Oblique illumination of a sample imaged using flow through a microscope

Abstract

Techniques are presented for oblique illumination of a sample in microscopy imaging. A sample can be illuminated obliquely by directing light from an emitter toward the sample at an oblique angle rather than directly along the optical axis of the imager. The oblique angle can be formed using an aperture mask having an aperture that is not coaxial with the optical axis of the imager. The oblique angle can also be formed using an emitter and collector lens that are not coaxial with the optical axis of the imager, such that a condenser lens directs light toward the sample. Images acquired using oblique illumination can provide improved contrast of semi-transparent particles and have an embossed three-dimensional appearance.

Description

[Technical field]

[0001] background Imaging of cells and particles in a sample fluid stream can be used to identify cells and particles and determine whether an individual is healthy or diseased. To gather the necessary information from the image, the image must be clear and clearly show the particles and cells in the sample fluid stream. Currently available imaging in sample fluid systems often provides low contrast for, for example, semi-transparent particles and cells in the sample fluid stream, making it difficult to detect some particles and cells in the image. Summary of the Invention [Means for solving the problem]

[0002] One general aspect may include a system for microscopy, the system including a flow cell including a sample. The system may also include an oblique illumination system configured to illuminate the sample in the flow cell obliquely. The system may also include an imaging device configured to acquire an image of the sample during illumination of the sample. The oblique illumination system may include a light emitter configured to form an illumination of the sample by emitting light, the illumination being used to acquire an image of the sample. The oblique illumination system may also include a collector lens disposed between the light emitter and the flow cell to collect light from the light emitter. The oblique illumination system may also include a condenser lens disposed between the flow cell and the collector lens to receive and direct light from the collector lens toward the sample in the flow cell. Illuminating the sample obliquely may refer to illuminating the sample at an oblique angle. An oblique angle means a non-zero angle other than 90°. The oblique angle may be an angle relative to an optical axis of the imaging device, a normal to the sample, or a plane of the sample. The oblique angle may be, for example, in the range of 5-85°, 10-80°, or 20-70°. Other embodiments of this general aspect include corresponding computer systems, devices, and computer programs recorded on one or more computer storage devices, each configured to perform operations of a microscopy method corresponding to the described system.

[0003] Each implementation may include one or more of the following features: In some embodiments, the sample is a urine sample, a blood sample, a cerebrospinal fluid sample, a synovial fluid sample, a serous fluid sample, a pleural fluid sample, a pericardial fluid sample, a peritoneal fluid sample, or an amniotic fluid sample. In some embodiments, the light emitter is a light emitting diode. In some embodiments, the light emitter is an arc lamp. In some embodiments, the sample in the flow cell is in motion and the illumination from the light emitter is a light pulse. The use of light pulses to illuminate the moving sample advantageously prevents blurring in the acquired image. In some embodiments, the sample in the flow cell is not in motion and the illumination from the light emitter is continuous. Each implementation of the described technology may include hardware, methods or processes on a computer-accessible medium, or computer software.

[0004] In some embodiments, the oblique illumination system may further include an aperture mask including an aperture that allows light from the collector lens to pass through the aperture, where the aperture is not centered relative to the aperture mask such that the illumination of the sample is asymmetric. The aperture mask may preferably be an opaque mask that blocks light from the collector lens in all positions except through the aperture. Optionally, the light emitter, collector lens, aperture mask, condenser lens, and sample are each centered relative to the optical axis of the imaging device. Optionally, the diameter of the aperture may be based on the type of sample.

[0005] In some embodiments, the condenser lens and the sample can each be centered with respect to the optical axis of the imaging device, such that light from the center of the condenser lens illuminates the sample without tilt (e.g., directly or straight on) and light from the edge of the condenser lens illuminates the sample at an oblique angle. In other words, the optical path of the light from the center of the condenser lens can be coaxial with the optical axis of the imaging device, and therefore can be parallel (with respect to the plane of the sample) and coaxial (with respect to the plane of the sample) to the normal of the sample. Thus, the light from the center of the condenser lens can illuminate the sample directly (without tilt (at substantially 0°) with respect to the optical axis or the normal of the sample). Such direct illumination of the sample can be referred to as on-axis illumination or normal illumination. The optical path of the light from the edge of the condenser lens can be non-axial with respect to the optical axis of the imaging device and can have a non-zero angle with respect to the optical axis. Thus, the light from the edge of the condenser lens illuminates the sample at an oblique angle. By oblique angle is meant a non-zero angle other than 90°. The oblique angle may be an angle relative to the optical axis, a normal to the sample, or a plane of the sample. The oblique angle may be, for example, in the range of 5° to 85°, 10° to 80°, or 20° to 70°. The collector lens may have a different size than the condenser lens, and the collector lens may not be coaxial with the optical axis of the imaging device. For example, the condenser lens may have a different diameter than the collector lens. As yet another example, the condenser lens may have a smaller diameter than the collector lens. Optionally, the light emitter is centered with respect to the collector lens. Optionally, the oblique illumination system includes a second light emitter configured to form the illumination of the sample by emitting light. The light emitter may be configured to form the illumination of the sample by emitting light having a first color, and the second light emitter may be configured to form the illumination of the sample by emitting light having a second color different from the first color.Optionally, the oblique illumination system includes a second collector lens disposed between the second light emitter and the flow cell to collect light from the second light emitter, the second light emitter may be centered relative to the second collector lens, the second collector lens may have a different size than the condenser lens, the second collector lens may not be coaxial with the optical axis of the imaging device, and the light emitter may not be centered relative to the second collector lens.

[0006] Another general aspect includes a method of microscopy. The method may include illuminating a sample in a flow cell at an oblique angle using a light emitter. The method may also include acquiring an image of the sample using an image acquisition device as the sample is illuminated at an oblique angle. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the method. Features described with respect to the system herein apply equally to the method of microscopy.

[0007] Each implementation may include one or more of the following features: In some embodiments, the light emitter is a light emitting diode and the illumination from the light emitter is a light pulse. The use of light pulses to illuminate the moving sample advantageously prevents blurring in the acquired image (e.g., when the acquired image is of a moving sample). In some embodiments, illuminating the sample obliquely includes capturing light from the light emitter with a collector lens, blocking light from the collector lens at all positions of the aperture mask except through the aperture that is not coaxial with the optical axis of the image acquisition device, and directing light from the aperture toward the sample with a condenser lens to form the oblique illumination. Optionally, the light emitter, condenser lens, and collector lens are each centered with respect to the optical axis of the image acquisition device. In some embodiments, illuminating the sample obliquely includes positioning the light emitter off-axis with respect to the optical axis of the image capture device, collecting light from the light emitter with a collector lens centered with respect to the axis of the light emitter, and directing light from the collector lens toward the sample with a condenser lens centered with respect to the optical axis of the image capture device to form the oblique illumination. Optionally, the collector lens has a different size than the condenser lens. Each implementation of the described techniques may include hardware, methods or processes on a computer-accessible medium, or computer software. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 illustrates aspects of a microscopy analysis system according to some embodiments. [Diagram 2] FIG. 1 illustrates an oblique illumination system according to some embodiments. [Diagram 3] FIG. 2 illustrates another oblique illumination system according to some embodiments. [Figure 4] FIG. 1 illustrates a method for oblique illumination, according to some embodiments. [Diagram 5]FIG. 1 illustrates another method for oblique illumination, according to some embodiments. [Figure 6] 1A-1C show exemplary images from an oblique illumination system, according to some embodiments. [Figure 7] FIG. 1 is a block diagram illustrating an exemplary computer system according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Analysis of cells or particles from living tissues (e.g., human, animal and plant) can be used as a medical diagnostic tool used to identify disease and cellular defects as well as healthy cells. Capturing cells or particles for analysis can be done, for example, by collecting particles from living tissue (i.e., biological samples) via a fluid. For example, a blood sample or urine sample from a human each contains cells, and the types and amounts of these cells can be analyzed and provided to a medical professional for analysis. Additionally, other particles within the sample can also be identified and provided for review.

[0010] Routine urinalysis is the third most frequently used patient laboratory profile by physicians worldwide. Urine testing is commonly required to screen new patients for subclinical metabolic or renal / urinary tract disease, to evaluate the status of patients with chronic renal / urinary tract disease, or to evaluate pre- or post-operative status.

[0011] Routine laboratory urinalysis may include a panel of semiquantitative chemical assays using impregnated test strips to detect the presence of hemoglobin, glucose, bilirubin and other substances, "macroscopic" examinations to visually determine color (related to disease or drug metabolites present) and clarity (based on particulate content), and microscopic examination of particulate matter, the presence of which is formed in the urine after it is concentrated to maximize sensitivity, directly reflects the condition of the kidneys, urinary tract and bladder.

[0012] Types of particles that are often present in normal urine include red and white blood cells (produced by vascular leakage), various types of epithelial cells lining the renal urinary tract, crystals of dissolved metabolites or drug crystals that can form under certain acid / base conditions, microorganisms such as bacteria, yeast, and Trichomonas, and "casts" formed from sediment proteins released from the renal glomeruli during stress. Kidney disease can cause renal hemorrhage and thus an increase in the observed concentration of blood cells, increased cast formation caused by leakage of the glomerular membrane, and increased desquamation of the renal endometrial epithelial cells.

[0013] Diseases of the lower urinary tract can be characterized by blood cells due to inflammatory bleeding, detachment of transitional epithelium, and evidence of infectious organisms. Because some urinary analytes are present in both healthy and diseased states, and others are associated only with pathological conditions, both detection and quantification of urinary particles per reference ranges are essential.

[0014] In general, the presence of formed urinary sediment components provides different diagnostic information than the urine chemistry profile and macroscopic examination information because only the chemistries measuring hemoglobin and leukocyte esterase directly relate to sediment findings (red and white blood cells).

[0015] Blood cell analysis is one of the most commonly performed medical tests to provide an overview of a patient's health. A blood sample may be taken from a patient's body and stored in a test tube containing an anticoagulant to prevent clotting. A whole blood sample usually consists of three major classes of blood cells, including red blood cells (i.e., erythrocytes), white blood cells (i.e., leukocytes), and platelets (i.e., thrombocytes). Each class may be further divided into subclasses of members. For example, the five major types or subclasses of white blood cells each have different shapes and functions. White blood cells may include neutrophils, lymphocytes, monocytes, eosinophils, and basophils. There are also subclasses of red blood cell types. The appearance of particles in a sample may vary according to disease states, cell maturity, and other causes. Subclasses of red blood cells may include reticulocytes and nucleated red blood cells.

[0016] Analysis of urine cells and particles is also common and provides information about the health of a patient. A patient can provide a urine sample without invasive procedures. Because the procedure is non-invasive, it can be repeated without shock to the individual providing the sample. Urine can contain many cells and particles, many of which are translucent and can be difficult to distinguish from the rest of the liquid sample. The techniques described herein allow for more accurate and effective differentiation and identification of these translucent particles.

[0017] Unless otherwise specified, references made in this disclosure to a "particle" or "particles" are understood to encompass any separate or formed object dispersed in a fluid. As used herein, a "particle" can include any component in a biological fluid that is measurable and detectable (e.g., by image and / or other measurable parameters). A particle can be of any material, any shape and any size. A particle can include a cell. Examples of particles include, but are not limited to, cells, including blood cells, fetal cells, epithelial cells, stem cells, tumor cells, or bacteria, parasites or any of the above or other fragments in a biological fluid. A blood cell can be any normal or abnormal, mature or immature cell that may be present in a biological fluid, including red blood cells ("RBCs"), white blood cells ("WBCs"), platelets ("PLTs") and other cells. Members herein also include immature or abnormal cells. Immature WBCs can include metamyelocytes, myelocytes, promyelocytes, and blasts. In addition to mature RBCs, RBC members may include nucleated RBCs ("NTRCs") and reticulocytes. PLTs may include "giant" PLTs and PLT clumps. Throughout this specification, images are described as images of cells or particles. Although often referred to as cells, images may be of any particle. Platelets, reticulocytes, nucleated RBCs, as well as WBCs including neutrophils, lymphocytes, monocytes, eosinophils, basophils, and immature WBCs including blasts, promyelocytes, myelocytes, or metamyelocytes are counted and analyzed as particles.

[0018] Exemplary urine particles may include urine sediment particles. Exemplary urine sediment particles may include red blood cells (i.e., RBCs), dysmorphic red blood cells, white blood cells (i.e., WBCs), neutrophils, lymphocytes, phagocytes, eosinophils, basophils, squamous epithelial cells, transitional epithelial cells, decoy cells, renal tubular epithelial cells, casts, crystals, bacteria, yeast, parasites, oval fat bodies, lipid droplets, sperm, mucus, trichomonas, cell clumps, and cell fragments. Exemplary cells may include red blood cells, white blood cells, and epithelial cells. Exemplary casts may include acellular casts, hyaline casts, unclassified casts, granular casts, waxy casts, broad casts, fatty casts, crystalline casts, RBC casts, WBC casts, cellular casts, and the like. Exemplary crystals may include, for example, calcium oxalate, triphosphate, calcium phosphate, uric acid, calcium carbonate, leucine, cystine, tyrosine, and amorphous crystals. Exemplary non-squamous epithelial cells may include, for example, renal tubular epithelial cells and transitional epithelial cells. Exemplary yeasts may include, for example, Saccharomyces cerevisiae and yeasts with pseudohyphae. Exemplary urinary sediment particles may also include RBC clumps, fat, oval fat bodies, and trichomonas. Certain of these particles may be difficult to see in imaging, for example, due to their translucent appearance. For example, hyaline casts are translucent and may be difficult to see in an image. Using oblique illumination as described herein allows particles that are otherwise difficult to see in an image, such as hyaline casts, to be more clearly seen in images acquired using oblique illumination.

[0019] Blood cell analysis, such as analysis of urine or other body fluids, can be performed using counting techniques. In imaging-based counting techniques, a pixel data image of a prepared sample that can pass through an observation area is acquired using a microscope objective connected to a digital camera. The pixel image data can be analyzed using data processing techniques and can also be displayed on a monitor. The pixel data image can be further stored in a storage device within the system or transmitted to an external location.

[0020] The term high optical resolution imaging device may include devices capable of acquiring particle images with sufficient visual distinction to distinguish morphological features and / or changes. Exemplary high optical resolution imaging devices may include devices having an optical resolution of 1 μm or less, including, for example, an optical resolution of 0.2 μm to 0.5 μm, e.g., 0.345 μm.

[0021] In some embodiments, the image acquired in any of the compositions and / or methods of the present invention may be a digital image. Optionally, at least part of the procedure for acquiring the image is automated. In some embodiments, the image may be acquired using a visual analysis device with a flow cell, a high optical resolution imaging device, or a digital image acquisition device.

[0022] Optionally, the image provides information regarding the cytoplasm, the nucleus and / or nuclear components of the cells. Optionally, the image provides information regarding the granular components and / or other morphological features of the cells. Optionally, the image provides information regarding the cytoplasm, the nuclear components and / or granular components of the cells. Granular and / or nuclear images and / or features can be determined independently or in combination with each other for both class and subclass classification of cells.

[0023] In the following description, for purposes of explanation, specific details are set forth in order to provide a thorough understanding of the embodiments of the invention. However, it will be apparent that various embodiments may be practiced without these specific details. The drawings and description are not intended to be limiting.

[0024] The systems depicted in some of the figures can be provided in a variety of configurations. Optionally, the system can be configured as a distributed system in which one or more components of the system are distributed across one or more networks in a cloud computing system. All features of the described system can be applied mutatis mutandis to the described method and conversely, all features of the method can be applied mutatis mutandis to the described system.

[0025] 1 illustrates an embodiment of a system 100 for imaging particles in a fluid sample. The fluid sample can be a bodily fluid sample, such as a blood sample or a urine sample. As illustrated herein, the system 100 includes a sample fluid injection system 110, a sheath fluid injection system 150, a processor 140, a flow cell 120, an image acquisition device 130, and an oblique illumination system 160. The flow cell 120 optionally combines with the sample fluid to form a flow path 122 that conveys a sheath fluid flow. Within the flow cell 120, the sample can flow continuously or in motion during image acquisition. In some embodiments, the sample can be stationary during image acquisition so that the sheath fluid flow can be paused to form a particular portion of the sample for imaging at an image acquisition location 132. According to some embodiments, the sample fluid injection system 110 can include or be connected to a cannula or tube 112. The sample fluid injection system 110 can be fluidly connected to the flow path 122 (e.g., via the sample fluid inlet 102) and is operable to inject sample fluid 124 through the distal exit port 113 of the cannula 112 into the sheath fluid 126 flowing within the flow cell 120 to form a sample fluid flow 128.

[0026] The processor 140 may include, or may be operatively associated with, a storage medium having a computer application configured, when executed by the processor, to cause the sample fluid injection system 110 to inject the sample fluid 124 into the flowing sheath fluid 126. As shown here, the sheath fluid 126 may be introduced into the flow cell 120 by a sheath fluid injection system 150 (e.g., via the sheath fluid inlet 101). For example, the processor 140 may include, or may be operatively associated with, a storage medium having a computer application configured, when executed by the processor, to cause the sheath fluid injection system 150 to inject the sheath fluid 126 into the flow cell 120.

[0027] The sample fluid stream 128 has a first thickness T1 adjacent the inlet tube 112. The channel 122 of the flow cell 120 reduces in channel size such that the thickness of the sample fluid stream 128 decreases from the initial thickness T1 to a second thickness T2 adjacent an image acquisition location 132. The image acquisition device 130 is aligned with respect to the image acquisition location 132 such that a plurality of particles of the sample fluid are imaged at the image acquisition location 132 of the flow cell 120.

[0028] The processor 140 is connected to the sample fluid injection system 110, the image acquisition device 130, and the optional sheath fluid injection system 150. The processor 140 is configured to initiate acquisition of an image of a plurality of particles from the sample fluid at the image acquisition location 132 of the flow cell 120. For example, the processor 140 may include or be operatively associated with a storage medium having a computer application configured, when executed by the processor, to cause the image acquisition device 130 to initiate acquisition of an image of a second plurality of particles from a second sample fluid at the image acquisition location 132 of the flow cell 120 within a period of imaging the first plurality of particles after the sample fluid flow has passed the image acquisition location 132.

[0029] The processor 140 can further control the oblique illumination system 160. The oblique illumination system 160 can provide illumination to the image acquisition location 132 for the image acquisition device 130 to acquire images of cells and particles in the sample fluid. The oblique illumination system 160 can provide illumination that is directed toward the sample fluid stream 128 at an oblique angle. At the image acquisition location 132, the flow cell 120 is transparent, so that illumination from the oblique illumination system 160 can illuminate the sample fluid and image acquisition device 130 can acquire images. The oblique illumination system 160 is described in more detail with reference to Figures 2 and 3. The oblique illumination system 160 can provide illumination pulses (i.e., light pulses) that make the sample with the sample fluid stream 128 appear frozen, allowing the image acquisition device 130 to acquire still images without the blurring effect that is normally caused by the motion of the sample. The processor 140 can coordinate the pulsing of light emitters in the oblique illumination system 160 and in the image acquisition device 130 while the oblique illumination system 160 is illuminating the sample fluid stream 128 to cause the image acquisition device 130 to acquire an image. In some embodiments, the sample fluid stream 128 does not need to be in motion during image acquisition, in which case the oblique illumination system 160 can provide continuous illumination of the sample. For example, the sample fluid stream 128 can be moved to place a portion of the sample at the image acquisition location 132 and paused until the image acquisition device 130 takes the image. In this manner, the sample fluid stream 128 can move a portion of the sample during image acquisition so that it can be observed at the image acquisition location 132. The processor 140 can coordinate the motion of the sample fluid stream 128 and the image acquisition device 130 to ensure that the image acquisition device 130 acquires an image when the sample fluid stream 128 is not moving. In this embodiment, the oblique illumination system 160 can provide continuous illumination because no pulses are required to "freeze" the motion of the sample. In some embodiments, even if the sample fluid stream 128 is not moving during image acquisition, the oblique illumination system 160 can provide illumination only during image acquisition, such that light pulses are formed by the oblique illumination system 160 to illuminate the sample only during image acquisition.

[0030] 2 shows a block diagram of an oblique illumination system 160 for illuminating a sample 230. Having described the oblique illumination system 160 with respect to FIG. 1, an embodiment of how oblique illumination can be achieved will now be described. The sample 230 may be part of the sample fluid stream 128 within the image acquisition location 132, as described with respect to FIG.

[0031] The oblique illumination system 160 may include a light emitter 205, a collector lens 210, an aperture mask 215, and a condenser lens 225. Although not shown here for clarity of the components shown and described, the oblique illumination system may include other components such as an interface to the processor 140, a housing unit, coupling parts for coupling the oblique illumination system 160 to a housing of a microscope system, a stabilizing coupling member to ensure that the oblique illumination system 160 does not move relative to the flow cell 120, and an alignment mechanism that allows for adjustment of the precise positioning and alignment of the oblique illumination system relative to the image acquisition device.

[0032] The light emitter 205 may be any suitable light source. The light emitter 205 may be a pulsed light emitter 205 to "freeze" the sample for the image capture device 130. When light from the light emitter 205 hits the image capture location 132, the image capture device 130 images the sample 230 and can capture a clear image without motion blur. In some embodiments, the light emitter 205 can provide continuous illumination of the sample 230, for example, when the sample 230 is not moving. The light emitter 205 can be an arc lamp, a light emitting diode (LED), or any other suitable light emitter configured to pulse or flash with a very short duration (e.g., 1-10 microseconds) and a short off time (e.g., 10-20 milliseconds), as well as any other suitable light emitter configured to provide continuous illumination. The light emitter 205 can receive a signal from the processor 140 indicating when to turn the flash on and off, which can be synchronized with the image capture device 130 to capture an image during the light flash. As faster processors and imagers are developed, imaging at higher frame rates may be possible, but the exemplary time frames of use are not intended to limit the functionality of the system. In some embodiments, the light emitter 205 may be a continuous light source to provide continuous illumination at the image capture location 132. For example, the sample 230 may be a stationary sample (i.e., one that does not move during image capture), and thus no light pulses are required to "freeze" the sample for image capture.

[0033] The light emitter 205 can emit light directly outward along the optical axis 235, as well as at angles including light beams 240, 245, and 250 and all positions in between. The light emitter 205 can be centered with respect to the optical axis 235. The optical axis 235 can be determined based on the image capture device 130. The image capture device 130 has the optical axis 235 about which its rotational symmetry is centered.

[0034] The collector lens 210 may be any collector lens suitable for collecting the light emitted from the light emitter 205. The collector lens 210 may have a diameter determined based on the geometry of the light emitter 205. The geometry of the light emitter 205 may allow the light to be emitted, for example, as a predetermined point (e.g., if an LED emits point-like light) or as a predetermined line (e.g., if an arc lamp emits line-like light). The size of the collector lens is determined based on the geometry of the light so that the light is captured and then a uniform light transmission occurs. For example, if the light emitter 205 is an LED, the collector lens 210 may have a smaller diameter than if the light emitter 205 is an arc lamp. The collector lens 210 may collect the light emitted from the light emitter 205 and transmit the light uniformly to the aperture mask 215. The light beams 240, 245, and 250 show such a uniform transmission. The collector lens 210 may be centered with respect to the optical axis 235. In some embodiments, the collector lens 210 and / or other components, including the aperture mask 215, the condenser lens 225, the light emitter 205, and the sample 230, may be tilted and still be centered with respect to the optical axis 235. Regardless of whether one or more components are tilted, the illumination provided by the oblique illumination system 160 is still an oblique illumination of the sample 230.

[0035] The aperture mask 215 can include an aperture 220. The aperture 220 can be a void or hole in the aperture mask 215. The aperture mask 215 can be an opaque mask that blocks light from the collector lens everywhere except through the aperture 220. The aperture mask 215 can have the same diameter as the collector lens 210 or a larger diameter and is centered about the optical axis 235, which allows uniform transmission of light from the collector lens 210 to be blocked by the aperture mask 215 or to pass through the aperture 220. For example, light beams 240 and 245 can pass through the aperture 220. However, light beam 250 is blocked by the aperture mask 215. The aperture 220 can be a circular aperture, the size of which is determined based on the type of sample 230. For example, a sample 230 containing particles that are difficult to see (e.g., translucent particles) can be more easily seen if the aperture 220 has a smaller size than the aperture size required when the particles are less difficult to see (e.g., opaque particles). The geometry of the aperture 220 can be any suitable shape, including, for example, circular, elliptical, teardrop, heart, square, triangular, octagonal, etc. The selection of the shape of the aperture 220 can be based on the type of sample 230. In oblique illumination, the aperture 220 is not coaxial with the optical axis 235. In other words, the aperture 220 is not centered with respect to the optical axis 235. The light transmitted through the aperture 220 then reaches the condenser lens in a non-centered state with respect to the optical axis 235. In FIG. 2, this is illustrated by the exemplary light beams 240 and 245 that are not coaxial with respect to the optical axis 235. It should be noted with respect to aperture mask 215 that conventional systems have a loss of approximately ninety-nine percent (99%) of light from the light emitter. The significant loss of light occurs for at least two reasons. First, conventional systems use arc lamps, and the shape of the light arc is linear, and point light is much better suited for manipulating light than line light because it is easier to achieve uniform illumination with point light.A diffuser is provided in the illumination device to make the line light look more uniform, but the diffuser not only blurs the line light but also disperses the light in all of the multiple directing devices, resulting in a lot of light loss. In the present solution, a higher light efficiency is achieved by using LEDs. Secondly, the low numerical aperture used in the present solution system is larger than the low numerical aperture in the conventional system. The aperture in the conventional system needs to be very small for a low numerical aperture image that allows detailed observation. Therefore, the higher numerical aperture used in the present solution provides a higher light efficiency. According to the oblique illumination system 160 described above, about twenty-five percent (25%) of the light is retained, which is a significant efficiency improvement (i.e., only 75% loss compared to the conventional 99% loss). This efficiency gain allows the use of a lower power light emitter 205, which reduces the cost of the oblique illumination system 160 and therefore the entire microscope system. In addition, the smaller light emitter 205 creates more point-like illumination, which results in less light loss. This is because it is easier to create a uniform illumination pattern with point illumination from the light emitters 205. The cost savings here come from reduced cost of the low power electronics, reduced cost of powering the low power electronics, and reduced cost of replacing the light emitters 205 less frequently.

[0036] The condenser lens 225 is configured to tilt the received light towards the sample 230. All the light passing through the aperture 220 reaches the condenser lens 225 and is transmitted to the sample 230. The condenser lens 225 may have the same diameter as the collector lens 210. In some embodiments, the condenser lens 225 and the collector lens 210 are not the same size. For example, the condenser lens 225 may have a diameter smaller than that of the collector lens 210. The condenser lens 225 is centered with respect to the optical axis 235. The light from the aperture 220 is received by the condenser lens 225 and directed towards the sample 230, so that the sample 230 is illuminated by this light at an oblique angle. As shown in FIG. 2, the light beams 240 and 245 are directed towards the sample 230 so that the sample 230 is illuminated. According to FIG. 1, the image acquisition location 132 is illuminated. The resulting illumination of the sample 230 is an oblique illumination. 230 is oblique because the light beam from aperture 220 reaches sample 230 at an oblique angle. Light directly along optical axis 235 is blocked by aperture mask 215, so all of the illumination of sample 230 is oblique.

[0037] Figure 3 shows a block diagram of an alternative embodiment of an oblique illumination system 160 for illuminating a sample 230. Having described the oblique illumination system 160 with respect to Figure 1, an alternative embodiment of how oblique illumination can be achieved to the embodiment described with respect to Figure 2 is now described. The sample 230 may be part of the sample fluid stream 128 within the image acquisition location 132 as described with respect to Figure 1.

[0038] In this embodiment, the oblique illumination system 160 may include a light emitter 305, a collector lens 310, and a condenser lens 225. Although not shown here for clarity of the components shown and described, the oblique illumination system may include other components such as an interface to the processor 140, a housing unit, coupling parts for coupling the oblique illumination system 160 to a housing of a microscope system, a stabilizing coupling member to ensure that the oblique illumination system 160 does not move relative to the flow cell 120, and an alignment mechanism that allows for adjustment of the precise positioning and alignment of the oblique illumination system relative to the image acquisition device.

[0039] The light emitter 305 may be any suitable light source. The light emitter 305 may be a pulsed light emitter 305 to "freeze" the sample for the image capture device 130. The light emitter 305 may be the same as the light emitter 205, but located at a different position with respect to the optical axis 235. It should be noted that the light emitter 305 may be different from the light emitter 205. For example, the light emitter 305 may be smaller than the light emitter 205. When the light from the light emitter 305 hits the image capture location 132, the image capture device 130 images the sample 230 and can capture a clear image without motion blur. In some embodiments, the light emitter 305 can provide continuous illumination of the sample 230, for example when the sample 230 is not moving. The light emitter 305 may be an arc lamp, a light emitting diode (LED), or any other suitable light emitter configured to pulse or flash with very short duration (e.g., 1-10 microseconds) and short off time (e.g., 10-20 milliseconds), as well as any other suitable light emitter configured to provide continuous illumination. The light emitter 305 may receive a signal from the processor 140 indicating when to turn the flash on and off, and may synchronize the light flash or light pulse with the image capture device 130 to capture images during the light flash or light pulse. The light emitter 305 may also receive a signal from the processor 140 indicating when to turn on and off when continuous illumination is used. As faster processors and image capture devices are developed, imaging at higher frame rates may be possible, but this is not intended to limit the functionality of the system by the exemplary time frames of use. In some embodiments, the light emitter 305 may be a continuous light source that provides continuous illumination at the image capture location 132. For example, the sample 230 may be a stationary sample (ie, one that does not move during image acquisition), and thus no light pulse is required to "freeze" the sample for image acquisition.

[0040] The light emitter 305 can emit light parallel to the optical axis 235 (shown by light beam 320) and at angles including light beams 315 and 325 and all angles therebetween. The light emitter 305 does not have to be coaxial with the optical axis 235. The optical axis 235 can be determined based on the image capture device 130. The image capture device 130 has an optical axis 235 about which its rotational symmetry is centered. The light emitter 205 can be centered with respect to the optical axis 235.

[0041] The collector lens 310 may be any suitable collector lens for collecting the light emitted from the light emitter 305. The collector lens 310 may have a diameter determined based on the geometry of the light emitter 305. The geometry of the light emitter 305 may, for example, emit light as a predetermined point (e.g., if an LED emits point-like light) or as a predetermined line (e.g., if an arc lamp emits line-like light). The size of the collector lens is determined based on the geometry of the light so that the light is captured and then a uniform light transmission occurs. For example, if the light emitter 305 is an LED, the collector lens 310 may have a smaller diameter than if the light emitter 305 is an arc lamp. In some embodiments, the size of the collector lens 310 may be determined based on the type of sample 230. The collector lens 310 may collect the light emitted from the light emitter 305 and transmit the light uniformly to the condenser lens 225. Light beams 315, 320, and 325 illustrate such a uniform transmission. The collector lens 310 may not be coaxial with the optical axis 235, but is centered with respect to the light emitter 305. Such a non-coaxial arrangement provides the desired oblique illumination. In some embodiments, the collector lens 310 and / or other components, including the condenser lens 225, the light emitter 305, and the sample 230, may be tilted. Regardless of whether one or more components are tilted, the illumination provided by the oblique illumination system 160 is still an oblique illumination of the sample 230.

[0042] It should be noted that all of the light from the light emitter 305 is collected by the collector lens 310 and transmitted to the condenser lens 225. This embodiment is more efficient than the embodiment described with reference to Figure 2. Since all of the light from the light emitter 305 is directed towards the sample 230, very little light is lost. In this way, a lower power light emitter 305 can improve cost efficiency.

[0043] The condenser lens 225 has been described above with reference to FIG. 2. As described with reference to FIG. 2, the condenser lens 225 is configured to direct the light received from the collector lens 310 towards the sample 230. All the light emitted from the light emitter 305 and collected by the collector lens 310 reaches the condenser lens 225 and is directed towards the sample 230. The condenser lens 225 may have a larger diameter than the collector lens 310 as described above. The condenser lens 225 is centered with respect to the optical axis 235. The light from the collector lens 310, which is not coaxial with the optical axis 235, is directed towards the sample 230, so that the sample 230 is illuminated by the light at an oblique angle. As shown in FIG. 3, the light beams 315, 320 and 325 are directed towards the sample 230 so that the sample 230 is illuminated. According to FIG. 1, the image acquisition site 132 is illuminated. Because the light beam reaches the sample 230 at an oblique angle, the resulting illumination of the sample 230 is oblique illumination. Because no light is transmitted directly along the optical axis 235, all illumination of the sample 230 is oblique illumination.

[0044] In some embodiments, the area below light emitter 305 shown in Figure 3 can be used to provide, for example, a second oblique illumination emitter and a collector lens (not shown). In some embodiments, light emitter 305 and the second light emitter can each emit different colors of light.

[0045] FIG 4 illustrates a method 400 for oblique illumination of a sample and image acquisition of the sample. Method 400 may be performed, for example, using system 100 of FIG 1. More specifically, method 400 may be performed by oblique illumination system 160 and image acquisition device 130 described with respect to FIG 2. Method 400 may begin in step 405 with obliquely illuminating a sample in a flow cell using an illuminator. Oblique illumination system 160 may, for example, use illuminator 205 to obliquely illuminate sample 230 in flow cell 120.

[0046] In step 410, which is a sub-step of step 405, a collector lens can capture light from the light emitter. For example, the collector lens 210 can capture light emitted from the light emitter 205. For example, the processor 140 can pulse the light emitter 205, which can include processor-readable instructions for pulsing or flashing the light emitter 205, thereby allowing for blur-free imaging of moving objects. Thus, the sample can flow through the flow cell, and the pulsed light can provide a blur-free image. In some embodiments, the light emitter 205 can be a continuous illumination source that allows the sample to be stationary during imaging.

[0047] In a further sub-step of step 405, step 415, an aperture mask can block light from the collector lens everywhere except through an aperture that is not coaxial with the optical axis of the imager. For example, aperture mask 215 can block light from collector lens 210 everywhere except through aperture 220, which is not coaxial with the optical axis 235 of image acquisition device 130.

[0048] In a further substep of step 405, step 420, a condenser lens can direct the light from the aperture towards the sample to form the oblique illumination. For example, a condenser lens 225 can direct the light from the aperture 220 (light beams 240 and 245) towards the sample 230 to form the oblique illumination.

[0049] In step 425, the image acquisition device can acquire an image of the sample when the sample is illuminated at an oblique angle. For example, the image acquisition device 130 can acquire an image of the sample 230 in the flow cell 120 at the image acquisition location 132 when the sample is illuminated at an oblique angle by the oblique illumination system 160.

[0050] FIG 5 illustrates another method 500 for oblique illumination of a sample and image acquisition of the sample. Method 500 can be performed, for example, using system 100 of FIG 1. More specifically, method 500 can be performed by oblique illumination system 160 described with reference to FIG 3 and image acquisition device 130. Method 500 can begin at step 505 with obliquely illuminating a sample in a flow cell using a light emitter. Oblique illumination system 160 can, for example, illuminate sample 230 in flow cell 120 obliquely using light emitter 305.

[0051] In step 510, which is a sub-step of step 505, the light emitter is positioned off-axis relative to the optical axis of the imaging device. For example, the light emitter 305 can be positioned off-axis relative to the optical axis 235 of the image capture device 130. The light emitter 305 can be pulsed, for example, because the processor 140 includes processor-readable instructions for causing the light emitter 305 to pulse or flash, thereby coordinating with the image capture device 130 to capture clear images of the sample in the flow cell 120 while the sample is in motion. In some embodiments, the light emitter 305 can emit continuous illumination that is used to capture images of the sample while stationary.

[0052] In a further sub-step of step 505, step 515, the light from the light emitter can be collected by a collector lens. The collector lens may be centered with respect to the axis of the light emitter. For example, the collector lens 310 can collect the light from the light emitter 305. The collector lens 310 may be centered with respect to the light emitter 305. For example, an axis extending straight from the center of the light emitter 305, illustrated by the light beam 320 in FIG. 3, may be the axis about which the collector lens 310 is centered.

[0053] In a further substep of step 505, step 520, a condenser lens can direct the light from the collector lens towards the sample to form oblique illumination. For example, the condenser lens 225 can direct the light from the collector lens 310 (light beams 315, 320 and 325) towards the sample 230 to form oblique illumination.

[0054] In step 525, the image acquisition device may acquire an image of the sample when the sample is illuminated at an oblique angle. For example, the image acquisition device 130 may acquire an image of the sample 230 in the flow cell 120 at the image acquisition location 132 when the sample is illuminated at an oblique angle by the oblique illumination system 160.

[0055] An alternative exemplary image of a sample is presented in Fig. 6. In a microscope system, bright field illumination can be used to form the illumination of the sample. In such a system, the illumination source is behind the sample relative to the position of the image acquisition device. This indicates that, in the context of Fig. 1, the oblique illumination system 160 is on the opposite side of the image acquisition device 130 relative to the image acquisition location 132 of the flow cell 120.

[0056] The aperture size affects the resolution of the resulting image. For example, the smaller the aperture of the illumination system (e.g., aperture 220), the tighter the illumination of the sample can be focused, resulting in different imaging resolutions. In systems that do not use oblique illumination, the aperture is centered with respect to the optical axis of the imager. A low numerical aperture (meaning the aperture is smaller or has a smaller diameter) can result in a lower resolution for images to observe particles in the sample than a high numerical aperture (meaning the aperture is larger or as large as a condenser lens). In FIG. 6, three images 605, 610, and 615 taken during illumination of a particle 635 using illumination systems with varying numerical apertures are shown to illustrate the change in information available in each image. As shown, image 605 taken with an illumination system using a low numerical aperture of about 0.1 shows the particle 635 with some definition. Inset 620, shown as a dotted box, shows a detailed partial view of a portion of particle 635. The resolution and detail in image 605 shown in inset 620, as well as the particles within inset 620, are quite unclear.

[0057] Image 610, taken with an illumination system using a moderate numerical aperture of about 0.2, shows some definition of particle 635, which is more distinct than that shown in image 605. Inset 625, shown as a dotted box, shows a detailed partial view of a portion of particle 635, similar to inset 620. Inset 625 shows that some of the particles are more distinct than inset 620.

[0058] Image 615, taken with an illumination system using a high numerical aperture of about 0.4, shows particle 635 with the greatest amount of definition of images 605, 610, and 615. Inset 630, shown as a dotted box, shows a detailed partial view of some of particle 635, as dots 620 and 625. The details of particle 635 are much sharper in image 615.

[0059] Image 640 is an image acquired while illuminating particle 635 with oblique illumination using a system such as oblique illumination system 160. Image 640 has an embossed, three-dimensional appearance. While the image is not truly three-dimensional, the oblique illumination gives it a three-dimensional appearance. Details of the particles, particularly translucent particles such as particle 635, are much clearer in image 640 using oblique illumination than those shown in images 605, 610, and 615. The embossed, three-dimensional appearance gives depth to the uniform translucent particles, improving visibility for a human analyst and / or software analysis system.

[0060] As already mentioned, existing microscope systems typically use bright field illumination or other symmetrical illumination techniques. There are several reasons for this. The first reason is that the goal in engineering is often to find symmetry. Designing and analyzing optical systems is simpler with symmetry, so it is easier to work with symmetrical solutions. For this reason, symmetrical solutions are usually what is sought and used, and the search can end when such a working solution is found. Asymmetrical or asymmetrical solutions are more rarely sought. For this reason, oblique illumination, which deviates from symmetry by creating off-center illumination from the optical axis, is not an obvious choice to be used in the microscope systems described above. The second reason is that the change to an embossed three-dimensional appearance image is a big change from a typical two-dimensional image such as shown in images 605, 610, and 615. Microscope systems using symmetrical illumination have been around for many years. Images acquired by microscope systems are often analyzed by either software or humans to identify abnormalities or normalities to determine whether the patient from whom the sample was taken has a disease or health problem. Software used to analyze and evaluate images is often programmed using two-dimensional images. Similarly, human analysts are trained using two-dimensional images. Changing the imaging to an image with an embossed three-dimensional appearance is not an obvious option, as this would require reprogramming of the analysis software and additional training for the human analyst.

[0061] 7 illustrates a block diagram of an exemplary computing device 700 that can be used to perform the image acquisition and oblique illumination described herein. Computing device 700 may be or include, for example, a custom-purpose and / or custom-designed computing device, a server computer, a laptop computer, a desktop computer, a tablet, an e-reader, a smartphone or mobile device, a smart watch, a personal data assistant (PDA), or other electronic device.

[0062] Computing device 700 may include a processor 740 that interfaces to other hardware via a bus 705. Memory 710, which may include any suitable tangible (and non-transitory) computer-readable medium, such as RAM, ROM, EEPROM, etc., may embody program components (e.g., instructions 715) that configure the operation of computing device 700. In some examples, computing device 700 may include input / output ("I / O") interface components 725 (e.g., for interfacing with a display 745, keyboard, mouse, and / or the like) and additional storage devices 730.

[0063] The computing device 700 may include a network component 720. The network component 720 may be one or more of any components that enable a network connection. In some examples, the network component 720 may enable a wireless connection and may include a radio interface, such as IEEE 802.11, Bluetooth, or a radio interface for accessing a cellular network (e.g., a transceiver / antenna for accessing a CDMA, GSM, UMTS, or other mobile communication network). In other examples, the network component 720 may be wired and may include interfaces such as Ethernet, USB, IEEE 1394, RS232, and / or the like.

[0064] 7 shows a single computing device 700 with a single processor 740, the system may include any number of computing devices 700 and any number of processors 740. For example, multiple computing devices 700 or multiple processors 740 may be distributed over a wired or wireless network (e.g., a wide area network, a local area network, or the Internet). Multiple computing devices 700 or multiple processors 740 may perform any of the steps of the disclosure individually or in coordination with each other.

[0065] Each of the calculations or operations described herein can be performed using a computer or other processor having hardware, software and / or firmware. Various method steps can be performed by modules, which may include any of a wide variety of digital and / or analog data processing hardware and / or software configured to perform the method steps described herein. The modules optionally include data processing hardware adapted to perform one or more of the steps by providing appropriate machine programming code associated with the modules, with modules for two or more steps (or parts of two or more steps) being integrated into a single processor board or separated into different processor boards within any of a wide variety of integrated and / or distributed processing architectures. Such methods and systems often employ tangible media embodied with machine-readable code including instructions for performing the method steps described above. Suitable tangible media may include memory (including volatile and / or non-volatile memory), storage media (e.g., magnetic recording on a floppy disk, hard disk, tape, etc.; recording on optical memory such as a CD, CD-R / W, CD-ROM, DVD, etc.; recording on a flash drive or any other digital or analog storage medium), and the like.

[0066] Different arrangements of the components shown in the drawings or described above, as well as components and steps not shown or described, are possible. Similarly, some features and subcombinations are useful and can be used without reference to other features and subcombinations. Although embodiments of the invention have been described for non-limiting illustrative purposes, alternative embodiments will be apparent to the reader of this patent. In certain cases, method steps or actions can be performed or executed in a different order, or actions can be added, deleted, or modified. It will be understood that certain aspects of the invention can be substituted for a single component with multiple components and multiple components with a single component to provide an element or structure or to perform a given function or functions. Such substitutions are deemed to be within the scope of the invention, except where such substitutions are ineffective for the practice of a particular embodiment of the invention.

[0067] It should be understood that the drawings and descriptions of the embodiments of the present invention are simplified for the purpose of illustrating elements that are important for a clear understanding of the present invention. However, one skilled in the art would recognize that these and other elements may be desirable. However, because such elements are well known in the art and do not particularly aid in a better understanding of the present invention, descriptions of such elements are not provided herein. It should be understood that the figures are presented for illustrative purposes and not as structural drawings. Omitted details and modifications or alternative embodiments are within the scope of those skilled in the art.

[0068] The examples presented herein are intended to illustrate potential specific implementations of the present invention. It should be understood that the examples are primarily intended to explain the invention to those skilled in the art. Changes can be made to these diagrams or the operations described herein without departing from the spirit of the invention.

[0069] Moreover, while specific embodiments of the invention have been described herein for purposes of illustration and not limitation, those skilled in the art will recognize that many changes in the details, materials and arrangements of elements, steps, structures and / or parts may be made within the basic principles and scope of the invention without departing from the invention as set forth in the claims.

[0070] All patents, patent publications, patent applications, journal articles, books, technical literature, and the like discussed in this disclosure are hereby incorporated by reference in their entirety for all purposes.

[0071] The present technology may also be implemented as described in accordance with the following clauses.

[0072] Clause 1 1. A system for microscopy, comprising: a flow cell containing a sample; a light emitter configured to generate an illumination of the sample by emitting light, the illumination being used to acquire an image of the sample; a collector lens disposed between the light emitter and the flow cell, the collector lens collecting light from the light emitter; a condenser lens disposed between the flow cell and the collector lens to receive light from the collector lens and direct it toward the sample in the flow cell; an aperture mask including an aperture that allows at least a portion of the light from the collector lens to pass through the aperture, the aperture being non-centered with respect to the aperture mask such that illumination of the sample is asymmetric; and an imaging device for acquiring an image of the sample while illuminating the sample; A system for microscopy comprising:

[0073] Clause 2 2. The system of claim 1, wherein the sample is one of a urine sample, a blood sample, a cerebrospinal fluid sample, a synovial fluid sample, a serous fluid sample, a pleural fluid sample, a pericardial fluid sample, a peritoneal fluid sample and an amniotic fluid sample.

[0074] Clause 3 3. The system of claim 1 or 2, wherein the diameter of the aperture is based on the type of the sample.

[0075] Clause 4 4. The system of any one of clauses 1 to 3, wherein the light emitter is a light emitting diode.

[0076] Clause 5 4. The system of any one of clauses 1 to 3, wherein the light emitter is an arc lamp.

[0077] Clause 6 6. The system of any one of clauses 1 to 5, wherein the light emitter, the collector lens, the aperture mask, the condenser lens and the sample are each centered relative to the optical axis of the imaging device.

[0078] Clause 7 7. A system according to any one of clauses 1 to 6, wherein the sample in the flow cell is in motion and the illumination from the light emitter is a light pulse that prevents blurring in the acquired image.

[0079] Clause 8 7. The system of any one of clauses 1 to 6, wherein the sample in the flow cell is not moving and illumination from the light emitter is continuous.

[0080] Clause 9 1. A system for microscopy, comprising: a flow cell containing a sample; a light emitter configured to generate an illumination of the sample by emitting light, the illumination being used to acquire an image of the sample; a collector lens disposed between the flow cell and the light emitter and configured to collect light from the light emitter; a condenser lens disposed between the flow cell and the collector lens to receive light from the collector lens and direct it toward the sample in the flow cell; an imaging device for acquiring an image of the sample while illuminating the sample; Equipped with the sample and the condenser lens are each centered with respect to an optical axis of the imaging device, such that light from a center of the condenser lens illuminates the sample without tilt and light from an edge of the condenser lens illuminates the sample at an oblique angle; the collector lens has a different size than the condenser lens, and the collector lens is not coaxial with respect to an optical axis of the imaging device. system.

[0081] Clause 10 10. The system of clause 9, wherein the sample is one of a urine sample, a blood sample, a cerebrospinal fluid sample, a synovial fluid sample, a serous fluid sample, a pleural fluid sample, a pericardial fluid sample, a peritoneal fluid sample and an amniotic fluid sample.

[0082] Clause 11 11. The system of claim 9 or 10, wherein a diameter of the collector lens is based on a type of the sample.

[0083] Clause 12 12. The system of any one of clauses 9 to 11, wherein the light emitter is centered relative to the collector lens.

[0084] Clause 13 13. The system of any one of clauses 9 to 12, wherein the light emitter is a light emitting diode.

[0085] Clause 14 14. The system of any one of clauses 9 to 13, wherein the light emitter is an arc lamp.

[0086] Clause 15 15. A system according to any one of clauses 9 to 14, wherein the sample in the flow cell is in motion and the illumination from the light emitter is a light pulse that prevents blurring in the acquired image.

[0087] Clause 16 15. The system of any one of clauses 9 to 14, wherein the sample in the flow cell is not moving and illumination from the light emitter is continuous.

[0088] Clause 17 1. A method of microscopy comprising the steps of: using a light emitter to obliquely illuminate the sample in the flow cell; acquiring an image of the sample using an image acquisition device while the sample is obliquely illuminated; A method comprising:

[0089] Clause 18 18. The method of claim 17, wherein the sample in the flow cell is in motion, the light emitter is a light emitting diode, and the illumination from the light emitter is a light pulse that prevents blurring in the acquired image.

[0090] Clause 19 Obliquely illuminating the sample comprises: receiving light from the light emitter with a collector lens; blocking light from the collector lens at all positions of an aperture mask except through an aperture that is not coaxial with an optical axis of the image capture device; directing light from the aperture towards the sample with a condenser lens to form oblique illumination; 19. The method of clause 17 or 18, comprising:

[0091] Clause 20 Obliquely illuminating the sample comprises: positioning the light emitter non-coaxially with respect to an optical axis of the image capture device; collecting light from the light emitter with a collector lens positioned centered about an axis of the light emitter; directing light from the collector lens towards the sample with a condenser lens centered on an optical axis of the image acquisition device to form an oblique illumination; 19. The method of clause 17 or 18, comprising:

Claims

1. 1. A system for microscopy, the system comprising: a flow cell containing a sample; an oblique illumination system configured to obliquely illuminate a sample within the flow cell; an imaging device configured to acquire an image of the sample during illumination of the sample; Equipped with The oblique illumination system comprises: a light emitter configured to generate an illumination of the sample by emitting light, the illumination being used to obtain an image of the sample; a collector lens disposed between the light emitter and the flow cell, the collector lens collecting light from the light emitter; a condenser lens disposed between the flow cell and the collector lens to receive light from the collector lens and direct it toward the sample in the flow cell; Equipped with system.

2. The sample is one of a urine sample, a blood sample, a cerebrospinal fluid sample, a synovial fluid sample, a serous fluid sample, a pleural fluid sample, a pericardial fluid sample, a peritoneal fluid sample, and an amniotic fluid sample. The system of claim 1 .

3. The light emitter is a light emitting diode. The system of claim 1 .

4. The light emitter is an arc lamp. The system of claim 1 .

5. The sample in the flow cell is in motion; The illumination from the light emitter is a light pulse that prevents blurring in the captured image. The system of claim 1 .

6. The sample in the flow cell is not moving, Illumination from the light emitter is continuous. The system of claim 1 .

7. the oblique illumination system further includes an aperture mask including an aperture, the aperture mask allowing at least a portion of the light from the collector lens to pass through the aperture, the aperture being non-centered with respect to the aperture mask such that illumination of the specimen is asymmetric. The system of claim 1 .

8. the light emitter, the collector lens, the aperture mask, the condenser lens and the sample are each centered with respect to an optical axis of the imaging device; The system of claim 7.

9. The diameter of the aperture is based on the type of sample. The system of claim 7.

10. the sample and the condenser lens are each centered with respect to an optical axis of the imaging device, such that light from a center of the condenser lens illuminates the sample without tilt and light from an edge of the condenser lens illuminates the sample at an oblique angle; the collector lens has a different size than the condenser lens, and the collector lens is not coaxial with respect to an optical axis of the imaging device. The system of claim 1 .

11. the light emitter is centered with respect to the collector lens; The system of claim 10.

12. the oblique illumination system includes a second light emitter configured to emit light to provide illumination of the sample; The system of claim 10.

13. the light emitter is configured to provide illumination of the sample by emitting light having a first color; the second light emitter is configured to provide illumination of the sample by emitting light having a second color different from the first color.

13. The system of claim 12.

14. the oblique illumination system includes a second collector lens disposed between the second light emitter and the flow cell, the second collector lens collecting light from the second light emitter; the second light emitter is centered with respect to the second collector lens; the second collector lens has a different size than the condenser lens, and the second collector lens is not coaxial with respect to an optical axis of the imaging device; the light emitter is not centered with respect to the second collector lens; 13. The system of claim 12.

15. 1. A method of microscopy, the method comprising: using a light emitter to obliquely illuminate the sample in the flow cell; acquiring an image of the sample using an image acquisition device while the sample is obliquely illuminated; The method includes:

16. the sample in the flow cell is in motion, the light emitter is a light emitting diode, and the illumination from the light emitter is a light pulse that prevents blurring in the acquired image.

16. The method of claim 15.

17. The step of obliquely illuminating the sample comprises: receiving light from the light emitter with a collector lens; blocking light from the collector lens at all positions of an aperture mask except through an aperture that is not coaxial with an optical axis of the image capture device; directing light from the aperture toward the sample with a condenser lens to form an oblique illumination; Including, 16. The method of claim 15.

18. the light emitter, the condenser lens and the collector lens are each centered with respect to an optical axis of the image capture device; 20. The method of claim 17.

19. The step of obliquely illuminating the sample comprises: positioning the light emitter non-coaxially with respect to an optical axis of the image capture device; collecting light from the light emitter with a collector lens centered about an axis of the light emitter; directing light from the collector lens towards the sample with a condenser lens centered on an optical axis of the image acquisition device to form an oblique illumination; Including, 16. The method of claim 15.

20. the collector lens has a different size than the condenser lens; 20. The method of claim 19.

Images