Flow cell holder and biological analysis system and method

Abstract

The biological imaging analyzer includes a flow cell (22) configured to flow biological cells therethrough and including an imaging region where images of the biological cells are captured. The analyzer also includes an illumination module configured to generate light, the illumination module including a light guide configured to transmit light to the imaging region of the flow cell. The analyzer further includes an imaging module (24) configured to capture images of the biological cells in the imaging region of the flow cell. The analyzer also includes a flow cell holder in operative connection with the flow cell. A method of positioning a flow cell for biological analysis includes providing a flow cell configured to flow a biological sample for analysis and operatively connecting the flow cell holder to the flow cell.

Description

Technical Field

[0001] Priority This application claims the benefit of U.S. Patent Application No. 63 / 391,536, titled "Lighting Module for Biological Analysis", filed on July 22, 2022; U.S. Patent Application No. 63 / 391,545, titled "Biological Sample Staining Module", filed on July 22, 2022; and U.S. Patent Application No. 63 / 391,549, titled "Flowcell Holder", filed on July 22, 2022, the disclosures of which are incorporated herein by reference.

Background Art

[0002] Various types of tests related to patient diagnosis and treatment can be performed by analyzing patient samples. This can include the analysis of a patient's microorganisms (i.e., "microbes"), which are microscopic organisms such as bacteria, fungi, or viruses that can be single-celled or multi-celled, as well as the analysis of samples to determine chemical structures, antigens, antibodies, blood cell counts, microparticles (e.g., deposits), and other factors that can affect patient health. When analyzing microorganisms, a biological sample containing the patient's microorganisms can be taken from the patient's infective body, body fluid, or abscess, placed within a test panel or array, combined with various reagents, incubated, and analyzed to assist in the patient's treatment. Analysis of patient chemical structures, immunoassays, blood cell counts, microparticles, and other characteristics can be performed similarly. For these various analyses, automated biochemical analyzers or biological test systems have been developed to facilitate the analysis of patient samples and to improve the accuracy and reliability of results compared to analysis using manual procedures, to assist in determining the effectiveness of various antimicrobial agents, to meet the needs of medical facilities and other facilities.

[0003] Blood cell analysis is one of the most commonly performed medical tests to provide an overview of a patient's health status. A blood sample can be taken from a patient's body and stored in a test tube containing an anticoagulant to prevent clotting. A whole blood sample typically contains three major classes of blood cells, namely red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Each class can be further divided into subclasses of its kind. For example, the five major types or subclasses of white blood cells (WBCs) 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 particles in a sample can vary due to pathological conditions, cell maturity, and other causes. Red blood cell subclasses can include reticulocytes and nucleated red blood cells.

[0004] Conventional methods of blood analysis have involved using indirect methods of analysis (e.g., impedance, light scattering, fluorescence intensity profile) to gather information about blood cells, such as cell count and cell type. However, these techniques can have limitations in the quality of information obtained about cells due to these indirect methods.

[0005] Newer analysis techniques can utilize imaging as part of the analysis process. However, there can be challenges in obtaining high-quality images sufficient to determine accurate cell counts and cell types. Therefore, it is necessary to ensure appropriate quality of images to obtain sufficient cell information useful in blood and biological analysis. SUMMARY OF THE INVENTION

[0006] Described herein are devices, systems, and methods for classifying objects, such as cells, within an image captured by an analysis device, such as a bioassay system that captures images of blood cells from a blood sample.

[0007] In some embodiments, a biological imaging analysis apparatus is described. The analysis apparatus includes a flow cell configured to flow biological cells therethrough and including an imaging region where images of the biological cells are captured. The analysis apparatus also includes an illumination module configured to generate light and including an optical guide configured to transmit the light to the imaging region of the flow cell. The analysis apparatus further includes an imaging module configured to capture images of the biological cells in the imaging region of the flow cell. The analysis apparatus also includes a flow cell holder in operable connection with the flow cell.

[0008] In some embodiments, the biological cells are stained blood cells. The light may be pulsed light. Additionally or alternatively, the optical guide may be in operable connection with the flow cell holder. The distance between the optical guide and the flow cell may be constant.

[0009] In some embodiments, the flow cell holder includes a connector that contacts a portion of the imaging region of the flow cell. The connector may include a pair of arcuate ridges. The pair of arcuate ridges may each include an engagement surface configured to frictionally engage a portion of the imaging region of the flow cell. The engagement surface may be configured to frictionally engage a portion of the imaging region of the flow cell on both sides of the flow path of the flow cell. The connector may include a metallic material. For example, the connector may include aluminum. Additionally or alternatively, a portion of the imaging region of the flow cell may include glass.

[0010] In some embodiments, a method of positioning a flow cell for biological analysis using a biological imaging analysis apparatus includes providing a flow cell configured to flow a biological sample therethrough and operably connecting a flow cell holder to the flow cell. The method may further include connecting an optical guide to the holder. Additionally or alternatively, a portion of the holder may abut the imaging region of the flow cell.

[0011] Although multiple examples are described in this specification, it is still to be understood by those skilled in the art that other examples of the described subject matter will become apparent from the following detailed description and the drawings which illustrate and describe exemplary examples of the disclosed subject matter. As will be understood, the disclosed subject matter is amenable to various modifications in various aspects 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.

[0012] This specification concludes with claims that particularly point out and distinctly claim the invention, but the invention is better understood from the following description of specific examples in conjunction with the accompanying drawings, in which like numbers identify the same elements.

Brief Description of the Drawings

[0013]

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DETAILED DESCRIPTION OF THE INVENTION

[0014] The drawings are not intended to be limiting in any way, and it is contemplated that the various embodiments of the present invention can be implemented 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 this specification, illustrate some aspects of the present invention and, together with the description, serve to explain the principles of the present invention, but it should be understood that the present invention is not limited to the exact arrangements shown.

[0015] The present disclosure relates to an apparatus, system, composition, and method for analyzing a sample containing particles. In one embodiment, the present invention relates to an automated particle imaging system comprising an analyzer, which can be, for example, a visual analyzer. In some embodiments, the visual analyzer can further comprise a processor to facilitate automated analysis of images.

[0016] According to some aspects of the present disclosure, a system comprising a visual analyzer can be provided to obtain an image of a sample containing particles suspended in a liquid. Such a system can be useful in characterizing particles in a biological fluid, such as, for example, detecting and quantifying red blood cells, reticulocytes, nucleated red blood cells, platelets, and white blood cells, including white blood cell differential counting, categorization and sub-categorization, and analysis. Other similar applications, such as characterizing blood cells from other fluids, are also contemplated.

[0017] The discrimination and / or classification of blood cells in a blood sample is an exemplary use for which the subject matter is particularly well suited, although other types of body fluid samples may also be used. For example, aspects of the disclosed technology may be used for the analysis of non-blood body fluid samples containing blood cells (e.g., white blood cells and / or red blood cells), such as serum, bone marrow, lavage fluid, serous fluid, exudate, cerebrospinal fluid, pleural fluid, ascites, and amniotic fluid. It is also possible that the sample can be a solid tissue sample, e.g., a biopsy sample that has been processed to yield a cell suspension. The sample may also be a suspension obtained from processing a stool sample. The sample may also be a laboratory or production line sample containing particles, such as a cell culture sample. The term sample can be used to refer to a sample obtained from a patient or laboratory or any fraction, portion, or aliquot thereof. The sample can be diluted, divided into portions, or stained in some processes.

[0018] In some embodiments, the sample is automatically presented, imaged, or analyzed. In the case of a blood sample, the sample can be substantially diluted with a suitable diluent or saline, thereby reducing the extent to which some cells can be hidden from view by other cells in an undiluted or low-dilution sample. The cells can be treated with agents that enhance the contrast of some cell aspects, using, for example, a permeabilization agent to make the cell membrane permeable, as well as a tissue dye that adheres to features such as granules and nuclei and reveals them. In some cases, it may be desirable to stain an aliquot of the sample for counting and characterizing particles, including reticulocytes, nucleated red blood cells, and platelets, as well as for white blood cell differential, characterization, and analysis. In other cases, a sample containing red blood cells can be diluted prior to introduction into and / or imaging within a flow cell or otherwise.

[0019] As previously explained, imaging cells can provide enhanced benefits for characterization and represent an advancement over conventional non-imaging techniques. However, it can be difficult to image the sample with sufficient quality and resolution to enable such characterization. Aspects herein include the use of flow imaging, i.e., the use of a sample passed through a flow cell having an imaging region. Capturing a “static” image of a cell sample within a biological sample as the sample moves through the analysis region of the flow cell is a challenge. For example, the sample may be moving at a rate of about 20 cm / s as it moves through the analysis region of the flow cell, and as a result, it can be difficult to capture a clear static image of the sample cells.

[0020] In some embodiments presented herein, and as described in further detail herein, a biological sample can be passed through a flow cell of a biological test system for analysis. For example, such a test system can include a microscopy imaging system 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 the analysis region of the flow cell. Such a microscopy 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, onto the imaging sensor of the camera in an image formation plane by a tube lens.

[0021] I. System Overview Turning now to the drawings, FIG. 1 schematically shows an exemplary flow cell 22 for carrying a sample fluid through a field-of-view region 23 of a high optical resolution imaging device 24 configured to image fine particles in a sample fluid stream 32 using digital image processing. The flow cell 22 is coupled to a source 25 of a sample fluid that may have been subjected to processes such as contact with and heating by a particle contrast agent composition. The flow cell 22 is also coupled to one or more sources 27 of a particle and / or intracellular organelle orientation liquid (PIOAL), such as a transparent glycerin solution having a viscosity greater than the viscosity of the sample fluid.

[0022] The sample fluid is injected into the inside of the flow cell 22 at a point where the PIOAL flow is substantially established through a flat opening at the distal end 28 of the sample feed tube 29, resulting in a stable and symmetric laminar flow of PIOAL above and below (or on both sides) of the ribbon-shaped sample stream. The sample and PIOAL streams can be supplied by a precision metering pump that moves the PIOAL along with the injected sample fluid along a substantially narrowing flow path. The PIOAL compresses over the sample fluid in the region 21 where the flow path narrows. Therefore, the reduction in the flow path thickness in the region 21 can contribute to the geometric focusing of the sample stream 32. The sample fluid ribbon 32 passes in front of or separately through the field of view region 23 of the high optical resolution imaging device 24 and is carried covered with the PIOAL downstream of the narrow region 21. In this high optical resolution imaging device 24, the image is collected using, for example, the CCD 48. In this way, flow imaging is performed and an image from the flowing sample stream and the cellular material contained therein is collected. The processor 18 can receive pixel data from the CCD 48 as an input. The sample fluid ribbon flows to the discharge section 33 together with the PIOAL.

[0023] As shown here, the narrow 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 smaller than the proximal thickness PT. The sample fluid can thus be injected through the distal end 28 of the sample tube 29 at a location that is distal to the proximal portion 21a and proximal to the distal portion 21b. Therefore, the sample fluid can enter the PIOAL envelope when the PIOAL stream is compressed by the region 21. 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 is restricted by the reduction in the flow path size of the flow cell.

[0024] The digital high-resolution imaging device 24 having the objective lens 46 is directed along the optical axis that intersects the ribbon-shaped sample stream 32. The relative distance between the objective 46 and the flow cell 22 is variable by the operation of the motor drive 54 for resolving and collecting the focused digitized image on the optical sensor array. Additional information regarding the construction and operation of an exemplary flow cell as shown in FIG. 1 is provided in U.S. Patent No. 9,322,752, entitled "Flowcell Systems and Methods for Particle Analysis in Blood Samples," issued on April 26, 2016, the disclosure of which is hereby incorporated by reference in its entirety, and / or U.S. Patent No. 9,470,618, entitled "Sheath Fluid Systems and Methods for Particle Analysis in Blood Samples," issued on October 18, 2016, the disclosure of which is hereby incorporated by reference in its entirety.

[0025] Aspects of the disclosed technology may also be applicable in contexts other than flow cell systems such as that shown in FIG. 1. For example, FIG. 2 illustrates a slide-based vision inspection system 200 in which aspects of the disclosed technology may be used. In the system shown in FIG. 2, a slide 202 containing a sample, such as a blood sample, is placed within a slide holder 204. The slide holder 204 may be adapted to hold several slides, or, as illustrated in FIG. 2, only one slide. An image capture device 206 comprising an optical system 208 and an image sensor 210 is adapted to capture image data depicting the sample within the slide 202.

[0026] Image data captured by image capture device 206 may be transferred to image processing device 212. Image processing device 212 may be an external device, such as a personal computer, connected to image capture device 206. Alternatively, image processing device 212 may be incorporated into image capture device 206. Image processing device 212 may include a processor 214 associated with memory 216 configured to determine the change needed to determine the difference between the actual focus and the correct focus of image capture device 206. When the difference is determined, instructions may be transferred to steering motor system 218. Steering motor system 218 may change the distance z between slide 202 and optical system 208 based on instructions from image processing device 212. Descriptions of techniques that may be used for focusing using this type of setup are provided in U.S. Pat. No. 9,857,361, entitled "Flowcell, Sheath Fluid, and Autofocus Systems and Methods for Particle Analysis in Urine Samples," issued January 2, 2018, the disclosure of which is incorporated herein by reference in its entirety, and / or U.S. Pat. No. 10,705,008, entitled "Autofocus Systems and Methods for Particle Analysis in Blood Samples," issued July 7, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0027] II. Examples of Lighting Modules In the context of imaging, including the flow imaging concepts discussed for biological imaging, proper illumination is important to enable proper visualization of biological material (e.g., blood cells). Illumination is an important criterion for an image capture device (e.g., a camera) to capture clear and bright images of a sample, for example, for algorithms to properly identify cell types.

[0028] In a system as shown in FIG. 1 or FIG. 2, an illumination module (also referred to as an illumination system or a lighting device) 300 as shown in FIG. 3 can be used to illuminate cells imaged by a camera, such as the high-resolution imaging device 24 of FIG. 1 or the image sensor 210 of FIG. 2. For example, the illumination module 300 can be incorporated in place of the light source 42 shown in FIG. 1. FIG. 3 shows the illumination module 300 in conjunction with an exemplary flow cell 302 that can be configured and operable like the flow cell 22 shown in FIG. 1, and a high-resolution imaging device 304 that can be configured and operable like the high-resolution imaging device 24 shown in FIG. 1. As shown, the illumination module 300 is positioned on the side of the flow cell 302 opposite the high-resolution imaging device 304 to illuminate an analysis region (also referred to as an image capture region or an imaging region), such as the field of view region of the flow cell 302, as the sample moves through the analysis region to facilitate capture of an image of the sample by the high-resolution imaging device 304. The cells of the sample can be stained, for example, via a staining module such as either of the staining modules 400, 500 described below, before moving through the flow cell 302.

[0029] In the example shown, the illumination 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. Each of the light emitters 312a, 312b, 312c can be any suitable light source, including, for example, an arc lamp, a light emitting diode (LED), or any other suitable light emitter for providing either pulsed illumination or continuous illumination. In some embodiments, each of the light emitters 312a, 312b, 312c can be configured to emit light of a different color than the other light emitters 312a, 312b, 312c. For example, the first light emitter 312a can include a red LED configured to emit red light having a wavelength of from about 600 nanometers to about 650 nanometers, such as about 620 nanometers, the second light emitter 312b can include a green LED configured to emit green light having a wavelength of from about 470 nanometers to about 600 nanometers, such as about 525 nanometers, and / or the third light emitter 312c can include a blue LED configured to emit blue light having a wavelength of from about 400 nanometers to about 470 nanometers, such as about 450 nanometers.

[0030] Each of the light emitters 312a, 312b, 312c is mounted on a side surface of the housing 310 in a row that extends substantially parallel to the optical axis of the high-resolution imaging device 304 such that the light emitted by each light emitter 312a, 312b, 312c can first be projected into the housing 310 in a direction substantially perpendicular to the optical axis of the high-resolution imaging device 304. As shown, each focusing lens 314a, 314b, 314c is mounted within the housing 310 and is axially aligned with the corresponding one of the light emitters 312a, 312b, 312c to focus the light emitted by the corresponding light emitter 312a, 312b, 312c.

[0031] Each of the dichroic elements 316a, 316b, 316c is mounted within the housing 310 in alignment with the corresponding one of the light emitters 312a, 312b, 312c to reflect and / or filter light emitted by one or more of the light emitters 312a, 312b, 312c (and focused by the corresponding focusing lenses 314a, 314b, 314c). In this regard, each of the dichroic elements 316a, 316b, 316c of the present example includes a corresponding reflective side 320a, 320b, 320c and a corresponding filtering side 322a, 322b, 322c. Each of the dichroic elements 316a, 316b, 316c is oriented obliquely with respect to the optical axis of the high optical resolution imaging device 304 and with respect to the light received from the corresponding focusing lenses 314a, 314b, 314c. For example, each of the dichroic elements 316a, 316b, 316c can be oriented at an angle of approximately 45 degrees with respect to the optical axis of the high optical resolution imaging device 304. More specifically, each of the dichroic elements 316a, 316b, 316c is oriented such that the corresponding reflective side 320a, 320b, 320c generally faces both the corresponding focusing lenses 314a, 314b, 314c and the high optical resolution imaging device 304, while the corresponding filtering side 322a, 322b, 322c generally faces away from both the corresponding focusing lenses 314a, 314b, 314c and the high optical resolution imaging device 304.

[0032] In this mode, the reflective sides 320a, 320b, 320c of each dichroic element 316a, 316b, 316c are configured to reflect the light emitted from the corresponding light emitters 312a, 312b, 312c (and focused by the corresponding focusing lenses 314a, 314b, 314c) so that the reflected light travels substantially parallel to the optical axis of the high optical resolution imaging device 304 and travels substantially perpendicular to the optical axis of the high optical resolution imaging device 304. For example, the reflective side 320a of the first dichroic element 316a can be configured to reflect the light emitted from the first light emitter 312a (and focused by the first focusing lens 314a) so that the reflected light travels substantially parallel to the optical axis of the high optical resolution imaging device 304, and the reflective side 320b of the second dichroic element 316b can be configured to reflect the light emitted from the second light emitter 312b (and focused by the second focusing lens 314b) so that the reflected light travels substantially parallel to the optical axis of the high optical resolution imaging device 304, and / or the reflective side 320c of the third dichroic element 316c can be configured to reflect the light emitted from the third light emitter 312c (and focused by the third focusing lens 314c) so that the reflected light travels substantially parallel to the optical axis of the high optical resolution imaging device 304.

[0033] In addition, the filtering sides 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 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 322c of the third dichroic element 316c can be configured to filter light reflected from the second dichroic element 316b and / or light reflected from the first dichroic element 316a (and filtered by the second dichroic element 316b). In this regard, the filtering sides 322a, 322b, 322c of each dichroic element 316a, 316b, 316c can be configured to suppress light having a wavelength below a corresponding predetermined threshold from passing therethrough. For example, the filtering side 322b of the second dichroic element 316b can be configured to suppress light having a wavelength below a predetermined threshold of about 596 nanometers from passing therethrough, and / or the filtering side 322c of the third dichroic element 316c can be configured to suppress light having a wavelength below a predetermined threshold of about 484 nanometers from passing therethrough. In some cases, the filtering sides 322b, 322c of the second and third dichroic elements 316b, 316c can be configured to allow about 95% of the light having a wavelength above the corresponding predetermined threshold to pass therethrough and / or to suppress about 99% of the light having a wavelength below the corresponding predetermined threshold from passing therethrough.

[0034] Thus, light emitted by the first light emitter 312a may be focused by the first focusing lens 314a, reflected by the reflective side 320a of the first dichroic element 316a, filtered by the filtering side 322b of the second dichroic element 316b, and filtered by the filtering side 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 320b of the second dichroic element 316b, and filtered by the filtering side 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 320c of the third dichroic element 316c. In this manner, the light emitted by light emitters 312a, 312b, 312c can be conditioned via dichroic elements 316a, 316b, 316c to improve the whiteness of the light before being focused together by collimating lens 318 into a single parallel beam of white light, which can then be transmitted out of housing 310 towards flow cell 302.

[0035] It should be understood that when the first light emitter 312a includes a red LED, the red light emitted by the first light emitter 312a is substantially unaffected by the filtering sides 322b, 322c of the second and third dichroic elements 316b, 316c due to the relatively high wavelength of the red light, which may be greater than the threshold of either of the filtering sides 322b, 322c.

[0036] The parallel beam of white light formed by the collimating lens 318 can be transmitted through an optical pipe (also referred to as an illumination column or a light guide), such as a hexagonal light pipe, towards the flow cell 302. The optical pipe can be configured to collect the parallel beam of white light, randomize the parallel beam of white light, and / or converge the parallel beam of white light onto the flow cell 302 (e.g., onto the field of view region of the flow cell 302). The optical pipe can be positioned with respect to the collimating lens 318 such that the parallel beam converges at a point (e.g., a phase) at the entrance of the optical pipe. The optical pipe can be attached to a flow cell holder (not shown) that holds the flow cell 302, such as the flow cell holder 700 described below, to firmly fix the exit of the optical pipe with respect to the flow cell 302. In this way, the distance between the optical pipe and the flow cell 302 is constant because the optical pipe and the flow cell 302 are operably connected through the flow cell holder 700.

[0037] In some embodiments, the light emitters 312a, 312b, 312c may be configured to provide pulsed illumination in profile and in synchronization with each other (e.g., simultaneously) to capture a still image of a sample cell moving through the flow cell 302. For example, the objective lens of the high optical resolution imaging device 304 may be opened, then the light emitters 312a, 312b, 312c may emit pulses of light simultaneously, then an image of the sample cell may be captured by the high optical resolution imaging device 304, and then the objective lens of the high optical resolution imaging device 304 may be closed. This process may be repeated any suitable number of times. In some cases, the duration of each pulse may be from about 1 microsecond to about 3 microseconds, such as about 2 microseconds. The pulse frequency may depend on the camera frame acquisition frequency, which may be from about 220 frames per second to about 300 frames per second. For example, 220 frames per second may correspond to one photo every about 4.5 milliseconds. The objective lens may be open for about 100 milliseconds, which may be sufficient to capture one image. In some embodiments, a higher frame rate may be used, for example, with a reduced field of view. The increased speed may be used depending on the particular application, such as the type of camera used. The increased speed may provide more data (e.g., more images of more sample cells) in less time, while the smaller field of view may remove visual landmarks (e.g., "black lines") that may be used for focusing. It should also be understood that the pixel rate may contribute to the amount of data provided. For example, increasing the pixel rate may compensate for decreasing the frame rate to provide the same amount of data.

[0038] In some embodiments, the light emitters 312a, 312b, and 312c can be configured to emit pulses of light sequentially 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 instants along the sample cell's path. The pixel representation of distance can then be used to calculate the sample cell's velocity to determine whether the sample cell is accelerating or decelerating and / or whether the flow is too fast to acquire reliable data. This diagnostic mode can be selectively enabled or disabled. For example, after operating in the diagnostic mode, the light emitters 312a, 312b, and 312c can be configured to simultaneously emit pulses of light as described above in the basic operating mode.

[0039] III. Examples of Staining Modules In some embodiments, the flow imaging system incorporates dyes and associated staining modules to enhance visualization of biological material (e.g., blood cells). The stains can be useful, for example, to stain the interior cellular regions of white blood cells to help visualize internal nuclear structures and identify cell types (e.g., identification as a subset of white blood cell types—e.g., neutrophils, lymphocytes, monocytes, eosinophils, or basophils). In some instances, dyes are applied to the outer surface of cells to enhance visualization of the cells (e.g., outer staining of red blood cells or platelets).

[0040] A. Example of a Single-Chamber Staining Module In a system such as shown in FIG. 1 or FIG. 2, a staining module (also referred to as a staining device) 400, as shown in FIGS. 6A-6C, can be used both to mix a sample with a staining agent and to incubate the sample mixture by heating before cells in the sample mixture are imaged by a camera such as the high-resolution imaging device 24 of FIG. 1 or the image sensor 210 of FIG. 2. For example, the staining module 400 can be incorporated in place of the source 25 shown in FIG. 1, or between the source 25 shown in FIG. 1 and the sample feed tube 29, to facilitate mixing of the sample and the staining agent and incubation of the sample mixture prior to capture of an image of the sample by the high-resolution imaging device 24. The staining agent can comprise any suitable composition. For example, the staining agent can be configured according to any one or more teachings of U.S. Patent No. 9,279,750, entitled "Method and Composition for Staining and Sample Processing," issued on March 8, 2016, the disclosure of which is hereby incorporated by reference in its entirety, and / or U.S. Patent No. 9,322,753, entitled "Method and Composition for Staining and Processing a Urine Sample," issued on April 26, 2016, the disclosure of which is hereby incorporated by reference in its entirety, and / or U.S. Patent Publication No. 2021 / 0108994, entitled "Method and Composition for Staining and Sample Processing," published on April 15, 2021, the disclosure of which is hereby incorporated by reference in its entirety.

[0041] In the embodiment shown, the staining module 400 includes a housing 410, a pair of ferromagnetic sheets 412, and a heater in the form of a heating coil 414 (FIG. 6C). In various embodiments, the heating coil 414 may include a resistive coil or, alternatively, an induction coil. As best shown in FIG. 6A, the housing 410 includes a plurality (e.g., four) side walls 420 that jointly define an inner 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 an upper wall 424 and a port 426 that extends through the upper wall 424 to the inner chamber 422. The port 426 may enable a dye dispensing device (not shown) to deliver a stain to the inner chamber 422 and / or enable a sample dispensing device (not shown) to deliver a sample to the inner chamber 422 such that the sample is added to the stain.

[0042] In some embodiments, the housing 410 may include a metal material having a relatively high thermal conductivity, such as aluminum, to facilitate uniform heating of the housing 410 and likewise uniform heating of the contents of the inner chamber 422. In the embodiment shown, the side walls 420 of the housing 410 are laminated with the respective ferromagnetic sheets 412 to improve the efficiency of heating (e.g., resistive heating or, alternatively, inductive heating) performed by the staining module 400 (e.g., due to the relatively low magnetic properties of aluminum). More specifically, each ferromagnetic sheet 412 is fixed to the corresponding pair of outer surfaces of the side walls 420. It should be understood that any suitable number of ferromagnetic sheets 412 may be used to laminate the side walls 420. In the embodiment shown, a thermally conductive compound 430 is deposited on the outer surfaces of the side walls 420 to adhere the ferromagnetic sheets 412 to the side walls 420. As best shown in FIG. 6B, an adhesive tape 432 is tightly wound around the ferromagnetic sheets 412 to firmly engage the inner surfaces of the ferromagnetic sheets 412 with the outer surfaces of the side walls 420.

[0043] As best shown in FIG. 6C, 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 metallic material having 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. The heating coil 414 in one embodiment functions as an inductor or induction coil and is operably coupled to a power unit 450, which is configured to drive the heating coil 414 to a frequency at which the heating coil 414 behaves as a resonant circuit that, under excitation, provides an alternating current, thereby providing an alternating magnetic field to or near the heating coil 414. This magnetic field may generate an electromagnetic field (EMF) on the outer surface of the side wall 420, and this may cause an alternating current. This current, in conjunction with the resistance of the housing 410, may result in power loss and heat the outer surface of the side wall 420. Such heating may be transferred to the contents of the chamber 422, such as a stain and / or a sample. It should be understood that such inductive heating may be carried out using relatively low input power and / or may achieve uniform heating of the contents of the chamber 422, thereby improving staining and / or lysis performance. In this regard, exciting the circuit at the resonant frequency may deliver maximum power, and exciting the circuit at increasing frequencies may effectively regulate power delivery. Alternative embodiments may utilize a resistive heater / resistance heating coil for the heating coil 414.

[0044] In some embodiments, a temperature sensor, such as a thermistor (not shown), may be configured to continuously sense the temperature of the contents of chamber 422. The temperature sensor may be configured to transmit a feedback signal indicative of the sensed temperature to a controller (not shown), and this controller may be configured to transmit a control signal to a power unit 450 for selectively driving heating coil 414. In this manner, the controller may 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 elements of an algorithm or computer-driven instructions provided to power unit 450 and / or heating coil 414 to optimally control the temperature. In some embodiments, the controller may be configured to transmit a control signal to a maintenance heater (not shown) for maintaining the contents of chamber 422 at a threshold temperature.

[0045] In one embodiment, multiple staining modules are contemplated that each utilize the structures of FIGS. 6A-6C (i.e., the plurality of structural elements 400). In this way, multiple samples may be stained, incubated, or otherwise prepared at similar times. In one example, each staining module has its own unique heating element. In one example, a staining module has a plurality of chambers 422, each of which can receive a sample, and a common heating structure connected to a single housing 410 (e.g., having a common heating coil 414 surrounding the plurality of chambers 422 and housing 410) for the entire module.

[0046] B. Example of a Multi-Chamber Staining Module In a system such as shown in FIG. 1 or FIG. 2, a multi-chamber staining module (also referred to as a staining device) 500 as shown in FIGS. 7A and 7B can be used for both mixing the sample with a staining agent and incubating the sample mixture by induction heating before the cells in the sample mixture are imaged by a camera such as the high-resolution imaging device 24 of FIG. 1 or the image sensor 210 of FIG. 2. For example, the staining module 500 can be incorporated in place of the source 25 shown in FIG. 1, or between the source 25 shown in FIG. 1 and the sample feed tube 29, to facilitate mixing of the sample and the staining agent and incubation of the sample mixture prior to capture of an image of the sample by the high-resolution imaging device 24. The staining agent can comprise any suitable composition. For example, the staining agent can be configured according to any one or more teachings of U.S. Patent No. 9,279,750, titled "Method and Composition for Staining and Sample Processing", issued on March 8, 2016, the disclosure of which is hereby incorporated by reference in its entirety; and / or U.S. Patent No. 9,322,753, titled "Method and Composition for Staining and Processing a Urine Sample", issued on April 26, 2016, the disclosure of which is hereby incorporated by reference in its entirety; and / or U.S. Patent Application Publication No. 2021 / 0108994, titled "Method and Composition for Staining and Sample Processing", published on April 15, 2021, the disclosure of which is hereby incorporated by reference in its entirety. It should be noted that these staining agents generally describe staining agents containing a lysing agent for lysing red blood cells, a penetrating agent for penetrating white blood cells, a staining element for staining the internal contents of white blood cells, and a repair element for repairing white blood cells so that the dye does not escape.

[0047] In the embodiment shown, the staining module 500 includes a heater in the form of a housing 510, a bracket (also referred to as a sleeve) 512, and a heating coil 514. The housing 510 includes a plurality of inner 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 an upper wall 524 and a plurality of ports 526a, 526b, 526c, 526d that extend through the upper wall 524 to the corresponding inner chambers 522a, 522b, 522c, 522d. The ports 526a, 526b, 526c, 526d may enable a dye dispensing device (not shown) to deliver a stain to the corresponding inner chambers 522a, 522b, 522c, 522d and / or enable a sample dispensing device (not shown) to deliver a sample to the corresponding inner chambers 522a, 522b, 522c, 522d such that the sample is added to the stain. Although four inner chambers 522a, 522b, 522c, 522d and corresponding ports 526a, 526b, 526c, 526d are shown, it should be understood that any suitable number of inner chambers 522a, 522b, 522c, 522d and corresponding ports 526a, 526b, 526c, 526d, such as two, three, or more than four inner chambers 522a, 522b, 522c, 522d and corresponding ports 526a, 526b, 526c, 526d, may be used. In some variations, the first and second inner chambers 522a, 522b may be configured for use as white blood cell (WBC) chambers 522a, 522b, while the third inner chamber 522c may be configured for use as a red blood cell (RBC) chamber 522c.

[0048] In some embodiments, the housing 510 may include a metal material having a relatively high thermal conductivity, such as aluminum, to facilitate uniform heating of the housing 510 and also uniform heating of the contents of the inner chambers 522a, 522b, 522c, 522d.

[0049] In the embodiment shown, a bracket or sleeve 512 is positioned around housing 510. Bracket 512 includes an inner bore 530 sized and configured to receive at least a portion of housing 510. In some embodiments, inner bore 530 may be sized and configured to slidably receive a portion of housing 510 such that the portion of housing 510 can be selectively inserted into and / or removed from inner bore 530. Bracket 512 also includes upper and lower edges 532, 534 that define a recessed region 536 therebetween. Recessed region 536 is sized and configured to accommodate at least a portion of heating coil 514.

[0050] The heating coil 514 includes a wire 540 wound around a bracket 512 on a recessed region 536. The wire 540 can include a metallic material having a relatively high electrical conductivity, such as copper. The wire 540 can have any suitable cross-sectional area and / or thickness and can be wound to define any suitable number of turns for the heating coil 514. The heating coil 514 is operably coupled to a power supply unit 550, which can be configured to drive the heating coil 514 to a frequency at which the heating coil 514 behaves as a resonant circuit that, when excited, provides an alternating current and thereby provides an alternating magnetic field to or near the heating coil 514. In this way, the heating coil 514 can act as an inductor and can be configured as an induction coil or an induction heating coil. This magnetic field can generate an electromagnetic field (EMF) on the outer surface of the housing 510, and this can cause an alternating current. This current, in combination with the resistance of the housing 510, can result in power loss and can heat the outer surface of the housing 510. Such heating can be transferred to the contents of one or more chambers 522a, 522b, 522c, 522d, such as a stain and / or a sample. Such 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 dissolution performance. In this regard, exciting the circuit at the resonant frequency can deliver maximum power, and exciting the circuit at increasing frequencies can effectively regulate power delivery.

[0051] In some embodiments, the bracket or sleeve 512 is made of a conductive material (such as a metallic material such as aluminum) to enhance heat transfer to the housing and inner surface that receives the blood sample and dye. In some embodiments, the bracket or sleeve 512 is made of a ferromagnetic material. In some embodiments, the bracket or sleeve 512 is not utilized and instead the heater / heater coil 514 is directly attached to the outer surface of the housing 510.

[0052] In some embodiments, a temperature sensor, such as a thermistor (not shown), may be configured to continuously detect the temperature of the contents of one or more of the chambers 522a, 522b, 522c, 522d. The temperature sensor may be configured to send a feedback signal indicative of the detected temperature to a controller (not shown), and this controller may be configured to send a control signal to a power unit 550 for selectively driving the heating coil 514. In this manner, the controller may stop heating the contents of the chamber 522 when a threshold temperature is reached. In some embodiments, the controller may be configured to send a control signal to a maintenance heater (not shown) for maintaining the contents of one or more of the chambers 522a, 522b, 522c, 522d at a threshold temperature.

[0053] C. Example of a Sample Preparation Process In a system as shown in FIG. 1 or FIG. 2, the process as shown in FIG. 8 can be used to perform sample preparation before the cells are imaged by a camera such as the high-resolution imaging device 24 of FIG. 1 or the image sensor 210 of FIG. 2. First, in the process of FIG. 8, the stain can be delivered to a chamber in step 601, such as chamber 422 of the staining module 400 shown in FIGS. 6A - 6C, or one or both of the WBC chambers 522a, 522b of the staining module 500 shown in FIGS. 7A - 7B. This can include, for example, delivering the stain through corresponding ports 426, 526a, 526b via a dye dispensing device to chambers 422, 522a, 522b. The stain can then be preheated in chambers 422, 522a, 522b in step 602, such as by induction heating. Next, the sample can be delivered to chambers 422, 522a, 522b in step 603. This can include, for example, delivering the sample through corresponding ports 426, 526a, 526b via a dye dispensing device so as to be added to the stain to chambers 422, 522a, 522b. In some embodiments, the delivery of the sample to chambers 422, 522a, 522b can include mixing the sample with the preheated dye within chambers 422, 522a, 522b. In the process of FIG. 8, a uniform sample mixture can then be formed in chambers 422, 522a, 522b in step 604. This can include, for example, using fluid energy to mix the sample and the dye, such as by periodically withdrawing the sample from chambers 422, 522a, 522b through corresponding tangential ports of housings 410, 510 and pushing the sample back in to perform countercurrent mixing. Alternatively, this can include using a magnet to drive spherical ferromagnetic balls placed within chambers 422, 522a, 522b to perform agitation mixing. As another example, this can include introducing one or more bubbles to the bottom of chambers 422, 522a, 522b to create a vortex.

[0054] The homogeneous sample mixture can then be heated within chambers 422, 522a, 522b, such as by induction heating or resistive heating, in step 605. In some embodiments, the homogeneous sample mixture can be heated to a threshold temperature by induction heating or resistive heating and then maintained at the threshold temperature by a maintenance heater. In embodiments using the multi-chamber staining module 500, it should be understood that the sample mixtures within both WBC chambers 522a, 522b can be heated simultaneously by induction heating, and any sample within the RBC chamber 522c can also be heated by induction heating (even if such heating of the sample within the RBC chamber 522c is not required prior to imaging), and that the fourth chamber 522d can remain empty. In some other embodiments using the multi-chamber staining module 500, one or more of the 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, are being flushed simultaneously, and in such cases, increased power can be provided to the heating coil 540 to counter any cooling effect that the flushing of the RBC chamber 522c might have on the heating of the sample mixture within the first WBC chamber 522a (e.g., by adding the same amount of energy as the energy lost due to such a cooling effect).

[0055] After the homogeneous sample mixture has reached the threshold temperature, the sample mixture can be transported to a flow cell, such as flow cell 22 of FIG. 1, to be imaged by a camera, such as camera 24 of the high-resolution imaging device of FIG. 1. For example, the homogeneous sample mixture can be transported directly from chambers 422, 522a, 522b to flow cell 22 (e.g., without further preparation). It should be understood that the throughput of the staining process can be improved by heating the sample mixture within the same chambers 422, 522a, 522b in which the sample mixture is formed.

[0056] The formation and inductive heating of the sample mixture is described as occurring within chambers 422, 522 of housings 410, 510, but it should be understood that alternative arrangements may include a tube having a lumen (not shown), within which the sample mixture can be formed and inductively heated in a manner similar to that described above. Additionally, or alternatively, any one or more of the teachings herein can be combined with any one or more of the teachings disclosed in U.S. Patent No. 9,429,524, entitled "Systems and Methods for Imaging Fluid Samples," issued on August 30, 2016, the disclosure of which is hereby incorporated by reference in its entirety.

[0057] In some embodiments, the addition of the diluent is part of the preparation step, and the diluent is added to each of chambers 522a - 522d before, after, or both before and after the blood sample is added 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 dye.

[0058] It should be understood that the preparation steps for the RBC chamber may differ from those for the WBC chamber. For example, the RBC chamber utilizes a preparation step involving a) receiving the diluent followed by the blood sample, b) receiving the blood sample followed by the diluent, or c) receiving the diluent, followed by the blood sample, followed by an additional diluent, and does not receive the dye. In this way, the preparation time for the RBC chamber may be shorter and the workflow may involve passing the RBC sample through the imaging process while the WBC sample is still being prepared.

[0059] In some embodiments, the staining reagent utilizes both a lysing agent (to lyse red blood cells) and a staining agent (to penetrate the remaining white blood cells, stain the internal regions, and fix the white blood cells so that the dye does not escape). In this way, a single staining reagent can be used to process a particular type of cell (e.g., white blood cells) to remove red blood cells and stain the remaining white blood cells. Other embodiments may utilize multiple compositions, e.g., a first lysing reagent to lyse red blood cells and a second staining reagent to stain white blood cells, and the workflow involves a chamber (e.g., a WBC chamber) receiving a separate lysing reagent and a separate staining reagent to prepare a WBC sample for visualization.

[0060] In some embodiments, the various chambers (e.g., 522a - 522d) are not intended to strictly prepare dedicated cell types, i.e., cell types can be carried over. For example, a chamber can alternately be used for RBC preparation and for WBC preparation. In this manner, when a sample within the chamber is prepared for imaging, a cleaning cycle can be utilized to clean the chamber prior to receiving a subsequent blood sample (e.g., chamber 522a can be configured to first prepare a specific amount of WBC for the same preparation run and then a specific amount of RBC for a sample preparation run, e.g., one WBC preparation followed by one RBC preparation, or two WBC preparations followed by one RBC preparation, followed by two more WBC preparations, etc.). A cleaning reagent such as a diluent or a detergent can be used between sample runs to eliminate carryover. Even in situations where a particular chamber is used only for a particular cell type (e.g., 522a is used only as a WBC chamber), there can be a cleaning step run after the sample is prepared and imaged to eliminate carryover.

[0061] Other embodiments may still utilize multiple dyes as part of the preparation process. For example, a first dye configured to stain white blood cells in the manner described herein, and a second dye configured to stain at least one of platelets or reticulocytes may be used. These staining compositions can be uniquely used in various workflows. For example, the first chamber of housings 410, 510 can be used to prepare a white blood cell sample, including receiving at least a WBC dye and a lysis reagent, while the second chamber of housings 410, 510 can be used to prepare a platelet sample that is different from the WBC dye and the lysis reagent - this chamber receives at least a platelet reagent.

[0062] The terms white blood cell (WBC) chamber and red blood cell (RBC) chamber are used to refer to sample preparation chambers for imaging, but it should be noted that the samples imaged as a result of the preparation process may enable in vivo imaging of multiple cell types. For example, the WBC chamber utilizes lysis to exclude red blood cells, but since lysis can still retain platelets and reticulocytes, the sample prepared in the WBC chamber can still image, for example, at least white blood cells, platelets, and reticulocytes. Similarly, the RBC chamber can receive a different preparation procedure (e.g., without lysis, or without a dye / lysis combination reagent) from the WBC chamber, but the sample 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.

[0063] IV. Examples of Flow Cell Holders In a system as shown in FIG. 1 or FIG. 2, a flow cell holder (also referred to as a flow cell stage or a flow stage) 700 as shown in FIGS. 9 to 15 can be used to facilitate imaging of cells by a camera, such as the high-resolution imaging device 24 of FIG. 1 or the image sensor 210 of FIG. 2. For example, the flow cell holder 700 can be used to adjustably support the flow cell 22 shown in FIG. 1. FIG. 9 shows the flow cell holder 700 together with an exemplary flow cell 702 configured and operable like the flow cell 22 shown in FIG. 1, and a high-resolution imaging device 704 configured and operable like the high-resolution imaging device 24 shown in FIG. 1. The flow cell holder 700 and the flow cell 702 can collectively define a flow cell device. As shown in FIGS. 9 to 10, the flow cell holder 700 is operably attached to the output drive of a motor 706 so as to be precisely movable toward and away from a high-resolution imaging device 704 that can be firmly attached to a frame 708 so as not to move. Although not shown, an illumination module can be positioned on the side of the flow cell 702 opposite the high-resolution imaging device 704 to illuminate an analysis region (also referred to as an image capture region or an imaging region), such as the field of view region of the flow cell 702 when the sample moves through the analysis region, in order to facilitate capture of an image of the sample by the high-resolution imaging device 704. Such an illumination module can be configured and operable in the same manner as, for example, the illumination module 300 described above. In some embodiments, the flow cell 702 may be the same as the flow cell 302 described above, and / or the high-resolution imaging device 704 may be the same as the high-resolution imaging device 304 described above. In the embodiment shown, the flow cell 702 includes a first portion 702a constructed of a first material, such as plastic (e.g., acrylic), and a second portion 702b (FIGS. 13 to 14) constructed of a second material different from the first material, such as glass or sapphire glass. The glass portion 702b can define the analysis region of the flow cell 702, while the plastic portion 702a can define the non-analysis region of the flow cell 702.

[0064] As best shown in FIGS. 11-12, the flow cell holder 700 includes a stage plate 710, a first gimbal 712, and a second gimbal 714. The stage plate 710 is operably coupled to a motor 706 to facilitate translational movement of the stage plate 710 along the optical axis of the high optical resolution imaging device 704, also referred to as the z-axis. The first gimbal 712 is movably coupled to the stage plate 710 to facilitate yaw movement of the first gimbal 712 about a vertical axis orthogonal to the optical axis of the high optical resolution imaging device 704, also referred to as the y-axis, and / or to facilitate translational movement of the first gimbal 712 along the y-axis. The second gimbal 714 is pivotally coupled to the first gimbal 712 to facilitate pitch movement of the second gimbal 714 about a horizontal axis orthogonal to the optical axis of the high optical resolution imaging device 704, also referred to as the x-axis, and / or to facilitate translational movement of the second gimbal 714 along the x-axis.

[0065] More specifically, the first gimbal 712 is movably coupled to the stage 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 spring-loaded plunger 726 such that rightward advancement of the left-hand screw 724 can translate the second gimbal 714 rightward along the x-axis and leftward retraction of the left-hand screw 724 can translate the second gimbal 714 leftward along the x-axis.

[0066] As best shown in FIG. 12, the yaw adjustment screw 730 and the corresponding spring-loaded plunger 732 extend through the stage plate 710 to engage the back surface of the first gimbal 712 on both sides of the y-axis. As a result, the forward movement of the yaw adjustment screw 730 can cause the first gimbal 712 to yaw counterclockwise about the y-axis, and the retraction of the yaw adjustment screw 730 backward can cause the first gimbal 712 to yaw clockwise about the y-axis. Similarly, the pitch adjustment screw 734 and the corresponding spring-loaded plunger (not shown) extend through both the stage plate 710 and the first gimbal 712 to engage the back surface of the second gimbal 714 on both sides of the x-axis. As a result, the forward movement of the pitch adjustment screw 734 can cause the second gimbal 714 to pitch counterclockwise about the x-axis, and the retraction of the pitch adjustment screw 734 backward can cause the second gimbal 714 to pitch clockwise about the x-axis.

[0067] In the illustrated embodiment, the second gimbal 714 includes a pair of forwardly extending support arms 738, the purpose of which will be described below.

[0068] Referring now to FIGS. 13 - 15 and continuing to refer to FIGS. 11 - 12, the flow cell holder 700 of the illustrated embodiment further includes an adapter plate (also referred to as a connector) 740, a fixed block 742, a mounting bracket 744, a compression spring 746 in the form of a coil spring, and an optical pipe holding member 748 configured to rigidly hold an optical pipe (also referred to as an illumination column or a light guide) 750. The optical pipe 750 may be configured to transmit light from an illumination module (not shown), such as illumination module 300, to the analysis region of the flow cell 702. In one embodiment, the optical pipe 750 randomizes the light received at a first end of the optical pipe 750 (the end closer to the illumination module 300 that generates light as previously described), and this randomized light is then projected at a second end of the optical pipe 750 (the end closer to the flow cell 702) to illuminate the imaging region of the flow cell 702. The distance between the optical pipe 750 and the flow cell 702 is important for creating a similar illumination profile regardless of the positioning of the flow cell 702 (by the flow cell holder 700), and in this way, it is important to keep this distance fixed or as close to fixed as possible. By positioning the optical pipe 750 as part of the flow cell holder assembly 700, this ensures an operable connection with the flow cell 702 and also ensures that the distance between the optical pipe 750 and the flow cell 702 / imaging region of the flow cell 702 remains constant to ensure this consistent illumination profile.

[0069] As best shown in FIG. 15, the adapter plate 740 includes a circular hole 760 and a pair of forwardly extending arcuate ridges 762 positioned on both sides of the hole 760. The arcuate ridges 762 each include a respective forward engaging surface 764 configured to frictionally engage a corresponding portion of the flow cell 702. For example, the engaging surface 764 may be configured to frictionally engage the back surface of the flow cell 702 (e.g., facing the high optical resolution imaging device 704). More specifically, the engaging surface 764 may be configured to frictionally engage the back surface of the glass portion 702b of the flow cell 702 that defines the analysis region on both sides of the y-axis from each other and thus on both sides of the flow path of the flow cell 702. Thus, the engaging surface 764 may define an attachment plane along which the glass portion 702b of the back surface flow cell 702 contacts the flow cell holder 700.

[0070] As will be described in more detail below, the flow cell 702 is sandwiched between the arcuate ridge 762 and the fixed block 742, so that the engaging surface 764 of the arcuate ridge 762 abuts against the back surface of the glass portion 702b of the flow cell 702 that defines the analysis region, and the flow cell 702 can be fixed to the flow cell holder 700. It should be understood that the glass portion 702b of the flow cell 702 may have a relatively low coefficient of thermal expansion, at least as compared to that of the plastic portion 702a of the flow cell 702. Additionally, the material thickness between the flow path of the flow cell 702 and the back surface of the glass portion 702b of the flow cell 702 may be substantially smaller than the material thickness between the flow path of the flow cell 702 and the back surface of the plastic portion 702a of the flow cell 702. Thus, by fixing the back surface of the glass portion 702b of the flow cell 702 to the engaging surface 764, the risk that the field of view region of the flow cell 702 moves out of focus in response to temperature changes can be substantially reduced or eliminated. In some embodiments, the adapter plate 740 may include a metal material having a relatively low coefficient of thermal expansion, such as aluminum, to further reduce the risk that the field of view region of the flow cell 702 moves out of focus in response to temperature changes. Due to the positioning of the engaging surface 764 of the arcuate ridge 762 on both sides of the flow path of the flow cell 702, the engaging surface 764 does not bridge the flow path (e.g., extend over or under the flow path), and as a result, the engaging surface 764 can avoid applying pressure to the flow cell 702 along the flow path.

[0071] As shown in FIG. 15, the adapter plate 740 includes alignment holes 766 and alignment slots 768 configured to align with corresponding alignment holes and slots (not shown) of the flow cell 702 and / or to receive through respective alignment pins (not shown) of the second gimbal 714 to facilitate proper alignment of the adapter plate 740 with respect to the flow cell 702 and / or the second gimbal 714.

[0072] In the illustrated embodiment, the mounting bracket 744 is secured to the support arm 738 of the second gimbal 714 by corresponding screws 770. As best shown in FIGS. 13-14, the mounting bracket 744 includes an internal cavity 772 sized and configured to securely receive an optical pipe retaining member 748, as will be described in more detail below, and a generally annular collar portion 774 configured to slidably support a fixed block 742. In some embodiments, the fixed block 742 may be movably coupled to the mounting bracket 744 by one or more screws 775 or the like. As shown, a pair of screw holes 776 are provided through an upper portion of the mounting bracket 744 to receive corresponding set screws (not shown), whereby pressure can be applied to the optical pipe retaining member 748 when it is positioned within the cavity 772 to inhibit inadvertent movement of the optical 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 such that a front end of a compression spring 746 can be seated against the shoulder 778, as will be described in more detail below.

[0073] The optical pipe holding member 748 is sized and configured to snugly receive the optical pipe 750 while aligning the optical pipe 750 with the analysis region of the flow cell 702 and positioning the outlet of the optical pipe 750 substantially proximal to the analysis region, and includes a longitudinal hole 780 extending along the z-axis. As shown, at least one screw hole 782 (FIG. 12) is provided through an upper portion of the optical pipe holding member 748 to receive a corresponding set screw (not shown), whereby pressure can be applied when the optical pipe 750 is positioned within the hole 780 relative to the optical pipe 750 to inhibit inadvertent movement of the optical pipe 750 relative to the optical pipe holding member 748. In this manner, the optical pipe 750 can be firmly fixed relative to the flow cell 702 such that the outlet of the optical pipe 750 can be firmly fixed relative to the flow cell 702, thereby facilitating proper illumination of the analysis region of the flow cell 702 through the optical pipe 750 and inhibiting the outlet of the optical pipe 750 from inadvertently shifting or otherwise moving away from the analysis region of the flow cell 702, and can be firmly attached to or otherwise connected to the flow cell holder 700. For example, the position of the optical pipe 750 relative to the flow cell 702 (e.g., the distance between the outlet of the optical pipe 750 and the flow cell 702) can remain fixed during adjustment of the position and / or orientation of the flow cell 700 by 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. As described above and herein, such a fixed position of the optical pipe 750 relative to the flow cell 702 is important to ensure consistent illumination and imaging quality of the biological sample.

[0074] The fixed block 742 includes a generally annular rearward engaging surface 790 configured to frictionally engage a corresponding portion of the flow cell 702 opposite the engaging surface 764 of the arcuate ridge 762 of the adapter plate 740 to sandwich the flow cell 702 therebetween. In this regard, the fixed block 742 also includes a generally annular recess 792 configured to slidably receive the collar portion 774 of the mounting bracket 744, as well as a generally annular shoulder 794 sized and configured such that the rearward end of the compression spring 746 can be seated against the shoulder 794. Thus, the compression spring 746 can be configured to urge the fixed block 742 rearwardly along the z-axis to promote frictional engagement between the engaging surface 790 of the fixed block 790 and the corresponding portion of the flow cell 702, as well as frictional engagement between the engaging surface 764 of the arcuate ridge 762 of the adapter plate 740 and the corresponding portion of the flow cell 702 to firmly sandwich the flow cell 702 between the fixed block 742 and the adapter plate 740.

[0075] In some embodiments, the fixed block 742 can slide slightly along the collar portion 772 of the mounting bracket 744 in a forward direction, such as along the z-axis, to accommodate thermal expansion of the flow cell 702 (e.g., of the plastic portion 702a) along the z-axis. In such a case, it should be understood that the compression spring 746 can be configured to maintain frictional engagement between the engaging surface 790 of the fixed block 790 and the corresponding portion of the flow cell 702, as well as frictional engagement between the engaging surface 764 of the arcuate ridge 762 of the adapter plate 740 and the corresponding portion of the flow cell 702.

[0076] The arcuate ridges 762 of the embodiments described above are incorporated into the adapter plate 740 that is fixed to the second gimbal 714, while the arcuate ridges 762, including their respective engaging surfaces 764, can alternatively be incorporated directly into the second gimbal 714.

[0077] In this regard, FIG. 16 shows an alternative arrangement in which the adapter plate 740 is integrally formed as a single (e.g., monolithic) component together with the second gimbal 714. In the arrangement shown in FIG. 16, the compression spring 746 is provided in the form of a wave disk.

[0078] In the illustrated embodiment, the second gimbal 714 also 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 to facilitate proper alignment of the second gimbal 714 with respect to the flow cell 702. As shown, the second gimbal 714 further includes a forwardly extending stabilizing arm 796, and this forwardly extending stabilizing arm 796 has a threaded hole 797 extending therethrough for receiving a spring load plunger 798. The spring load plunger 798 can be configured to apply pressure to the side surface of the flow cell 702 to reduce or eliminate any mechanical play that might 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.

[0079] The stage plate 710 of the embodiment described above is operatively attached to the output drive of the motor 706 to facilitate translational movement of the stage plate 710 along the z-axis (i.e., the optical axis of the high optical resolution imaging device 704) such that the flow cell holder 700 is precisely movable toward and away from the high optical resolution imaging device 704, although such relative movement can be provided in any other suitable manner.

[0080] In this regard, FIG. 17 shows an alternative arrangement in which the stage plate 710 is firmly attached to the frame 708 so as not to move, and at least the objective lens of the high optical resolution imaging device 704 is operably attached to the output drive of the motor 706 to facilitate translational movement 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), i.e., precisely movable toward and away from the flow cell holder 700.

[0081] V. Additional Exemplary System Features In a system such as shown in FIG. 1 or FIG. 2, various features may be implemented in addition to, or alternatively to, any one or more of the features described above, as will be described in more detail below.

[0082] A. Example of an LED Driver In some cases, a light source driver in the form of an electronic circuit can be designed to pass a high-throughput current pulse through an LED light source (e.g., similar to light emitters 312a, 312b, 312c) to generate a current and a modulated light pulse. The pulse duration of the pulse can be controlled to generate a pulse width in the range of about 1 μs to about 10 μs or more, as needed to control the exposure time of the imaging device. The light pulse can be used to control the image capture exposure time so that a clear image of a moving cell or particle can be obtained. By illuminating the particles at a sufficient speed to capture a non-blurry image, it enables the acquisition of images of blood cells, latex beads, particles, crystals, bubbles, debris, and a wide range of particles flowing through the flow cell at high speed. The repetition rate of the light pulse can be increased by increasing the trigger rate as needed, from less than about 1 Hz to greater than about 1 kHz, to enable the acquisition of sufficient image throughput. The light source characteristics can enable discrimination of details in contrast, internal particle content, accurate color reproduction, and adjustable color spectrum adjustment using the DAC interface and illumination power control of all light sources to provide an unlimited variety of color combinations. The light source can enable optimal focusing performance by the optical system and the digitizing imaging device.

[0083] LED illumination can be coupled, for example, through optical means including a parabolic mirror light reflector, an optical fiber bundle, a solid optical light guide, an optical fiber cable, a bifurcated optical fiber bundle, a focusing lens, a light guide with an internal mirror, an optical prism, so as to guide, shape, and combine pulsed illumination from the LED into the viewing window of the flow cell where particles, cells, or other objects visible under a microscope are moving within a tissue area for imaging. Filters are used to optimize the interrogation spectrum illuminating the flow cell and the sample stream therein. A hexagonal homogenizer pipe can be implemented to provide an interface between the illumination sources and to randomize the light rays coupled to the flow cell. Brightfield illumination can be implemented on the imaging digitizer.

[0084] In this regard, a capacitive discharge circuit with a synchronized trigger input can be provided. Functional sub-modules include input voltage power regulation, input voltage pre-regulator, discharge capacitor regulator, discharge capacitor bank, discharge capacitor voltage regulator, trigger pulse generator, capacitive discharge transistor, slow timer, interfacing signal, DC power for the discharge capacitor bank, DC power for logic, DC power for analog devices, communication interface with addressing for discharge voltage control, discharge trigger signal, gate driver disable / enable signal, slow timer disable / enable signal, and / or discharge capacitor low inductance interface.

[0085] The following rise and fall times of the current pulse can be kept below 250 ns so that the modulated light pulse can illuminate and extinguish to produce a symmetric and square pulse. These rise and fall times can enable the total duration of the light pulse to produce a useful light pulse with a duration width greater than about 1 μs. A low ESL charge / discharge capacitor may be desired to obtain a fast-rising edge pulse. A low inductance throughout the LED current path may be desired to obtain a fast-rising edge pulse. A low ESR capacitor can be implemented in the capacitive discharge circuit to avoid power loss and heating of the capacitor. The lighting device may be characterized by an enable signal for the purpose of operating the light source during the imaging phase of the system operation and disabling it when the system is not in operation. The white LED may be of a high CRI type to accurately reproduce colors by the imaging system. A light diffuser with a wide angle can be used between the LED and an optical light guide, or an optical fiber, or a parabolic reflector, or an elliptical reflector to diffuse the LED illumination in such a way that shadows are reduced within the image. A color filter can be used to optimize the color response of the LED illumination in such a way that excessive blue light is suppressed to provide a uniform color throughout the visible light spectrum. A focusing lens can be used to couple the LED illumination and match its numerical aperture to various objective lenses with different numerical apertures. The electronic control circuit is characterized by voltage regulation, controlling the voltage across the discharge capacitor, and as a result, controlling the voltage across the capacitor, and as a result, controlling the LED illumination power input and the light intensity. The electronic circuit may be characterized by pulse width control, and as a result, the light pulse time can be controlled and the light pulse can be controlled. In a multi-LED configuration, a timer can be implemented to add a time delay between multi-LED flashes, and a known time delay between flashes can be provided to give the system the ability to measure particle or cell velocity.

[0086] B. Example of a 2-Cube Beam Splitter RGB LED Combiner FIG. 18 shows an example of another lighting module 1300 that can be easily incorporated into a system as shown in FIG. 1 or FIG. 2 instead 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 cube beam splitters 1311a, 1311b, and a plurality of light emitters 1312a, 1312b, 1312c. Each of the light emitters 1312a, 1312b, 1312c can be any suitable light source including, for example, a light emitting diode (LED), or any other suitable light emitter for providing either pulsed illumination or continuous illumination. In some embodiments, each of the light emitters 1312a, 1312b, 1312c can be configured to emit light of a different color than the other light emitters 1312a, 1312b, 1312c. For example, the first light emitter 1312a can include a blue LED configured to emit blue light having a wavelength of from about 400 nanometers to about 470 nanometers, such as about 450 nanometers, the second light emitter 1312b can include a green LED configured to emit green light having a wavelength of from about 470 nanometers to about 600 nanometers, such as about 525 nanometers, and / or the third light emitter 1312c can include a red LED configured to emit red light having a wavelength of from about 600 nanometers to about 650 nanometers, such as about 620 nanometers.

[0087] Each of the cube beam splitters 1311a, 1311b can include a corresponding pair of right angle prisms 1315a, 1315b, 1315c, 1315d. Each of the beam splitters 1311a, 1311b can be coated with a bandpass dichroic coating that can efficiently reflect one color while transmitting the other color with high efficiency. 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.

[0088] The first light emitter 1312a is attached to an edge of the first beam splitter 1311a such that light emitted by the first light emitter 1312a can first be projected into the first beam splitter 1311a in a direction substantially parallel to the optical axis of the high-resolution imaging device 304. The second light emitter 1312b is attached to an upper part of the first beam splitter 1311a such that light emitted by the second light emitter 1311b can first be projected into the first beam splitter 1311a in a direction substantially perpendicular to the optical axis of the high-resolution imaging device 304. The third light emitter 1312c is attached to an upper part of the second beam splitter 1311b such that light emitted by the third light emitter 1312c can first be projected into the second beam splitter 1311b in a direction substantially perpendicular to the optical axis of the high-resolution imaging device 304. In this manner, blue light can pass completely through the beam splitters 1311a, 1311b, and the light pipe 1310 with high transmittance. Green light can be selectively reflected into the optical axis of the high-resolution imaging device 304 and immediately mixed with the blue light. Red light can be selectively reflected into the optical axis of the high-resolution imaging device 304 and immediately mixed with the blue and green lights. The light pipe 1310 can be configured to mix the light received from the beam splitters 1311a, 1311b to make it uniform. In this manner, the light emitted by the light emitters 1312a, 1312b, 1312c can be adjusted to improve the whiteness of the light, and this light can then be transmitted out of the light pipe 1310 toward the flow cell 302.

[0089] FIG. 19 shows an alternative arrangement in which the third light emitter 1312c is attached to a lower part of the second beam splitter 1311b such that light emitted by the third light emitter 1312c can first be projected into the second beam splitter 1311b in a direction substantially perpendicular to the optical axis of the high-resolution imaging device 304.

[0090] C. Example of a White Light LED Lighting Module with Multiple LEDs FIG. 20 schematically shows an example of another illumination module 1400 that can be easily incorporated into a system such as that shown in FIG. 1 or FIG. 2 in place of the illumination module 300 described above. In the example shown, the illumination module 1400 includes a mounting portion and / or housing (not shown), a plurality of light emitters 1412a, 1412b, 1412c, 1412d, an LED combiner optical system 1415, an LED collimator or focusing lens assembly 1418, an LED hexagonal homogenizer light pipe 1419, a light diffuser wafer 1421, a color filter (not shown) for adjusting the final illumination output, and a stage alignment calibration device (not shown). The light emitters 1412a, 1412b, 1412c, 1412d can each be any suitable light source, including, for example, light emitting diodes (LEDs), or any other suitable light emitter for providing either 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 can include a red LED configured to emit red light having a wavelength of from about 600 nanometers to about 650 nanometers, such as about 620 nanometers, the second light emitter 1412b can include a green LED configured to emit green light having a wavelength of from about 470 nanometers to about 600 nanometers, such as about 525 nanometers, the third light emitter 1412c can include a blue LED configured to emit blue light having a wavelength of from about 400 nanometers to about 470 nanometers, such as about 450 nanometers, and / or the fourth light emitter 1412d can include a high power white LED configured to emit white light.

[0091] The monochromatic light emitters 1412a, 1412b, 1412c can be configured to enhance the final illumination light beam and fill in the color spectrum breaks provided by the white light emitter 1412d. Although three monochromatic light emitters 1412a, 1412b, 1412c are shown, it should be understood that only one, two, or four or more monochromatic light emitters 1412a, 1412b, 1412c can be used. Thus, the illumination module 1400 can be used for hematology flow imaging and may be capable of providing programmable monochromatic, multi-color, or white light illumination within the visible spectrum.

[0092] In some embodiments, the illumination module 1400 can be configured to provide multi-strobe illumination, such as double and triple strobe. The illumination module 1400 may be able to activate or deactivate any combination of the individual LEDs 1412a, 1412b, 1412c, 1412d. The illumination module 1400 may be able to adjust the individual LED outputs from 100% to 0%. The light output from the light pipe 1419 can be directed to the flow cell and can include a homogenized light output that is red, green, blue, or white by combining the light emitted from the RGB LED sources 1412a, 1412b, 1412c, and the W LED source 1412d, and can provide illumination of the sample with a continuous light spectrum.

[0093] In addition to the output LEDs 1412a, 412b, 1412c, 1412d, the electronics of the illumination module 1400 can include LED switching and capacitive discharge (e.g., one for each LED), a pulse width generator (e.g., one channel for all LEDs), and a digitally controlled discharge power controller (e.g., 4 channels).

[0094] FIG. 21 shows an alternative arrangement having only two monochromatic light emitters 1412a, 1412b.

[0095] FIG. 22 shows an alternative arrangement having dedicated collimator optical systems 1418a, 1418b, 1418c, 1418d for respective light emitters 1412a, 1412b, 1412c, 1412d.

[0096] D. Example of a Block Heater for Sample Incubation FIG. 23 shows an example of a heating module 1500 that includes a pair of metal blocks 1510 having high thermal conductivity and low heat retention. For example, each of the blocks 1510 may be formed of aluminum. To maintain a stable temperature, a large mass of the heater blocks 1510 is used to increase heat retention and reduce temperature variations due to active thermal control. To increase the efficiency of heat conduction to all cells in the sample, a small inner diameter solid tube portion 1512 is used as an incubation vessel to increase the heat exchange surface contact area. The tube portion 1512 is grooved inside the aluminum block 1510 in a meandering style to balance the amount of left and right turns that blood cells need to make to prevent centrifugation in sample integrity. Heating elements, such as one or more heater pads 1514, are located in a central cavity 1516 of the aluminum block 1510 to provide uniform heating to the module 1500 and reduce losses to the surrounding environment. A non-thermal conductive material 1518 is used to surround the blocks 1510 to prevent heat exchange to the environment in order to maintain a temperature gradient even at the boundaries of the module 1500. A thermistor 1520 for thermal control feedback is also located inside the block 1510.

[0097] FIG. 24 shows an alternative arrangement having a modified meandering tube portion 1512.

[0098] E. Example of Sample Countercurrent Mixing In some cases, it may be desirable to improve the method of power delivery mixing and bubble mixing in one or more chambers, such as the chamber 1600 shown in FIG. 25. For example, countercurrent mixing may use repeated sample introduction / expulsion and controlled bubbles to increase the mixing power in one or more chambers 1600.

[0099] Figures 26A - 26B show an example of such countercurrent mixing, where a high - volume air gap of about 2 - 3 inches opens and passes through the ventilation ports on N14 and N8 and is introduced into each of the reagent delivery lines for RBC and WBC. The tube length between the ventilation valve and the chamber can hold the same volume as the target reagent delivery volume (Figure 26A). The ventilation port can then be switched to the delivery line for dispensing (Figure 26B). As soon as the blood sample is delivered to chamber 1600, the diluted sample can be drawn back without passing the air gap through the ventilation valve and then extruded in a slightly larger volume along with some air to cream the bubble mixing. The same draw - in and extrusion movements can be repeated about 2 - 5 times in an increased volume to achieve cumulative agitation mixing. Gradually, most of the air gap is consumed with a small amount remaining between the reagent port and the reagent in the tube to prevent sample diffusion.

[0100] It should be understood that controlled bubbles assist mixing without causing dissolved reagent formation. Repeated power delivery can better mix small - volume samples and can be adjustable to the required degree of homogeneity. Mixing can be evaluated as efficient and homogeneous based on low - count variation throughout the acquisition time. Thus, a dedicated mixing component may not be required.

[0101] F. Example of a Dye AC Heater In some cases, it may be desirable to directly heat the dye / blood mixture instead of transferring heat from a heat source. In this regard, the dye can have a conductivity of 0.011 Siemens / cm and / or a resistivity of 0.909 Ω·m. Electrically, this creates a resistive element. The resistive element gets hot when current flows through it. In this situation, the fluid is the heat source, and the power loss is uniform along the length of the path because each differential length has the same resistivity. This results in consistent heating in the dye / blood mixture and obtains consistent cell staining.

[0102] The volume used in the imaging breadboard can be about 0.2 mL. The amount of heat (energy) required to produce this volume depends on the initial dye / blood mixture temperature, which is assumed to be ambient conditions: 26 J at an ambient environment of 15°C, 18 J at an ambient environment of 25°C, and 12 J at an ambient environment of 32°C.

[0103] One factor is to fabricate these electrodes with the minimum mass to minimize temperature loss and avoid the temperature gradient in the proximity of the electrodes. Another factor is to use alternating current or AC-coupled excitation to avoid electrolysis and contact pore corrosion.

[0104] Figures 27A - 27B show an example of the AC heater 1700, where the column of the dye / blood mixture is used as a resistor by incorporating conductive electrodes 1710 at each end of the tube region 1712 containing the mixture to be heated. The voltage source 1714 can be applied as shown in Figure 27A, where the switch 1716 symbolizes means for controlling the voltage source 1714 (e.g., PID control driven from a temperature feedback sensor to close a loop). When the mixture spreads and is introduced to both electrodes 1710, as shown in Figure 27B, the switch 1716 is closed and the mixture gets hot.

[0105] This method is applicable not only to fluids within a tube region, but also to any geometry that allows for the placement of electrodes across a given fluid volume. For example, the method can be applied to an alternative arrangement having an electrically insulated chamber 1722 as illustrated in FIG. 28. As shown, electrodes 1720 can each be thin conductive metal films that are constructed into chamber 1722 or deposited electrochemically on the inner surface of chamber 1722. This approach results in the ability to improve the homogeneity of the prepared sample in addition to preheating the dye and mixing and heating the dye / blood mixture prior to blood introduction. This approach saves time because the dye / blood mixture does not need to be transported to a separate heater, thereby improving the throughput of the staining process. Less transport can mean less stress on the cells, and as a result, morphological features can be better preserved.

[0106] It should be understood that the voltage required for any arrangement of the AC heater 1700 can be generated in various manners. For example, FIG. 29 shows a manner of generating the required voltage using a voltage-controlled amplitude amplifier to control the amplitude, FIG. 30 shows another manner of generating the required voltage using a voltage-controlled oscillator and a band-pass filter used to change the voltage amplitude, and FIG. 31 shows another manner of generating the required voltage by integrating a control loop into a microcontroller / DSP.

[0107] VI. Others Any one or more of the teachings within this specification can be combined with any one or more of the teachings disclosed in the specification of International Patent Application No. [Atty. Ref. 0133788.0770692] titled "Lighting Module for Biological Analysis and Biological Analysis Systems and Methods", filed on the same day as this specification, and / or the specification of International Patent Application No. [Atty. Ref. 0133788.0770693] titled "Biological Sample Staining Module and Biological Analysis Systems and Methods", filed on the same day as this specification. The disclosure of each of these international patent applications is hereby incorporated by reference in its entirety into this specification.

[0108] Different arrangements of the components depicted in the drawings or described above, as well as components and steps not shown or described, are possible. Similarly, some features and sub - combinations are useful and can be used without reference to other features and sub - combinations. Embodiments of the invention are described for purposes of illustration and not limitation, and alternative embodiments will be apparent to the reader of this patent. In certain cases, method steps or operations can be performed or executed in a different order, or operations can be added, deleted, or modified. In certain aspects of the invention, it should be understood that a single component can be replaced by a plurality of components to provide an element or structure, or to perform a given function(s), and a plurality of components can be replaced by a single component. Such replacements are considered within the scope of the invention, except where such replacement does not function to practice a particular embodiment of the invention. Accordingly, the invention is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications can be made without departing from the scope of the following claims.

Explanation of Reference Signs

[0109] 18 processors 21 narrow area 21a proximal flow path portion 21b distal flow path portion 22 flow cell 24 high optical resolution imaging device 25 source 28 distal end 29 sample feed tube 32 sample flow stream 42 light source 46 objective lens 48 charge-coupled device 54 motor drive 200 vision inspection system 202 slide 204 slide holder 206 image capture device 208 optical system 210 image sensor 212 image processing device 214 processor 216 memory 218 steering motor system 300 lighting module 302 flow cell 304 high optical resolution imaging device 310 housing 312a, 312b, 312c light emitters 314a, 314b, 314c focusing lenses 316a, 316b, 316c dichroic elements 318 collimating lens 320a, 320b, 320c reflection side 322a, 322b, 322c filtering side 400 staining module 410 housing 412 ferromagnetic sheet 414 heating coil 420 side wall 422 chamber 424 upper wall 426 port 430 Thermal Conductive Compound 440 Wire 450 Power Supply Unit 500 Multi-Chamber Dyeing Module 510 Housing 512 Bracket / Sleeve 514 Heating Coil 522a, 522b, 522c, 522d Chambers 524 Upper Wall 526a, 526b, 526c, 526d Ports 530 Inner Bore 532 Upper Edge 534 Lower Edge 536 Recessed Region 540 Wire / Heating Coil 700 Flow Cell Holder 702 Flow Cell 704 High-Resolution Imaging Device 708 Frame 706 Motor 702a First Part / Plastic Part 702b Second Part / Glass Part 710 Stage Plate 712 First Gimbal 714 Second Gimbal 720 Vertical Adjustment Screw 722 Load Plunger 724 Horizontal Adjustment Screw / Left Screw 726 Spring Loaded Plunger 730 Yaw Adjustment Screw 732 Load Plunger 734 Pitch Adjustment Screw 740 Adapter Plate 742 Fixed Block 744 Mounting Bracket 746 Compression Spring 748 Optical Pipe Holding Member 750 Optical Pipe 760 Circular Hole 762 Bowed Ridge 764 Forward Engaging Surface 766 Alignment Hole 768 Alignment Slot 772 Internal Cavity / Color Portion 742 Fixed Block 774 Sub - Annular Color Portion 775 One or More Screws 776 Pair of Screw Holes 778 Shoulder 780 Hole 790 Rearward Engaging Surface 794 Shoulder 795 Alignment Pin 796 Stabilizing Arm 797 Screw Hole 798 Spring - Loaded Plunger 1300 Lighting Module A rectangular glass light pipe 1311a, 1311b A pair of cube beam splitters 1315a, 1315b, 1315c, 1315d A pair of right - angle prisms 1312a, 1312b, 1312c Multiple light emitters 1400 Lighting Module 1412a, 1412b, 1412c, 1412d Multiple light emitters 1415 LED Combiner Optical System 1418a, 1418b, 1418c, 1418d LED collimator or focusing lens assembly 1419 LED hexagonal homogenizer light pipe 1421 Light diffuser wafer 1500 Heating Module 1510 Block 1512 Small - inner - diameter solid tube portion 1514 Heater pad 1516 Central cavity 1518 Non - heat - conducting material 1520 Thermistor 1600 Chamber 1700 AC Heater 1712 Pipe area 1714 Voltage source 1716 Switch 1710 Conductive electrode 1720 Electrode 1722 Chamber

Claims

1. A biological imaging analysis device, a flow cell configured to flow biological cells therethrough and including an imaging region where an image of the biological cells is captured; an illumination module configured to generate light and including an optical guide configured to transmit the light to the imaging region of the flow cell; an imaging module configured to capture an image of the biological cells in the imaging region of the flow cell; a flow cell holder in an operable connection state with the flow cell A biological imaging analysis device comprising the above components.

2. The biological imaging analysis device according to claim 1 or 2, wherein the biological cells are stained blood cells.

3. The biological imaging analysis device according to claim 1 or 2, wherein the light is pulsed light.

4. The biological imaging analysis device according to any one of claims 1 to 3, wherein the optical guide is in an operable connection state with the flow cell holder.

5. The biological imaging analysis device according to claim 4, wherein the distance between the optical guide and the flow cell is constant.

6. The biological imaging analysis device according to any one of claims 1 to 5, wherein the flow cell holder includes a connector that contacts a part of the imaging region of the flow cell.

7. The biological imaging analysis device according to claim 6, wherein the connector includes a pair of arcuate ridges.

8. The biological imaging analysis device according to claim 7, wherein each of the pair of arcuate ridges includes an engagement surface configured to frictionally engage with the part of the imaging region of the flow cell.

9. The biological imaging analysis device according to claim 8, wherein the engagement surface is configured to frictionally engage with the part of the imaging region of the flow cell on both sides of the flow path of the flow cell.

10. The biological imaging analysis device according to any one of claims 6 to 9, wherein the connector includes a metallic material.

11. The biological imaging analysis device according to claim 10, wherein the connector includes aluminum.

12. The biological imaging analysis device according to any one of claims 6 to 11, wherein the part of the imaging region of the flow cell includes glass.

13. A method of positioning a flow cell for biological analysis using the biological imaging analysis device according to any one of claims 1 to 12, the method comprising: providing a flow cell configured to flow a biological sample for analysis; operatively connecting a flow cell holder to the flow cell A method comprising [

14. ] The method according to claim 13, further comprising connecting an optical guide to the holder. [

15. ] The method according to claim 13 or 14, wherein a part of the holder abuts on the imaging region of the flow cell.

Images