the staining module 500 shown in . This can include, for example, delivering the stain to the chambers 422, 522a, 522b via a stain dispenser through corresponding ports 426, 526a, 526b. Then, in step 602, the stain can be preheated in the chambers 422, 522a, 522b, such as by induction heating. Next, in step 603, the sample can be delivered to the chambers 422, 522a, 522b. This can include, for example, delivering the sample to the chambers 422, 522a, 522b via a sample dispenser through corresponding ports 426, 526a, 526b so as to be added to the stain. In some embodiments, delivering the sample to the chambers 422, 522a, 522b can include mixing the sample with the preheated stain in the chambers 422, 522a, 522b. In Figure 8 In the process, at step 604, a uniform sample mixture can then be formed in chambers 422, 522a, 522b. This can include, for example, using fluid energy to mix the sample with the stain, such as by periodically pulling the sample out of chambers 422, 522a, 522b via corresponding tangential ports of housings 410, 510 and pushing the sample back into chambers 422, 522a, 522b to perform countercurrent mixing. Alternatively, this can include using a magnet to drive a spherical ferromagnetic ball placed in chambers 422, 522a, 522b to perform stirring mixing. As another example, this can include introducing one or more bubbles at the bottom of chambers 422, 522a, 522b to generate a vortex.

[0096] Then in step 605, the uniform sample mixture can be heated in chambers 422, 522a, 522b, such as by induction heating or resistance heating. In some embodiments, the uniform sample mixture can be heated to a threshold temperature via induction heating or resistance heating, and then the uniform sample mixture can be maintained at the threshold temperature via a maintenance heater. In an embodiment using a multi-chamber staining module 500, it will be understood that the sample mixture in both WBC chambers 522a, 522b can be heated simultaneously via induction heating, and any sample in RBC chamber 522c can also be heated via induction heating (even if such heating of the sample in RBC chamber 522c may not be needed before imaging), while the fourth chamber 522d can remain empty. In some other embodiments using a multi-chamber staining module 500, one or more chambers 522a, 522b, 522c, 522d, such as the first WBC chamber 522a, can be used to heat the sample mixture while one or more of the other chambers 522a, 522b, 522c, 522d, such as the RBC chamber 522c, is simultaneously flushed; in such a case, increased power can be provided to the heating coil 540 to offset any cooling effect that the flushing of the RBC chamber 522c might otherwise have on the heating of the sample mixture within the first WBC chamber 522a (e.g., by adding the same amount of energy that is lost by such cooling effect).

[0097] After the homogenous sample mixture reaches the threshold temperature, the sample mixture can be delivered to a flow cell, such as Figure 1 A flow cell 22 for use by, for example, Figure 1 The sample mixture can be imaged by a camera of a high optical resolution imaging device 24. For example, the homogenous sample mixture can be delivered directly from the chamber 422, 522a, 522b to the flow cell 22 (e.g., without undergoing further preparation). It will be appreciated that the throughput of the staining process can be increased by heating the sample mixture in the same chamber 422, 522a, 522b as the chamber in which the sample mixture is formed.

[0098] Although the formation and inductive heating of the sample mixture has been described as occurring within the chambers 422, 522 of the housings 410, 510, it will be appreciated that an alternative arrangement may include a tube having a lumen (not shown) wherein the sample mixture may be formed and inductively heated in a manner similar to that described above. Additionally or alternatively, any one or more of the teachings herein may be combined with any one or more of the teachings disclosed in U.S. Pat. No. 9,429,524, entitled “Systems and Methods for Imaging Fluid Samples,” issued Aug. 30, 2016, the entire disclosure of which is hereby incorporated by reference.

[0099] In some embodiments, the addition of the diluent is part of the preparation step, wherein the diluent is added to each chamber 522a to 522d before, after, or both before and after the addition of the blood sample to each chamber. For example, the RBC chamber may receive the diluent as the primary or only preparation reagent, while the WBC chamber may receive both the diluent and the stain.

[0100] It should be understood that the preparation steps of the RBC chamber can be different from those of the WBC chamber. For example, the RBC chamber will utilize a preparation step that involves: a) receiving a diluent followed by a blood sample, b) receiving a blood sample followed by a diluent, or c) receiving a diluent followed by a blood sample followed by an additional diluent; but will not receive a stain. In this way, the preparation time of the RBC chamber can be shorter, and the workflow can involve running the RBC sample through the imaging process while the WBC sample is still being prepared.

[0101] In some embodiments, the staining reagent utilizes both a lysing agent (to lyse the red blood cells) and a staining agent (to penetrate the remaining white blood cells, stain the internal area, and repair the white blood cells so that the staining agent does not escape). In this way, a single staining reagent can be used to treat certain types of cells (e.g., white blood cells) to eliminate red blood cells and stain the remaining white blood cells. Other embodiments can utilize multiple compositions, such as a first lysing reagent for lysing red blood cells and a second staining reagent for staining white blood cells, wherein the workflow will involve a chamber (e.g., a WBC chamber) that receives a separate lysing reagent and a separate staining reagent to prepare a WBC sample for visualization.

[0102] In some embodiments, different chambers (e.g., 522a to 522d) do not mean that a dedicated cell type is strictly prepared, or in other words, cell types can be swapped. For example, chambers can be used alternately for RBC and WBC preparation. In this way, once the sample in the chamber is ready for image, a cleaning cycle can be used to clean the chamber before receiving a subsequent blood sample (e.g., chamber 522a can be first configured to prepare WBC to perform a certain amount of the same preparation run, and then prepare RBC to perform a certain amount of sample preparation run--e.g., 1 WBC preparation, followed by 1 RBC preparation, or 2 WBC preparations, followed by 1 RBC preparation, followed by 2 WBC preparations, etc.). Cleaning agents, such as diluents or detergents, can be used between sample runs to eliminate residues. Even in the case where a specific chamber is only used for a specific cell type (e.g., 522a is used only as a WBC chamber), a cleaning step can be run after sample preparation and imaging to eliminate residues.

[0103] Other embodiments can still use multiple staining agents as a part of the preparation process. For example, the first staining agent is configured to dye white blood cells in a manner described herein, and the second staining agent is configured to dye at least one of platelets or reticulocytes. These staining compositions can be used uniquely in various workflows. For example, the first chamber of housing 410, 510 can be used to prepare a white blood cell sample, which includes receiving at least WBC staining agents and lysis reagents, and the second chamber of housing 410, 510 can be used to prepare a platelet sample--the chamber will receive at least platelet reagents--different from WBC staining agents and lysis reagents.

[0104] Note that although the terms white blood cell (WBC) chamber and red blood cell (RBC) chamber are used to represent sample preparation chambers for imaging, the samples imaged as a result of the preparation process can allow for biological imaging of multiple cell types. For example, the WBC chamber utilizes a lysate to eliminate red blood cells, yet the lysate can still retain platelets and reticulocytes, so samples prepared in the WBC chamber can still image at least, for example, white blood cells, platelets, and reticulocytes. Similarly, the RBC chamber can receive a different preparation procedure from the WBC chamber (e.g., no lysis, or no staining / lysis combination reagents), but samples prepared in the RBC chamber can still visualize multiple cell types, such as red blood cells and one or more of white blood cells, platelets, and reticulocytes.

[0105] IV. Example of a flow cell holder

[0106] In such Figure 1 or Figure 2 In the system shown in Figures 9 to 15The flow cell holder (also referred to as a flow cell platform or flow platform) 700 shown in FIG. 1 can be used to facilitate flow cell flow by, for example, Figure 1 A camera or a high optical resolution imaging device 24 Figure 2 For example, the flow cell holder 700 can be used to support the image sensor 210 in an adjustable manner. Figure 1 The flow cell 22 shown in FIG. Fig. 9 A flow cell holder 700 is shown in combination with an exemplary flow cell 702 that can be imaged and a high optical resolution imaging device 704. Figure 1 The high optical resolution imaging device 704 can be constructed and operated like the flow cell 22 shown in FIG. Figure 1 The flow cell holder 700 and the flow cell 702 may together define a flow cell device. Figures 9 and 10 As shown in , the flow cell holder 700 is operatively mounted to the output drive of the motor 706 so as to be able to move precisely toward and away from the high optical resolution imaging device 704, and the high optical resolution imaging device 704 can be fixedly mounted to the frame 708 so as to be stationary. Although not shown, an illumination module can be positioned on the side of the flow cell 702 opposite the high optical resolution imaging device 704 to illuminate the analysis area of ​​the flow cell 702, such as the observation area (also referred to as the image capture area or imaging area), as the sample moves through the analysis area, so as to capture an image of the sample by the high optical resolution imaging device 704. For example, such an illumination module can be constructed and operated like the illumination module 300 described above. In some embodiments, the flow cell 702 can be the same as the flow cell 302 described above, and / or the high optical resolution imaging device 704 can be the same as the high optical resolution imaging device 304 described above. In the illustrated embodiment, the flow cell 702 includes a first portion 702a composed of a first material such as plastic (e.g., acrylic) and a second portion 702b (e.g., glass or sapphire glass) composed of a second material different from the first material. Figure 13 to Figure 14 ). The glass portion 702b may define an analysis region of the flow cell 702, while the plastic portion 702a may define a non-analysis region of the flow cell 702.

[0107] like Figure 11 to Figure 12As best shown in FIG. 7 , the flow cell holder 700 includes a platform plate 710, a first gimbal 712, and a second gimbal 714. The platform plate 710 is operatively coupled to the motor 706 to facilitate translation of the platform plate 710 along the optical axis, also known as the z-axis, of the high optical resolution imaging device 704. The first gimbal 712 is movably coupled to the platform plate 710 to facilitate yaw of the first gimbal 712 about a vertical axis, also known as the y-axis, orthogonal to the optical axis of the high optical resolution imaging device 704, and / or to facilitate translation of the first gimbal 712 along the y-axis. The second gimbal 714 is pivotally coupled to the first gimbal 712 to facilitate pitch of the second gimbal 714 about a horizontal axis, also known as the x-axis, orthogonal to the optical axis of the high optical resolution imaging device 704, and / or to facilitate translation of the second gimbal 714 along the x-axis.

[0108] More specifically, the first gimbal 712 is movably coupled to the platform plate 710 via a vertical adjustment screw 720 and a corresponding spring-loaded plunger 722, such that downward advancement of the vertical adjustment screw 720 can translate the first gimbal 712 downward along the y-axis, and upward retraction of the vertical adjustment screw 720 can translate the first gimbal 712 upward along the y-axis. Similarly, the second gimbal 712 is movably coupled to the first gimbal 712 via a horizontal adjustment screw 724 and a corresponding spring-loaded plunger 726, such that rightward advancement of the left screw 724 can translate the second gimbal 714 rightward along the x-axis, and leftward retraction of the left screw 724 can translate the second gimbal 714 leftward along the x-axis.

[0109] like Fig.12 As best shown in FIG. 7 , a yaw adjustment screw 730 and a corresponding spring-loaded plunger 732 extend through the platform plate 710 for engaging a rear surface of the first gimbal 712 on the opposite side of the y-axis such that forward advancement of the yaw adjustment screw 730 can cause the first gimbal 712 to yaw counterclockwise about the y-axis, and rearward retraction of the yaw adjustment screw 730 can cause the first gimbal 712 to yaw clockwise about the y-axis. Similarly, a pitch adjustment screw 734 and a corresponding spring-loaded plunger (not shown) extend through both the platform plate 710 and the first gimbal 712 for engaging a rear surface of the second gimbal 714 on the opposite side of the x-axis such that forward advancement of the pitch adjustment screw 734 can cause the second gimbal 714 to pitch counterclockwise about the x-axis, and rearward retraction of the pitch adjustment screw 734 can cause the second gimbal 714 to pitch clockwise about the x-axis.

[0110] In the illustrated embodiment, the second gimbal 714 includes a pair of forwardly extending support arms 738, the purpose of which is described below.

[0111] Now refer to Figures 13 to 15 , and continue to refer to Figure 11 to Figure 12 , the flow cell holder 700 of the illustrated embodiment also includes an adapter plate (also referred to as a connector) 740, a fixing block 742, a mounting bracket 744, a compression spring 746 in the form of a coil spring, and a light pipe holding member 748 configured to securely hold a light pipe (also referred to as a light post or light guide) 750. The light pipe 750 can be configured to deliver light from an illumination module (not shown), such as the illumination module 300, to an analysis region of the flow cell 702. In one embodiment, the light pipe 750 randomizes light received at a first end of the light pipe 750 (the end closer to the illumination module 300, which produces light as previously described), wherein the randomized light is then projected at a second end of the light pipe 750 (the end closer to the flow cell 702) to illuminate an imaging region of the flow cell 702. Regardless of the positioning of the flow cell 702 (via the flow cell holder 700), the distance between the light pipe 750 and the flow cell 702 is important to produce a similar illumination distribution, and in this manner, it is important to keep the distance fixed or as close to fixed as possible. By positioning the light pipe 750 as part of the flow cell holder assembly 700, this ensures an operable connection to the flow cell 702 and ensures that the distance between the light pipe 750 and the flow cell 702 / imaging area of ​​the flow cell 702 remains constant to ensure this consistent illumination distribution.

[0112] like Fig.15 As best shown in , the adapter plate 740 includes a circular hole 760 and a pair of forwardly extending arcuate ridges 762 positioned on opposite sides of the hole 760. The arcuate ridges 762 include corresponding forward-facing engagement surfaces 764 that are configured to frictionally engage corresponding portions of the flow cell 702. For example, the engagement surfaces 764 can be configured to frictionally engage the rear surface of the flow cell 702 (e.g., which faces the high optical resolution imaging device 704). More specifically, the engagement surfaces 764 can be configured to frictionally engage the rear surface of the glass portion 702b of the flow cell 702 that defines the analysis area on opposite sides of the y-axis from each other and, therefore, on opposite sides of the flow path of the flow cell 702. Thus, the engagement surfaces 764 can define a mounting plane along which the rear surface of the glass portion 702b of the flow cell 702 contacts the flow cell holder 700.

[0113] As described in more detail below, the flow cell 702 can be sandwiched between the arcuate ridge 762 and the fixing block 742, thereby fixing the flow cell 702 to the flow cell holder 700, wherein the engagement surface 764 of the arcuate ridge 762 abuts the rear surface of the glass portion 702b of the flow cell 702 that defines the analysis area. It will be understood that the glass portion 702b of the flow cell 702 can have a relatively low coefficient of thermal expansion, at least compared to the coefficient of thermal expansion of the plastic portion 702a of the flow cell 702. In addition, the material thickness between the flow path of the flow cell 702 and the rear surface of the glass portion 702b of the flow cell 702 can be substantially less than the material thickness between the flow path of the flow cell 702 and the rear surface of the plastic portion 702a of the flow cell 702. Therefore, by fixing the rear surface of the glass portion 702b of the flow cell 702 against the engagement surface 764, the risk of the observation area of ​​the flow cell 702 moving out of focus in response to temperature changes can be substantially reduced or eliminated. In some embodiments, the adapter plate 740 can include a metal material having a relatively low coefficient of thermal expansion, such as aluminum, to further mitigate the risk of the observation area of ​​the flow cell 702 moving out of focus in response to temperature changes. Because the engagement surface 764 of the arcuate ridge 762 is positioned on the opposite side of the flow path of the flow cell 702, the engagement surface 764 does not span the flow path (e.g., extend above or below the flow path), so that the engagement surface 764 can avoid applying pressure to the flow cell 702 along the flow path.

[0114] like Fig.15 As shown in the figure, the adapter plate 740 includes an alignment hole 766 and an alignment slot 768, which are configured to align with corresponding alignment holes and slots (not shown) of the circulation pool 702 and / or receive corresponding alignment pins (not shown) of the second gimbal 714 therethrough to facilitate proper alignment of the adapter plate 740 relative to the circulation pool 702 and / or the second gimbal 714.

[0115] In the illustrated embodiment, the mounting bracket 744 is attached to the support arm 738 of the second gimbal 714 via corresponding screws 770. Figure 13 to Figure 14As best shown in FIG. 7 , the mounting bracket 744 includes an inner cavity 772 sized and configured to securely receive the light pipe retaining member 748 and a generally annular collar portion 774 configured to slidably support the fixing block 742, as described in more detail below. In some embodiments, the fixing block 742 can be movably coupled to the mounting bracket 744, such as via one or more screws 775. As shown, a pair of threaded holes 776 are provided through the upper portion of the mounting bracket 744 for receiving corresponding set screws (not shown), which can apply pressure to the light pipe retaining member 748 when the light pipe retaining member 748 is located in the cavity 772 to inhibit unintentional movement of the light pipe retaining member 748 relative to the mounting bracket 744. The mounting bracket 744 of the illustrated embodiment also includes a generally annular shoulder 778 sized and configured so that the front end of the compression spring 746 can seat against the shoulder 778, as described in more detail below.

[0116] The light pipe retaining member 748 includes a longitudinal bore 780 extending along the z-axis and sized and configured to securely receive the light pipe 750 while also aligning the light pipe 750 with the analysis region of the flow cell 702 and positioning the outlet of the light pipe 750 substantially proximate to the analysis region. As shown, at least one threaded hole 782 (not shown) is disposed through an upper portion of the light pipe retaining member 748 for receiving a corresponding set screw (not shown). Fig.12 ), which can apply pressure to the light pipe 750 when the light pipe 750 is located within the aperture 780 to inhibit unintentional movement of the light pipe 750 relative to the light pipe retaining member 748. In this manner, the light pipe 750 can be securely mounted or otherwise connected to the flow cell holder 700 so that the outlet of the light pipe 750 can be securely fixed relative to the flow cell 702, thereby promoting proper illumination of the analysis area of ​​the flow cell 702 via the light pipe 750 and inhibiting the outlet of the light pipe 750 from being unintentionally misaligned or otherwise moved away from the analysis area of ​​the flow cell 702. For example, during adjustment of the position and / or orientation of the flow cell 700 via the motor 706, the vertical adjustment screw 720, the horizontal adjustment screw 724, the yaw adjustment screw 730, and / or the pitch adjustment screw 734, the position of the light pipe 750 relative to the flow cell 702 (e.g., the distance between the outlet of the light pipe 750 and the flow cell 702) can remain fixed. As described above and herein, this fixed position of the light pipe 750 relative to the flow cell 702 is important for ensuring consistent illumination and imaging quality of biological specimens.

[0117] The fixing block 742 includes a generally annular rearwardly facing engagement surface 790 configured to frictionally engage a corresponding portion of the flow cell 702 opposite the engagement surface 764 of the arcuate ridge 762 of the adapter plate 740 to sandwich the flow cell 702 therebetween. In this regard, the fixing block 742 also includes a generally annular recess 792 configured to slidably receive the collar portion 774 of the mounting bracket 744 and a generally annular shoulder 794 sized and configured such that the rear end of the compression spring 746 can seat against the shoulder 794. Therefore, the compression spring 746 can be configured to push the fixed block 742 in the backward direction along the z-axis to promote frictional engagement between the mating surface 790 of the fixed block 790 and the corresponding portion of the circulation pool 702 and frictional engagement between the mating surface 764 of the arcuate ridge 762 of the adapter plate 740 and the corresponding portion of the circulation pool 702 to firmly clamp the circulation pool 702 between the fixed block 742 and the adapter plate 740.

[0118] In some embodiments, the fixing block 742 can slide slightly along the collar portion 772 of the mounting bracket 744, such as in a forward direction along the z-axis, to accommodate thermal expansion of the flow cell 702 (e.g., the plastic portion 702a) along the z-axis. In such a case, it will be understood that the compression spring 746 can be configured to maintain frictional engagement between the engagement surface 790 of the fixing block 790 and a corresponding portion of the flow cell 702 and frictional engagement between the engagement surface 764 of the arcuate ridge 762 of the adapter plate 740 and a corresponding portion of the flow cell 702.

[0119] While the arcuate ridges 762 of the above described embodiment are incorporated into an adapter plate 740 that is secured to the second gimbal 714 , the arcuate ridges 762 including their respective engagement surfaces 764 may alternatively be incorporated directly into the second gimbal 714 .

[0120] in this regard, Fig.16 An alternative arrangement is shown in which the adapter plate 740 is integrally formed with the second gimbal 714 as a unitary (eg, single-piece) piece. Fig.16 In the arrangement shown in , the compression spring 746 is provided in the form of a corrugated disc.

[0121] In the illustrated embodiment, the second gimbal 714 further includes a pair of forwardly extending alignment pins 795 configured to be received within corresponding alignment holes and / or slots (not shown) of the flow cell 702 for facilitating proper alignment of the second gimbal 714 relative to the flow cell 702. As shown, the second gimbal 714 further includes a forwardly extending stabilizing arm 796 having a threaded hole 797 extending therethrough for receiving a spring-loaded plunger 798. The spring-loaded plunger 798 can be configured to apply pressure to one side of the flow cell 702 to reduce or eliminate any mechanical play that may otherwise exist between the alignment pins 795 of the second gimbal 714 and the corresponding alignment holes and / or slots of the flow cell 702.

[0122] Although the platform plate 710 of the embodiment described above is operably mounted to the output drive of the motor 706 to facilitate translation of the platform plate 710 along the z-axis (i.e., the optical axis of the high optical resolution imaging device 704) so ​​that the circulation pool holder 700 can be precisely moved toward and away from the high optical resolution imaging device 704, such relative movement may be provided in any other suitable manner.

[0123] in this regard, Fig.17 An alternative arrangement is shown in which a platform plate 710 is fixedly mounted to a frame 708 so as to be stationary, and in which at least a portion of the high optical resolution imaging device 704, such as its objective lens, is operably mounted to an output drive of a motor 706 to facilitate translation of the objective lens of the high optical resolution imaging device 704 along the z-axis (i.e., the optical axis of the high optical resolution imaging device 704) so ​​that at least the objective lens of the high optical resolution imaging device 704 can be precisely moved toward and away from the circulation pool holder 700.

[0124] V. Additional Exemplary System Features

[0125] In such Figure 1 or Figure 2 In the system shown in , various additional features may be implemented in addition to or as an alternative to any one or more of the features described above, as described in more detail below.

[0126] A. Example of LED driver

[0127] In some cases, a light source driver in the form of an electronic circuit can be designed to drive a high conversion rate current pulse through an LED light source (e.g., similar to light emitters 312a, 312b, 312c) to generate current and modulated light pulses. The pulse duration of the pulse can be controlled to produce a pulse width range between about 1 μs and about 10 μs, or a larger pulse width range as needed to control the imager exposure time. The light pulse can be used to control the image capture exposure time so that a clear image of cells or particles in motion can be obtained. In order to allow the collection of images of blood cells, latex beads, particles, crystals, bubbles, debris, and a wide range of particles flowing through the circulation pool at high speed, it is intended to illuminate the particles at a sufficient speed to capture unblurred images. The repetition rate of the light pulse can be increased by increasing the trigger rate from less than about 1 Hz to greater than about 1 kHz as needed to allow sufficient image throughput to be obtained. The light source characteristics can allow for the distinction of fine details in terms of contrast, internal particle content, accurate color reproduction, tunable color spectrum adjustment by means of a DAC interface, and illumination power control of all light sources to provide an unlimited variety of color combinations. The light source allows for optimal focusing performance of the optics and digital imager.

[0128] LED illumination can be connected by optical devices including, for example, mirror parabolic light reflectors, fiber bundles, solid optical light guides, fiber optic cables, bifurcated fiber bundles, condenser lenses, light guides with internal mirrors, optical prisms, so that the pulsed illumination from LED is guided, shaped and connected to the observation window of the flow cell, wherein particles, cells or other microscopic objects move in organized areas for imaging. Filters are used to optimize the interrogation spectrum of the sample flow in the illumination flow cell and the flow cell. Hexagonal homogenizer tubes can be implemented to provide an interface between illumination sources and randomize the light connected to the flow cell. Bright field illumination can be implemented at the imaging digitizer.

[0129] In this regard, a capacitor discharge circuit with a synchronous trigger input may be provided. Functional submodules include power regulation of input voltage; input voltage pre-regulator; discharge capacitor regulator; discharge capacitor bank; discharge capacitor voltage regulator; trigger pulse generator; capacitor discharge transistor; delay timer; interface signals; DC power supply for discharge capacitor bank; DC power supply for logic; DC power supply for analog devices; communication interface with addressing for discharge voltage control; discharge trigger signal; gate driver disable / enable signal; delay timer disable / enable signal; and / or discharge capacitor low inductance interface.

[0130] The rise time and fall time of the current pulse can be kept below 250ns so that the modulated light pulse can illuminate and extinguish, thereby producing a symmetrical and square pulse. These rise times and fall times can allow the total duration of the light pulse to produce a useful light pulse with a duration width greater than about 1μs. Low ESL charging / discharging capacitors may be desired to obtain fast rising edge pulses. Low inductance through the entire LED current path may be desired in order to obtain fast rising edge pulses. Low ESR capacitors may be implemented in the capacitive discharge circuit to avoid power loss and heating of the capacitor. The lighting device may feature an enable signal, which is intended to operate the light source during the imaging phase of system operation and disable the light source when the system is idle. The white LED may be of a high CRI type in order to reproduce accurate colors through the imaging system. An optical diffuser with a wide angle may be used between the LED and the optical light guide or optical fiber or a parabolic reflector or an elliptical reflector in order to diffuse the LED illumination so that shadows in the image are reduced. Color filters may be used to optimize the color response of the LED illumination so that excessive blue light is suppressed to provide uniform color through the visible spectrum. A condenser lens can be used to couple the LED illumination to match the numerical aperture to various objectives with varying numerical apertures. The electronic control circuit can feature voltage regulation to control the voltage across the discharge capacitor, and therefore control the charge across the capacitor, and therefore control the LED illumination power input and light intensity. The electronic circuit can feature pulse width control, and therefore can control the light pulse time, and can control the light pulses. In multiple LED configurations, a timer can be implemented to add a time delay between multiple LED flashes and provide the system with the ability to measure particle or cell velocity, provided there is a known time delay between the flashes.

[0131] B. Example of a dual-cube beam splitter RGB LED combiner

[0132] Fig.18 Another example of a lighting module 1300 is shown, which can be easily incorporated into a device such as Figure 1 or Figure 2, in place of the lighting module 300 described above. In the example shown, the lighting module 1300 includes a rectangular glass light pipe 1310, a pair of cubic beam splitters 1311a, 1311b, and a plurality of light emitters 1312a, 1312b, 1312c. The light emitters 1312a, 1312b, 1312c can each be any suitable light source, including, for example, a light emitting diode (LED) or any other suitable light emitter for providing pulsed or continuous illumination. In some embodiments, the light emitters 1312a, 1312b, 1312c can each be configured to emit light of a different color than the other light emitters 1312a, 1312b, 1312c. For example, the first light emitter 1312a may include a blue LED configured to emit blue light having a wavelength between about 400 nanometers and about 470 nanometers, such as about 450 nanometers; the second light emitter 1312b may include a green LED configured to emit green light having a wavelength between about 470 nanometers and about 600 nanometers, such as about 525 nanometers; and / or the third light emitter 1312c may include a red LED configured to emit red light having a wavelength between about 600 nanometers and about 650 nanometers, such as about 620 nanometers.

[0133] Each cube beam splitter 1311a, 1311b can include a corresponding pair of right angle prisms 1315a, 1315b, 1315c, 1315d. Each beam splitter 1311a, 1311b can be coated with a bandpass dichroic coating that effectively reflects one color but also efficiently transmits other colors. In this regard, the first beam splitter 1311a can be coated with a green light reflecting dichroic coating, while the second beam splitter 1311b can be coated with a red light reflecting dichroic coating.

[0134] The first light emitter 1312a is mounted to the end of the first beam splitter 1311a so that the light emitted by the first light emitter 1312a can be initially projected into the first beam splitter 1311a in a direction substantially parallel to the optical axis of the high optical resolution imaging device 304; the second light emitter 1312b is mounted to the top of the first beam splitter 1311a so that the light emitted by the second light emitter 1311b can be initially projected into the first beam splitter 1311a in a direction substantially perpendicular to the optical axis of the high optical resolution imaging device 304; and the third light emitter 1312c is mounted to the top of the second beam splitter 1311b so that the light emitted by the third light emitter 1312c can be initially projected into the second beam splitter 1311b in a direction substantially perpendicular to the optical axis of the high optical resolution imaging device 304. In this way, blue light can pass completely through beam splitters 1311a, 1311b and light pipe 1310 with high transmittance; green light can be selectively reflected into the optical axis of high optical resolution imaging device 304 and immediately mixed with blue light; and red light can be selectively reflected into the optical axis of high optical resolution imaging device 304 and immediately mixed with blue light and green light. Light pipe 1310 can be configured to mix and homogenize the light received from beam splitters 1311a, 1311b. In this way, the light emitted by light emitters 1312a, 1312b, 1312c can be tuned to improve the whiteness of the light, and the light can then be transmitted out of light pipe 1310 toward flow cell 302.

[0135] Fig.19 An alternative arrangement is shown in which a third light emitter 1312c is mounted to the bottom of the second beam splitter 1311b so that light emitted by the third light emitter 1312c can be initially projected into the second beam splitter 1311b in a direction substantially perpendicular to the optical axis of the high optical resolution imaging device 304.

[0136] C. Example of a White Light LED Lighting Module with Multiple LEDs

[0137] Fig. 20 Schematically shows another example of a lighting module 1400 that can be easily incorporated into a computer system such as Figure 1 or Figure 2, in place of the illumination module 300 described above. In the example shown, the illumination module 1400 includes a mount and / or housing (not shown), a plurality of light emitters 1412a, 1412b, 1412c, 1412d, an LED combiner optical device 1415, an LED collimator or focusing lens assembly 1418, an LED hexagonal homogenizer light pipe 1419, an optical diffuser wafer 1421, a color filter (not shown) for adjusting the final illumination output, and a platform alignment calibrator (not shown). The light emitters 1412a, 1412b, 1412c, 1412d can each be any suitable light source, including, for example, a light emitting diode (LED) or any other suitable light emitter for providing pulsed or continuous illumination. In some embodiments, the light emitters 1412a, 1412b, 1412c, 1412d can each be configured to emit light of a different color than the other light emitters 1412a, 1412b, 1412c, 1412d. For example, the first light emitter 1412a may include a red LED configured to emit red light having a wavelength between about 600 nanometers and about 650 nanometers, such as about 620 nanometers; the second light emitter 1412b may include a green LED configured to emit green light having a wavelength between about 470 nanometers and about 600 nanometers, such as about 525 nanometers; the third light emitter 1412c may include a blue LED configured to emit blue light having a wavelength between about 400 nanometers and about 470 nanometers, such as about 450 nanometers; and / or the fourth light emitter 1412d may include a high-power white LED configured to emit white light.

[0138] The monochromatic color light emitters 1412a, 1412b, 1412c can be configured to enhance the final illumination beam and fill in the color spectrum interruption provided by the white light emitter 1412d. Although three monochromatic color light emitters 1412a, 1412b, 1412c are shown, it will be understood that only one, two, or more than three monochromatic color light emitters 1412a, 1412b, 1412c can be used. Thus, the illumination module 1400 can be used for hematology flow imaging and can be capable of providing programmable monochromatic, polychromatic, or white light illumination in the visible spectrum.

[0139] In some embodiments, the lighting module 1400 can be configured to provide multi-strobe lighting, such as double strobe and triple strobe. The lighting module 1400 can be capable of enabling or disabling any combination of individual LEDs 1412a, 1412b, 1412c, 1412d. The lighting module 1400 can be capable of adjusting individual LED powers from 100% down to 0%. The light output from the light pipe 1419 can be directed to the circulation pool, and can include a uniform light output, i.e. red, green, blue or white, by combining the light emitted from the RGB LED source 1412a, 1412b, 1412c and the W LED source 1412d, and can provide illumination of a sample with a continuous color spectrum.

[0140] In addition to power LEDs 1412a, 1412b, 1412c, 1412d, the electronics of lighting module 1400 may include: LED switches and capacitor discharges (e.g., 1 per LED); a pulse width generator (e.g., 1 channel for all LEDs); and a digitally controlled discharge power controller (e.g., 4 channels).

[0141] Fig.21 An alternative arrangement is shown having only two monochromatic color light emitters 1412a, 1412b.

[0142] Fig. 22 An alternative arrangement is shown having dedicated collimator optics 1418a, 1418b, 1418c, 1418d for each light emitter 1412a, 1412b, 1412c, 1412d.

[0143] D. Example of a block heater for sample incubation

[0144] Fig.23An example of a heating module 1500 is shown, which includes a pair of metal blocks 1510 with high thermal conductivity and low heat retention. For example, the blocks 1510 can each be formed by aluminum. In order to maintain a stable temperature, a large number of heater blocks 1510 are used to increase heat retention and reduce temperature fluctuations caused by active heating control. In order to improve the heat conduction efficiency of all cells in the sample, a small inner diameter solid tube 1512 is used as an incubation container to increase the heat exchange surface contact area. The tube 1512 is grooved in a serpentine manner inside the aluminum block 1510 to balance the amount of left and right turns that the blood cells need to travel through to prevent centrifugation of sample integrity. A heating element such as one or more heater pads 1514 is located in the central cavity 1516 of the aluminum block 1510 to provide uniform heating to the module 1500 and reduce environmental losses. In order to maintain a temperature gradient even at the boundary of the module 1500, a non-thermally conductive material 1518 is used to enclose the block 1510 to prevent heat exchange with the environment. A thermistor 1520 for thermal control feedback is also located within block 1510 .

[0145] Fig.24 An alternative arrangement having a modified serpentine tube 1512 is shown.

[0146] E. Example of sample backflow mixing

[0147] In some cases, it may be desirable to improve Fig.25 The method of power delivery mixing and bubble mixing in one or more chambers of chamber 1600 shown in FIG. For example, counter-current mixing can use repeated sample pull / push and controlled bubbles to increase the mixing power on one or more chambers 1600.

[0148] FIG. 26A to FIG. 26B An example of such backflow mixing is shown where a large volume air gap of approximately 2 inches to 3 inches is introduced into each of the reagent delivery lines for the RBCs and WBCs by opening and pulling the vent ports on N14 and N8. The length of tubing between the vent valve and the chamber can be maintained at the same volume as the target reagent delivery volume ( Fig.26A The ventilation port can then be switched to the delivery line for pre-dispensing ( Fig.26B ). Once the blood sample is delivered to chamber 1600, the diluted sample can be pulled back without passing the air gap through the exhaust valve, and then pushed out with a slightly larger volume along with some air to mix the bubbles. The same pulling and pushing motion can be repeated about 2 to 5 times with increasing volumes to achieve cumulative agitation mixing. Eventually, most of the air gap in the air gap is consumed, leaving little air gap between the reagent port and the reagent in the tube to prevent sample diffusion.

[0149] It will be appreciated that controlled bubbles aid mixing without causing lysing reagent formation. Repeated power delivery can better mix small volume samples and can be adjusted to the desired uniformity. Based on the low count variation throughout the acquisition time, the mixing can be assessed as effective and uniform. Therefore, a dedicated mixing component may not be required.

[0150] F. Example of a dye AC heater

[0151] In some cases, it may be desirable to heat the dye / blood mixture directly rather than transferring heat from a heating source. In this regard, the dye may have a conductivity of about 0.011*Siemens / cm and / or a resistivity of 0.909*Ω*m. Electrically, this constitutes a resistive element. A resistive element heats up when current flows through it. In this case, the fluid is the heat source, and the power dissipation is uniform along the length of the path because each differential length has the same resistivity. This produces consistent heating in the dye / blood mixture, thereby producing consistent cell staining.

[0152] The volume used in the imaging test plate can be about 0.2 mL. The amount of heat (energy) required to raise this volume depends on the initial stain / blood mixture temperature, assuming ambient conditions: 26 J, ambient is 15C; 18 J, where the ambient is 25C; 12 J, where the ambient is 32C.

[0153] One factor is to have these electrodes with minimal mass to minimize temperature losses and avoid temperature gradients near the electrodes. Another factor is to use alternating current or AC coupled excitation to avoid electrolysis and contact pitting.

[0154] FIG. 27A to FIG. 27B An example of an AC heater 1700 is shown in which a column of dye / blood mixture is used as a resistor by incorporating conductive electrodes 1710 at each end of a tube segment 1712 containing the mixture to be heated. Fig.27A As shown in , a voltage source 1714 may be applied, wherein switch 1716 represents a means for controlling the voltage source 1714 (e.g., driven by a temperature feedback sensor to close a loop PID control). When the mixture is introduced to cover the two electrodes 1710, switch 1716 closes and the mixture heats, as shown in FIG. Fig.27B as shown in .

[0155] The method can be applied not only to fluids in pipe segments, but also to any geometry that allows placement of electrodes within a given fluid volume. For example, the method can be applied to alternative arrangements with electrically insulating chambers 1722, such as Fig.28As illustrated in . As shown, the electrodes 1720 can each be a thin conductive metal film built into the chamber 1722 or deposited on the inner surface of the chamber 1722 via an electrochemical method. This method creates the ability to preheat the stain before introducing the blood, and in addition mix and heat the stain / blood mixture to improve the uniformity of the prepared sample. This method can save time because the stain / blood mixture does not need to be transported to a separate heater, thereby increasing the throughput of the staining process. Less transportation can result in less stress on the cells, so that morphological features can be better preserved.

[0156] It will be appreciated that the voltage required for either arrangement of AC heater 1700 may be generated in a variety of ways. For example, Fig.29 The method of using a voltage-controlled amplitude amplifier to control the amplitude to generate the required voltage is shown; Fig.30 shows another way of generating the required voltage using a voltage controlled oscillator and a band pass filter to vary the voltage amplitude; and Fig.31 Another way of generating the required voltage by integrating the control loop in the microcontroller / DSP is shown.

[0157] VI. Other matters

[0158] Any one or more of the teachings of this document may be combined with any one or more of the teachings disclosed in the following patent applications: International Patent Application No. [Atty. Ref. 0133788.0770692], entitled "Lighting Module for Biological Analysis and Biological Analysis Systems and Methods" filed on the same day as the present application; and / or International Patent Application No. [Atty. Ref. 0133788.0770694], entitled "Flowcell Holder and Biological Analysis Systems and Methods" filed on the same day as the present application. The disclosure of each of these international patent applications is incorporated herein by reference in its entirety.

[0159] Different arrangements of the components depicted in the drawings or described above and components and steps not shown or described are possible. Similarly, some features and sub-combinations are useful and can be adopted without reference to other features and sub-combinations. Embodiments of the present invention have been described for illustrative and non-restrictive purposes, and alternative embodiments will become apparent to readers of this patent. In some cases, method steps or operations may be performed or implemented in different orders, or operations may be added, deleted or modified. It is understood that in certain aspects of the present invention, a single component may be replaced by multiple components, and multiple components may be replaced by a single component to provide an element or structure or to perform a given one or more functions. Unless such an alternative is inoperable for practicing certain embodiments of the present invention, such an alternative is considered to be within the scope of the present invention. Therefore, the present invention is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications may be performed without departing from the scope of the appended claims.

Claims

1. A biological imaging analyzer, comprising: A staining module, the staining module is configured to produce stained cells, the staining module comprising: one or more chambers configured to receive a biological sample and a staining composition, and a heater configured to heat the biological sample and the staining composition; an illumination module configured to illuminate the stained cells; and An imaging module is configured to capture images of the stained cells.

2. The bio-imaging analyzer according to claim 1, further comprising a flow cell configured to allow the stained cells to flow therethrough, The flow cell also includes an imaging region in which the image is captured.

3. The biological imaging analyzer according to any one of claims 1 to 2, wherein: The imaging module includes a camera.

4. The biological imaging analyzer according to any one of claims 1 to 3, wherein: The one or more chambers are also configured to receive a lysis composition.

5. The biological imaging analyzer according to any one of claims 1 to 4, wherein: The staining composition also includes a lytic compound.

6. The biological imaging analyzer according to any one of claims 1 to 5, wherein: The stained cells are leukocytes having stained nuclear regions.

7. The biological imaging analyzer according to any one of claims 1 to 6, wherein: The heater includes an induction coil.

8. The biological imaging analyzer according to any one of claims 1 to 7, wherein: The one or more chambers include two or more chambers, and the heater is connected to all of the two or more chambers.

9. The biological imaging analyzer according to claim 8, wherein: The heater includes a mounting bracket connected to all of the two or more chambers and a heating element mounted to the mounting bracket.

10. A method for staining a biological sample using the biological imaging analyzer according to any one of claims 1 to 9, the method comprising: depositing the staining composition into the one or more chambers; preheating the dyeing composition in the one or more chambers; depositing the biological specimen into the one or more chambers; as well as The biological specimen and the staining composition are heated in the one or more chambers.

11. The method of claim 10, further comprising mixing the biological sample and the staining composition in the one or more chambers.

12. The method according to any one of claims 10 to 11, wherein: The staining composition also includes a lytic compound.

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