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WO2025120470A1 - Variation de cohérence spatiale dans un système de microscope - Google Patents

Variation de cohérence spatiale dans un système de microscope Download PDF

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Publication number
WO2025120470A1
WO2025120470A1 PCT/IB2024/062064 IB2024062064W WO2025120470A1 WO 2025120470 A1 WO2025120470 A1 WO 2025120470A1 IB 2024062064 W IB2024062064 W IB 2024062064W WO 2025120470 A1 WO2025120470 A1 WO 2025120470A1
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WO
WIPO (PCT)
Prior art keywords
light
sample
spatial coherence
respect
illumination module
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2024/062064
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English (en)
Inventor
Amir ZAIT
Eran RUBENS
Doron Malka
Neria OSHRY
Shani Rosen
Yochay Shlomo ESHEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SD Sight Diagnostics Ltd
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SD Sight Diagnostics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SD Sight Diagnostics Ltd filed Critical SD Sight Diagnostics Ltd
Publication of WO2025120470A1 publication Critical patent/WO2025120470A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/086—Condensers for transillumination only
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/12—Condensers affording bright-field illumination
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Definitions

  • the present invention relates to microscope systems, such as microscope systems used for the analysis of biological samples.
  • a property of a biological sample is determined by performing an optical measurement.
  • the density of a component e.g., a count of the component per unit volume
  • the concentration and/or density of a component may be measured by performing optical absorption, transmittance, fluorescence, and/or luminescence measurements upon the sample.
  • the sample is placed into a sample carrier and the measurements are performed with respect to a portion of the sample that is contained within a chamber of the sample carrier. The results of the measurements are analyzed in order to determine a property of the sample.
  • incoherent light provides better contrast and a larger depth of focus, and better facilitates finding the focal plane.
  • incoherent light is superior to partially- coherent light in other ways, e.g., by virtue of producing fewer artifacts.
  • inventions of the present invention provide an apparatus for microscopic imaging of a sample.
  • the apparatus includes a brightfield illumination module configured for multiple modes of operation in which the illumination module emits light at different respective degrees of spatial coherence such that the light passes through the sample.
  • the modes of operation vary with respect to the range of angles from which the sample is illuminated, the spatial coherence being a function of this range of angles.
  • the illumination module is configured for an incoherent mode, in which the emitted light is incoherent, and a partially-coherent mode, in which the emitted light is partially coherent.
  • the apparatus further includes an imaging sensor and an objective, which is configured to focus the light passing through the sample onto the imaging sensor.
  • the imaging sensor is configured to output multiple images of the sample based on the light focused onto the imaging sensor, the images varying from each other as a result of the different degrees of spatial coherence.
  • a processor switches between the modes of operation relatively rapidly; for example, light may be emitted at a second degree of spatial coherence less than 100 ms (e.g., less than 50, 10, 5, or 1 ms) after light is emitted at a first degree of spatial coherence.
  • Another apparatus for microscopic imaging of a sample includes a brightfield illumination module configured to emit light with a spatial coherence that varies with respect to different respective axes, such that the light passes through the sample.
  • the apparatus further includes an imaging sensor and an objective, which is configured to focus the light passing through the sample onto the imaging sensor.
  • the imaging sensor is configured to output an image of the sample based on the light focused onto the imaging sensor.
  • an apparatus for microscopic imaging of a sample includes a brightfield illumination module configured for multiple modes of operation in which the illumination module emits light at different respective degrees of spatial coherence such that the light passes through the sample.
  • the apparatus further includes an imaging sensor and an objective, which is configured to focus the light passing through the sample onto the imaging sensor.
  • the imaging sensor is configured to output multiple images of the sample based on the light focused onto the imaging sensor, the images varying from each other as a result of the different degrees of spatial coherence.
  • the modes of operation vary with respect to a range of angles from which the sample is illuminated.
  • the illumination module is configured for an incoherent mode, in which the emitted light is incoherent, and a partially-coherent mode, in which the emitted light is partially coherent.
  • the illumination module includes multiple light sources configured to emit the light, and the modes of operation differ from each other with respect to a number of the light sources that emit the light.
  • the light sources are arranged in a two-dimensional array.
  • the illumination module includes: an optical element including an aperture having a variable size; and one or more light sources configured to emit the light through the aperture, and the modes of operation differ from each other with respect to the size of the aperture.
  • the optical element includes a stop
  • the aperture is a stop aperture of the stop.
  • the optical element includes an optical filter including a filter aperture, part of which is configured to block the light at at least one of the wavelengths and pass the light through at at least one other one of the wavelengths, and the aperture is the filter aperture.
  • the illumination module further includes a diffuser disposed between the light sources and the optical element and configured to diffuse the light.
  • the illumination module further includes: a collimator disposed between the light sources and the diffuser, and configured to collimate the light; and a focusing lens disposed between the optical element and the sample, and configured to focus the light onto the sample.
  • the illumination module includes multiple illumination units configured to emit the light with the different respective degrees of spatial coherence, and the modes of operation differ from each other with respect to the illumination unit that emits the light.
  • the illumination units include: a first illumination unit, including at least one first light source configured to emit the light; and a second illumination unit, including: at least one second light source configured to emit the light; and a coherence-increasing optical element configured to increase the spatial coherence of the light emitted from the second light source, such that the spatial coherence of the light emitted from the second illumination unit is greater than the spatial coherence of the light emitted from the first illumination unit.
  • the first illumination unit further includes a collimator configured to collimate the light emitted from the first light source, and the illumination module further includes a focusing lens configured to focus the light onto the sample.
  • a method for microscopic imaging of a sample includes, by switching, by a processor, between multiple modes of operation of a brightfield illumination module, emitting light, from the illumination module, at different respective degrees of spatial coherence such that the light passes through the sample and is then focused, by an objective, onto an imaging sensor.
  • the method further includes outputting, from the imaging sensor, multiple images of the sample based on the light focused onto the imaging sensor, the images varying from each other as a result of the different degrees of spatial coherence.
  • an apparatus for microscopic imaging of a sample includes a brightfield illumination module configured to emit light with a spatial coherence that varies with respect to different respective axes, such that the light passes through the sample.
  • the apparatus further includes an imaging sensor and an objective, which is configured to focus the light passing through the sample onto the imaging sensor.
  • the imaging sensor is configured to output an image of the sample based on the light focused onto the imaging sensor.
  • the spatial coherence varies by virtue of a range of angles, from which the sample is illuminated, varying with respect to the axes.
  • the emitted light is incoherent with respect to a first one of the axes and partially-coherent with respect to a second one of the axes.
  • the illumination module includes multiple light sources configured to emit the light, and the spatial coherence varies by virtue of a number of the light sources that emit the light varying with respect to the axes.
  • the illumination module includes: a stop including a stop aperture that is shaped asymmetrically with respect to the axes; and one or more light sources configured to emit the light through the stop aperture, and the spatial coherence varies by virtue of the light passing through the stop aperture.
  • a method for microscopic imaging of a sample includes emitting light, from a brightfield illumination module, with a spatial coherence that varies with respect to different respective axes, such that the light passes through the sample and is then focused, by an objective, onto an imaging sensor.
  • the method further includes outputting, from the imaging sensor, an image of the sample based on the light focused onto the imaging sensor.
  • Fig. 1 is a block diagram showing components of a biological sample analysis system, in accordance with some applications of the present invention
  • FIGS. 2A, 2B, and 2C are schematic illustrations of an optical measurement unit, in accordance with some applications of the present invention.
  • Figs. 3A, 3B, and 3C are schematic illustrations of respective views of a sample carrier that is used for performing both microscopic measurements and optical density measurements, in accordance with some applications of the present invention
  • Fig. 4A is a schematic illustration of an illumination module operating in an incoherent mode, in accordance with some embodiments of the present invention.
  • Fig. 4B is a schematic illustration of an illumination module operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • Fig. 5A is a schematic illustration of an illumination module operating in an incoherent mode, in accordance with some embodiments of the present invention
  • Fig. 5B is a schematic illustration of an illumination module operating in a partially-coherent mode, in accordance with some embodiments of the present invention
  • Fig. 6A is a schematic illustration of an illumination module operating in an incoherent mode, in accordance with some embodiments of the present invention.
  • Fig. 6B is a schematic illustration of an illumination module operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • Fig. 7A is a schematic illustration of an illumination module operating in an incoherent mode, in accordance with some embodiments of the present invention.
  • Fig. 7B is a schematic illustration of an illumination module operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • Fig. 8A is a schematic illustration of an illumination module operating in an incoherent mode, in accordance with some embodiments of the present invention.
  • Fig. 8B is a schematic illustration of an illumination module operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • Fig. 9A is a schematic illustration of an illumination module operating in an incoherent mode, in accordance with some embodiments of the present invention.
  • Fig. 9B is a schematic illustration of an illumination module operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • Figs. 10A and 10B are schematic illustrations of an illumination module comprising a photodiode, in accordance with some embodiments of the present invention.
  • the spatial coherence of an illuminating light wave is typically quantified as the ratio between the coherence length of the wave and the approximate size of the smallest feature of interest.
  • Coherent light is defined as light for which this ratio is greater than a first threshold, which may be between 3 and 12 (e.g., 10), for example.
  • Incoherent light is defined as light for which this ratio is less than a second threshold, which may be between 0.01 and 0.3 (e.g., 0.1), for example.
  • Partially coherent light is defined as light for which this ratio is between the two thresholds.
  • the smallest feature of interest may have an approximate size of 1 pm, such that, for example, coherent light may have a coherence length greater than 3-12
  • jm e.g., 10 m
  • incoherent light may have a coherence length less than 0.01-0.3 pm (e.g., 0.1 pm)
  • partially coherent light may have a coherence length of 0.01-12 pm (e.g., 0.1-10 pm, 0.01-3 pm, 0.3-3 pm, or 0.3-12 pm).
  • incoherent light provides better contrast and a larger depth of focus, and better facilitates finding the focal plane.
  • incoherent light is superior to partially- coherent light in other ways, e.g., by virtue of producing fewer artifacts.
  • embodiments of the present invention provide a brightfield illumination module configured to illuminate the sample with multiple different degrees of spatial coherence.
  • a computer processor which is connected to the illumination module over any suitable wired or wireless interface, switches between the degrees of spatial coherence.
  • the processor may rapidly alternate the illumination module between an incoherent mode, in which the illumination module emits incoherent light, and a partially-coherent mode, in which the illumination module emits partially-coherent light.
  • the processor acquires multiple microscope images of the same portion of the sample, the images differing from each other by virtue of the different degrees of spatial coherence.
  • the processor capitalizes on the advantages that each degree of spatial coherence offers. In contrast, were the sample to be illuminated with a uniform degree of spatial coherence, some of these advantages would be missed.
  • the spatial coherence in an illumination system is affected by the numerical aperture of the system, which in turn depends on the range of illumination angles.
  • the different degrees of spatial coherence are obtained by varying the range of angles from which the sample is illuminated. A wider range of angles provides less coherence, relative to a narrower range.
  • the varying spatial coherence is achieved by varying the number of activated light sources, as described below with reference to Figs. 4A-B. In other embodiments, the varying spatial coherence is achieved by varying the size of a stop (or “shutter”) aperture, as described below with reference to Figs. 5A-B and 6A-B, or by varying the wavelength of the emitted light in the presence of a wavelength- selective filter, as described below with reference to Figs. 7A-B and 8A-B. In yet other embodiments, the varying spatial coherence is achieved using separate illumination units with suitable optics, as described below with reference to Figs. 9A-B.
  • the spatial coherence is varied with respect to different respective axes, such that the light illuminates the sample with multiple degrees of spatial coherence simultaneously.
  • Fig. 1 is a block diagram showing components of a biological sample analysis system 20, in accordance with some applications of the present invention.
  • a biological sample e.g., a blood sample
  • a sample carrier 22 While the sample is disposed in the sample carrier, optical measurements are performed upon the sample using one or more optical measurement devices 24.
  • the optical measurement devices may include a microscope (e.g., a digital microscope), a spectrophotometer, a photometer, a spectrometer, an imaging sensor such as a charge-coupled device (CCD) or complementary metal-oxide semiconductor (CMOS) sensor, a camera (which may comprise, for example, a CCD or CMOS sensor, and/or which may be configured for multispectral or hyperspectral imaging), a fluorometer, a spectrofluorometer, and/or a photodetector (such as a photodiode, a photoresistor, and/or a phototransistor).
  • the optical measurement devices include dedicated light sources (such as light emitting diodes, incandescent light sources, etc.) and/or optical elements for manipulating light collection and/or light emission (such as lenses, diffusers, filters, etc.).
  • a computer processor 28 typically receives and processes optical measurements that are performed by the optical measurement device. Further typically, the computer processor controls the acquisition of optical measurements that are performed by the one or more optical measurement devices. The computer processor communicates with a memory 30.
  • a user e.g., a laboratory technician, or an individual from whom the sample was drawn
  • the user interface includes a keyboard, a mouse, a joystick, a touchscreen device (such as a smartphone or a tablet computer), a touchpad, a trackball, a voice-command interface, and/or other types of user interfaces that are known in the art.
  • the computer processor generates an output via an output device 34.
  • the output device includes a display, such as a monitor, and the output includes an output that is displayed on the display.
  • the processor generates an output on a different type of visual, text, graphics, tactile, audio, and/or video output device, e.g., speakers, headphones, a smartphone, or a tablet computer.
  • user interface 32 acts as both an input interface and an output interface, i.e., it acts as an input/output interface.
  • the processor generates an output on a computer-readable medium (e.g., a non-transitory computer-readable medium), such as a disk, or a portable USB drive, and/or generates an output on a printer.
  • processor 28 may be embodied as a single processor, or as a cooperatively networked or clustered set of processors.
  • the functionality of processor 28 may be implemented solely in hardware, e.g., using one or more fixed-function or general-purpose integrated circuits, Application-Specific Integrated Circuits (ASICs), and/or Field-Programmable Gate Arrays (FPGAs).
  • this functionality may be implemented at least partly in software.
  • processor 28 may be embodied as a programmed processor comprising, for example, a central processing unit (CPU) and/or a Graphics Processing Unit (GPU).
  • Program code including software programs, and/or data may be loaded for execution and processing by the CPU and/or GPU.
  • the program code and/or data may be downloaded to the processor in electronic form, over a network, for example.
  • the program code and/or data may be provided and/or stored on non-transitory tangible media, such as magnetic, optical, or electronic memory.
  • Such program code and/or data when provided to the processor, produce a machine or special-purpose computer, configured to perform the tasks described herein.
  • FIGs. 2A, 2B, and 2C are schematic illustrations of an optical measurement unit 31, in accordance with some applications of the present invention.
  • Fig. 2A shows an oblique view of the exterior of the fully assembled device
  • Figs. 2B and 2C shows respective oblique views of the device with the cover having been made transparent, such components within the device are visible.
  • at least one optical measurement device 24 (and/or computer processor 28 and memory 30) is housed inside optical measurement unit 31.
  • sample carrier 22 is placed inside the optical measurement unit.
  • the optical measurement unit may define a slot 36, via which the sample carrier is inserted into the optical measurement unit.
  • the optical measurement unit includes a stage 64, which is configured to support sample carrier 22 within the optical measurement unit.
  • a screen 63 on the cover of the optical measurement unit e.g., a screen on the front cover of the optical measurement unit, as shown
  • microscope system 37 comprises a microscope system 37 (shown in Figs. 2B-C) configured to perform microscopic imaging of at least a portion of the sample.
  • microscope system 37 comprises a brightfield illumination module 65, which comprises one or more light sources (e.g. light emitting diodes (LEDs)) configured for brightfield illumination of the sample.
  • microscope system 37 comprises one or more fluorescence-excitation light sources configured for exciting fluorescence in the sample.
  • Microscope system 37 further comprises an imaging sensor 67, such as a CCD or CMOS sensor, and an objective 66 configured to focus brightfield light passing through the sample, or fluorescent light emitted from the sample, onto imaging sensor 67.
  • illumination module 65 is configured for multiple modes of operation in which the illumination module emits light at different respective degrees of spatial coherence. Typically, the modes of operation vary with respect to the range of angles from which the sample is illuminated, a larger range corresponding to less spatial coherence.
  • the illumination module may be configured for an incoherent mode, in which the illumination module emits incoherent light, and a partially-coherent mode, in which the illumination module emits partially-coherent light.
  • Processor 28 switches between the modes so as to emit the light at the different respective degrees of spatial coherence.
  • the light passes through the sample and is then focused, by the objective (together with the other microscope elements), onto the imaging sensor.
  • the imaging sensor outputs multiple images of the sample based on the light focused onto the imaging sensor, the images varying from each other as a result of the different degrees of spatial coherence.
  • the processor switches between the modes relatively rapidly; for example, light may be emitted at a second degree of spatial coherence less than 100 ms (e.g., less than 50, 10, 5, or 1 ms) after light is emitted at a first degree of spatial coherence.
  • the processor rapidly alternates between the modes in this manner, thereby rapidly acquiring multiple images, which have different respective properties due to the varying spatial coherence, for the same sample portion and the same focus level.
  • the optical measurement unit also includes an optical-density-measurement unit 39 (shown in Fig. 2C) configured to perform optical density measurements (e.g., optical absorption measurements) on a second portion of the sample.
  • the optical-densitymeasurement unit includes a set of optical-density-measurement light sources (e.g., light emitting diodes) and light detectors, which are configured for performing optical density measurements on the sample.
  • At least some of the brightfield light sources, at least some of the fluorescence-excitation light sources, and/or at least some of the optical-density-measurement light sources differ from each other with respect to the wavelength of emitted light.
  • a first brightfield light source, fluorescence-excitation light source, or optical-density-measurement light source emits light at a first wavelength
  • a second brightfield light source, fluorescenceexcitation light source, or optical-density-measurement light source emits light at a second wavelength.
  • Figs. 3 A and 3B are schematic illustrations of respective views of sample carrier 22, in accordance with some applications of the present invention.
  • Fig. 3A shows a top view of the sample carrier (the top cover of the sample carrier being shown as being opaque in Fig. 3A, for illustrative purposes), and
  • Fig. 3B shows a bottom view (in which the sample carrier has been rotated around its short edge with respect to the view shown in Fig. 3A).
  • the sample carrier includes a first set 52 of one or more sample chambers, which are used for performing microscopic analysis upon the sample, and a second set 54 of sample chambers, which are used for performing optical density measurements upon the sample.
  • the sample chambers of the sample carrier are filled with a biological sample, such as blood via sample inlet holes 38.
  • the sample chambers define one or more outlet holes 40.
  • the outlet holes are configured to facilitate filling of the sample chambers with the biological sample, by allowing air that is present in the sample chambers to be released from the sample chambers.
  • the outlet holes are located longitudinally opposite the inlet holes (with respect to a sample chamber of the sample carrier). For some applications, the outlet holes thus provide a more efficient mechanism of air escape than if the outlet holes were to be disposed closer to the inlet holes.
  • the sample carrier includes at least three components: a molded component 42, a glass layer 44 (e.g., glass sheet), and an adhesive layer 46 configured to adhere the glass layer to an underside of the molded component.
  • the molded component is typically made of a polymer (e.g., a plastic) that is molded (e.g., via injection molding) to provide the sample chambers with a desired geometrical shape.
  • the molded component is typically molded to define inlet holes 38, outlet holes 40, and gutters 48 which surround the central portion of each of the sample chambers.
  • the gutters typically facilitate filling of the sample chambers with the biological sample, by allowing air to flow to the outlet holes, and/or by allowing the biological sample to flow around the central portion of the sample chamber.
  • a sample carrier as shown in Figs. 3A-C is used when performing a complete blood count and/or biomarker detection on a blood sample.
  • the sample carrier is used with optical measurement unit 31 configured as generally shown and described with reference to Figs. 2A-C.
  • a first portion of the blood sample is placed inside first set 52 of sample chambers (which are used for performing microscopic analysis upon the sample, e.g., using microscope system 37 (shown in Figs. 2B-C)), and a second portion of the blood sample is placed inside second set 54 of sample chambers (which are used for performing optical density measurements upon the sample, e.g., using optical-density-measurement unit 39 (shown in Fig.
  • first set 52 of sample chambers includes a plurality of sample chambers
  • second set 54 of sample chambers includes only a single sample chamber, as shown.
  • the scope of the present application includes using any number of sample chambers (e.g., a single sample chamber or a plurality of sample chambers) within either the first set of sample chambers or within the second set of sample chambers, or any combination thereof.
  • the first portion of the blood sample is typically diluted with respect to the second portion of the blood sample.
  • the diluent may contain pH buffers, stains, fluorescent stains, antibodies, sphering agents, lysing agents, etc.
  • the second portion of the blood sample which is placed inside second set 54 of sample chambers is a natural, undiluted blood sample.
  • the second portion of the blood sample may be a sample that underwent some modification, including, for example, one or more of dilution (e.g., dilution in a controlled fashion), addition of a component or reagent, or fractionation.
  • one or more staining substances are used to stain the first portion of the blood sample (which is placed inside first set 52 of sample chambers) before the sample is imaged microscopically.
  • the staining substance may be configured to stain DNA with preference over staining of other cellular components.
  • the staining substance may be configured to stain all cellular nucleic acids with preference over staining of other cellular components.
  • the sample may be stained with Acridine Orange reagent, Hoechst reagent (i.e., a bis-benzimide dye and/or a blue, fluorescent dye), and/or any other staining substance that is configured to preferentially stain DNA and/or RNA within the blood sample.
  • the staining substance is configured to stain all cellular nucleic acids but the staining of DNA and RNA are each more prominently visible under some lighting and filter conditions, as is known, for example, for Acridine Orange.
  • Images of the sample may be acquired using imaging conditions that allow detection of cells (e.g., brightfield) and/or imaging conditions that allow visualization of stained bodies (e.g., appropriate fluorescent illumination).
  • the first portion of the sample is stained with Acridine Orange and with a Hoechst reagent.
  • the first (diluted) portion of the blood sample may be prepared using techniques as described in US 9,329,129 to Pollak, which is incorporated herein by reference, and which describes a method for preparation of blood samples for analysis that involves a dilution step, the dilution step facilitating the identification and/or counting of components within microscopic images of the sample.
  • the first portion of the sample is stained with one or more stains that cause platelets within the sample to be visible under brightfield imaging conditions and/or under fluorescent imaging conditions, e.g., as described hereinabove.
  • the first portion of the sample may be stained with methylene blue and/or Romanowsky stains.
  • the sample is a fine needle aspirate sample, and the first portion of the sample is stained with stains that cause one or more of the following entities to fluoresce: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, and/or parasites.
  • sample carrier 22 is supported within the optical measurement unit by stage 64.
  • the stage has a forked design, such that the sample carrier is supported by the stage around the edges of the sample carrier, but such that the stage does not interfere with the visibility of the sample chambers of the sample carrier by the optical measurement devices.
  • the sample carrier is held within the stage, such that molded component 42 of the sample carrier is disposed above the glass layer 44, and such that objective 66 is disposed below the glass layer of the sample carrier.
  • illumination module 65 illuminates the sample carrier from above the molded component.
  • additional light sources such as fluorescence-excitation light sources, illuminate the sample carrier from below the sample carrier (e.g., via objective 66).
  • the first portion of blood (which is placed in first set 52 of sample chambers) is allowed to settle such as to form a monolayer of cells, e.g., using techniques as described in US 9,329,129 to Pollak, which is incorporated herein by reference.
  • the first portion of blood is a cell suspension and the chambers belonging to the first set 52 of chambers each define a cavity 55 that includes a base surface 57 (shown in Fig. 3C).
  • the cells in the cell suspension are allowed to settle on the base surface of the sample chamber of the carrier to form a monolayer of cells on the base surface of the sample chamber.
  • At least one microscopic image of at least a portion of the monolayer of cells is typically acquired.
  • a plurality of images of the monolayer are acquired, each of the images corresponding to an imaging field that is located at a respective, different area within the imaging plane of the monolayer.
  • an optimum depth level at which to focus the microscope in order to image the monolayer is determined, e.g., using techniques as described in US Patent US 10,176,565 to Greenfield, which is incorporated herein by reference.
  • respective imaging fields have different optimum depth levels from each other.
  • the term monolayer is used to mean a layer of cells that have settled, such as to be disposed within a single focus level of the microscope (referred to herein as “the monolayer focus level").
  • the monolayer focus level there may be some overlap of cells, such that within certain areas there are two or more overlapping layers of cells.
  • red blood cells may overlap with each other within the monolayer, and/or platelets may overlap with, or be disposed above, red blood cells within the monolayer.
  • the microscopic analysis of the first portion of the blood sample is performed with respect to the monolayer of cells.
  • the first portion of the blood sample is imaged under brightfield imaging.
  • the first portion of the blood sample is additionally imaged under fluorescent imaging.
  • the fluorescent imaging is performed by exciting stained objects (i.e., objects that have absorbed the stain(s)) within the sample by directing light toward the sample at known excitation wavelengths (i.e., wavelengths at which it is known that stained objects emit fluorescent light if excited with light at those wavelengths), and detecting the fluorescent light.
  • a separate set of light sources e.g., one or more light emitting diodes
  • the sample is stained with Acridine Orange reagent and Hoechst reagent.
  • the sample is illuminated with light that is at least partially within the UV range (e.g., 300-400 nm), and/or with light that is at least partially within the blue light range (e.g., 450-520 nm), in order to excite the stained objects.
  • the sample is mixed with one or more fluorescently-labeled antibodies.
  • the sample is illuminated with light at a wavelength that excites the fluorescent stains with which the antibodies are labeled.
  • sample chambers belonging to set 52 have different heights from each other, in order to facilitate different measurands being measured using microscope images of respective sample chambers, and/or different sample chambers being used for microscopic analysis of respective sample types.
  • a blood sample, and/or a monolayer formed by the sample has a relatively low density of red blood cells
  • measurements may be performed within a sample chamber of the sample carrier having a greater height (i.e., a sample chamber of the sample carrier having a greater height relative to a different sample chamber having a relatively lower height), such that there is a sufficient density of cells, and/or such that there is a sufficient density of cells within the monolayer formed by the sample, to provide statistically reliable data.
  • Such measurements may include, for example, red blood cell density measurements, measurements of other cellular attributes, (such as counts of abnormal red blood cells, red blood cells that include intracellular bodies (e.g., pathogens, Howell-Jolly bodies), etc.), and/or hemoglobin concentration.
  • red blood cell density measurements measurements of other cellular attributes, such as counts of abnormal red blood cells, red blood cells that include intracellular bodies (e.g., pathogens, Howell-Jolly bodies), etc.
  • hemoglobin concentration e.g., hemoglobin concentration
  • the sample chamber within the sample carrier upon which to perform optical measurements is selected.
  • a sample chamber of the sample carrier having a greater height may be used to perform a white blood cell count (e.g., to reduce statistical errors which may result from a low count in a shallower region), white blood cell differentiation, and/or to detect more rare forms of white blood cells.
  • microscopic images may be obtained from a sample chamber of the sample carrier having a relatively low height, since in such sample chambers the cells are relatively sparsely distributed across the area of the region, and/or form a monolayer in which the cells are relatively sparsely distributed.
  • microscopic images may be obtained from a sample chamber of the sample carrier having a relatively low height, since within such sample chambers there are fewer red blood cells which overlap (fully or partially) with the platelets in microscopic images, and/or in a monolayer.
  • a sample chamber of the sample carrier having a lower height for performing optical measurements for measuring some measurands within a sample (such as a blood sample), whereas it is preferable to use a sample chamber of the sample carrier having a greater height for performing optical measurements for measuring other measurands within such a sample.
  • a first measurand within a sample is measured, by performing a first optical measurement upon (e.g., by acquiring microscopic images of) a portion of the sample that is disposed within a first sample chamber belonging to set 52 of the sample carrier, and a second measurand of the same sample is measured, by performing a second optical measurement upon (e.g., by acquiring microscopic images of) a portion of the sample that is disposed within a second sample chamber of set 52 of the sample carrier.
  • the first and second measurands are normalized with respect to each other, for example, using techniques as described in US 2019/0145963 to Zait, which is incorporated herein by reference.
  • an optical density measurement is performed on the second portion of the sample (which is typically placed into second set 54 of sample chambers in an undiluted form).
  • concentration and/or density of a component may be measured by performing optical absorption, transmittance, fluorescence, and/or luminescence measurements upon the sample.
  • sample chambers belonging to set 54 define at least a first region 56 (which is typically deeper) and a second region 58 (which is typically shallower), the height of the sample chambers varying between the first and second regions in a predefined manner, e.g., as described in US 2019/0302099 to Pollak, which is incorporated herein by reference.
  • the heights of first region 56 and second region 58 of the sample chamber are defined by a lower surface that is defined by the glass layer and by an upper surface that is defined by the molded component.
  • the upper surface at the second region is stepped with respect to the upper surface at the first region.
  • the step between the upper surface at the first and second regions provides a predefined height difference Ah between the regions, such that even if the absolute height of the regions is not known to a sufficient degree of accuracy (for example, due to tolerances in the manufacturing process), the height difference Ah is known to a sufficient degree of accuracy to determine a parameter of the sample, using the techniques described herein, and as described in US 2019/0302099 to Pollak, which is incorporated herein by reference.
  • the height of the sample chamber varies from the first region 56 to the second region 58, and the height then varies again from the second region to a third region 59, such that, along the sample chamber, first region 56 defines a maximum height region, second region 58 defines a medium height region, and third region 59 defines a minimum height region.
  • additional variations in height occur along the length of the sample chamber, and/or the height varies gradually along the length of the sample chamber.
  • optical measurements are performed upon the sample using one or more optical measurement devices 24 (Fig. 1).
  • the sample is viewed by the optical measurement devices via the glass layer, glass being transparent at least to wavelengths that are typically used by the optical measurement device.
  • the sample carrier is inserted into optical measurement unit 31, which houses the optical measurement device while the optical measurements are performed.
  • the optical measurement unit houses the sample carrier such that the molded layer is disposed above the glass layer.
  • the sample carrier is formed by adhering the glass layer to the molded component.
  • the glass layer and the molded component may be bonded to each other during manufacture or assembly (e.g.
  • the glass layer and the molded component are bonded to each other during manufacture or assembly using adhesive layer 46.
  • the apparatus and methods described herein are configured to automatically identify components in the blood such as platelets, white blood cells, anomalous white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howell-Jolly bodies, etc.
  • the apparatus and methods described herein are configured to determine parameters relating to one or more of the components.
  • the apparatus and methods described herein are configured to determine parameters such as corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red blood cell distribution width (RDW), red blood cell morphologic features, clumping, and/or red blood cell abnormalities.
  • the apparatus and methods described herein are configured to determine parameters such as absolute and relative numbers of neutrophils, lymphocytes, monocytes, eosinophils and basophils.
  • the apparatus and methods described herein are configured to perform normal and abnormal leukocyte differentiation, including detecting the existence of immature or hyper segmented cells, white blood cell agglutination or fragmentation, blasts, and/or atypical or abnormal lymphocytes.
  • the apparatus and methods described herein are configured to detect leukocyte subpopulations (such as B, T-cells), and/or morphological cell activation, and/or or any other cell or population based biomarkers (e.g. monocyte distribution width (MDW).
  • leukocyte subpopulations such as B, T-cells
  • morphological cell activation e.g. monocyte distribution width (MDW).
  • MDW monocyte distribution width
  • the apparatus and methods described herein are configured to determine parameters such as the presence of giant platelets, platelets clumps or abnormal platelets distribution, immature (i.e., reticulated) platelets fraction, average platelet size (MPV), platelet distribution width (PDW), platelet clumping, and/or platelet activation levels.
  • MPV average platelet size
  • PDW platelet distribution width
  • the apparatus and methods described herein are configured to detect parasites, bacteria, fungi, and/or any other abnormal biomarkers.
  • the apparatus and methods described herein are configured to determine parameters such as the relative number of blast cells, nucleated red blood cells, abnormal or atypical lymphocyte cells, immature granulocyte cells and/or reticulocytes.
  • the apparatus and methods described herein are configured to detect biomarkers, such as CD64, CD3, CD 14, and/or CD16.
  • biomarkers such as CD64, CD3, CD 14, and/or CD16.
  • the bodily sample is mixed with antibodies against one or more of the aforementioned biomarkers and the apparatus and methods described herein are configured to detect the antibodies.
  • the apparatus and methods described herein are applied to a fine needle aspirate sample.
  • one or more of the following entities within the sample are made to fluoresce: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, and/or parasites.
  • illumination module 65 is configured for multiple modes of operation, and processor 28 is configured to switch between the operational modes.
  • Fig. 4A is a schematic illustration of illumination module 65 operating in an incoherent mode
  • Fig. 4B is a schematic illustration of illumination module 65 operating in a partially -coherent mode, in accordance with some embodiments of the present invention.
  • illumination module 65 comprises multiple light sources 68, such as multiple LEDs.
  • illumination module 65 further comprises illumination optics 74, comprising a condenser and collector for example, which focus light onto the sample.
  • the modes of operation of illumination module 65 differ from each other with respect to the number of light sources 68 that are active, i.e., that are emitting light.
  • the processor varies the degree of spatial coherence by varying the number of activated light sources.
  • Fig. 4A shows five beams 72 of light emitted from respective light sources 68.
  • the beams impinge on sample carrier 22 from a relatively wide range of angles, such that the sample is incoherently illuminated.
  • Fig. 4B shows only a single beam 72 emitted from a single light source. This beam impinges on sample carrier 22 from a smaller range of angles, such that the illumination of the sample is partially coherent. More generally, any other number of light sources may be activated, such that the illumination provided by illumination module 65 may have another degree of spatial coherence not depicted in Figs. 4A-B.
  • light sources 68 are arranged in a two-dimensional (2D) array 70. In some such embodiments, for incoherence, multiple rows of light sources 68 are simultaneously activated.
  • illumination module 65 comprises multiple sets of light sources 68, each of the sets configured to emit light at a different respective wavelength.
  • each of the sets is arranged in a respective 2D array 70.
  • these 2D arrays are merged, i.e., the illumination module comprises a “composite” 2D array in which the sets alternate with each other.
  • the light sources may be arranged in the following arrangement:
  • the processor in addition to varying the spatial coherence of the emitted light, varies the wavelength so as to additionally capitalize on the advantages provided by each wavelength.
  • the processor may acquire a respective image for each wavelength in combination with each operational mode, optionally with less than 1 ms between the acquisition of successive images. For example, given three wavelengths and two degrees of spatial coherence, the processor may acquire six images of each portion of the sample.
  • FIGs. 5A and 6A are schematic illustrations of illumination module 65 operating in an incoherent mode
  • Figs. 5B and 6B are schematic illustrations of illumination module 65 operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • illumination module 65 comprises an optical element 82 comprising an aperture 84 having a variable size, and one or more light sources 68, such as LEDs, configured to emit light through aperture 84.
  • the modes of operation of the illumination module differ from each other with respect to the size of the aperture.
  • optical element 82 comprises a stop 88 comprising a stop aperture 90.
  • the size of stop aperture 90 is controlled using circuitry 86, i.e., circuitry 86 is configured to open or close the stop.
  • the processor varies the degree of spatial coherence by, using circuitry 86, varying the size of stop aperture 90.
  • Figs. 5A and 6A show a beam 72 of light emitted by a light source 68.
  • the beam impinges on sample carrier 22 from a relatively wide range of angles, such that the sample is incoherently illuminated.
  • the stop aperture may have any size, such that the illumination provided by illumination module 65 may have another degree of spatial coherence not depicted in Figs. 5A-B or 6A-B.
  • illumination module 65 may comprise a set 98 of multiple light sources 68, at least some of which may emit light at different respective wavelengths.
  • the processor may vary the wavelength, as described above with reference to Figs. 4A-B. (The differences between Figs. 5A and 6A and between Figs. 5B and 6B are explained below.)
  • FIGs. 7A and 8A are schematic illustrations of illumination module 65 operating in an incoherent mode
  • Figs. 7B and 8B are schematic illustrations of illumination module 65 operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • Figs. 7A-B and 8A-B are similar to Figs. 5A-B and 6A-B in that the operating mode of illumination module 65 is controlled by varying the size of aperture 84.
  • Figs. 7A-B and 8A-B differ from Figs. 5A-B and 6A-B in that, rather than stop 88, optical element 82 comprises an optical filter 92 comprising a filter aperture 96, part of which is configured to block (i.e., to filter out) one or more wavelengths of light while passing through one or more other wavelengths.
  • the size of the aperture is varied not electromechanically as in Figs. 5A-B and 6A-B, but rather, by varying the wavelength of the emitted light.
  • the modes of operation of illumination module 65 differ from each other with respect to the wavelength at which the light is emitted, i.e., the processor varies the degree of spatial coherence by varying the wavelength of the emitted light.
  • illumination module 65 comprises multiple light sources, at least some of which are configured to emit light at different respective wavelengths.
  • a portion of filter aperture 96 is configured to pass the light through at at least one of the emitted wavelengths (as in Figs. 7A and 8A) and to block the light at at least one other one of the emitted wavelengths (as in Figs. 7B and 8B).
  • the filter is transparent, or nearly transparent, at each of the pass-through wavelengths.
  • a first light source 68a may provide incoherent illumination at a first wavelength (as in Figs.
  • a second light source 68b may provide partially-coherent illumination at a second wavelength (as in Figs. 7B and 8B).
  • a second wavelength as in Figs. 7B and 8B.
  • the mode of operation is not a function of the wavelength at which the light is emitted, i.e., both incoherent and partially-coherent illumination may be provided at the same wavelength.
  • optical filter 92 provides the same level of spatial coherence for multiple wavelengths.
  • the optical filter may provide incoherent red and blue illumination, but partially-coherent green illumination.
  • the optical filter provides more than two degrees of spatial coherence, by virtue of comprising multiple portions having different filtering properties. For example, a first portion of the filter may block red light but pass blue light and green light through, while a second portion surrounding the first portion may block red light and blue light but pass green light through, such that the degree of spatial coherence is greatest for red, less for blue, and least for green.
  • first light source 68a may provide incoherent illumination at a first wavelength while second light source 68b simultaneously provides partially-coherent illumination at a second wavelength.
  • illumination module 65 further comprises a diffuser 78 disposed between light source(s) 68 and optical element 82 and configured to diffuse the light.
  • the illumination module further comprises a collimator 76 disposed between the light source(s) and diffuser 78, and configured to collimate the light, and a focusing lens 80 disposed between optical element 82 and the sample, and configured to focus the light onto the sample.
  • a collimator 76 disposed between the light source(s) and diffuser 78, and configured to collimate the light
  • a focusing lens 80 disposed between optical element 82 and the sample, and configured to focus the light onto the sample.
  • FIG. 9A is a schematic illustration of illumination module 65 operating in an incoherent mode
  • Fig. 9B is a schematic illustration of illumination module 65 operating in a partially-coherent mode, in accordance with some embodiments of the present invention.
  • illumination module 65 comprises multiple illumination units 100 configured to emit light with different respective degrees of spatial coherence.
  • Each illumination unit 100 comprises one or more light sources, along with, optionally, one or more optical elements.
  • the modes of operation differ from each other with respect to the illumination unit that emits the light.
  • the processor varies the degree of spatial coherence by varying the activated illumination unit 100.
  • illumination units 100 may comprise a first illumination unit 100a, comprising at least one first light source 68a, and a second illumination unit 100b, comprising at least one second light source 68b and a coherence-increasing optical element 102.
  • Optical element 102 is configured to increase the spatial coherence of the light emitted from second light source 68b, such that the spatial coherence of the light emitted from second illumination unit 100b (as shown in Fig. 9B) is greater than the spatial coherence of the light emitted from first illumination unit 100a (as shown in Fig. 9A).
  • first illumination unit 100a further comprises a collimator 76 configured to collimate the light emitted from first light source 68a, and the illumination module further comprises a focusing lens 80 configured to focus the light (either from the first light source or from the second light source) onto the sample.
  • first illumination unit 100a comprises a homogenizer (not shown). (For embodiments in which the first illumination unit comprises both collimator 76 and a homogenizer, the homogenizer is typically disposed between first light source 68a and the collimator.)
  • optical element 102 comprises relay optics. Regardless of the components of optical element 102, the optical element comprises a relatively small exit aperture 104, i.e., an exit aperture 104 having a low numerical aperture (NA).
  • NA numerical aperture
  • illumination module 65 comprises additional optics configured to direct beams 72 at focusing lens 80 (or directly at the sample carrier).
  • a mirror 106 may reflect beams from the first illumination unit toward the focusing lens.
  • a multichroic mirror 108 reflects beams from the second illumination unit toward the focusing lens, while passing beams from the first illumination unit through.
  • illumination module 65 comprises a beamsplitter, which reflects a portion of each beam from the second illumination unit toward the focusing lens, and passes through a portion of each beam from the first illumination unit.
  • additional optics such as one or more mirrors, e.g., multichroic mirrors, and/or beamsplitters.
  • illumination module 65 comprises more than two illumination units, via which the illumination module provides more than two degrees of spatial coherence.
  • a single illumination unit comprises multiple light sources that provide illumination in different respective wavelengths.
  • multiple illumination units provide illumination in the same wavelength.
  • the processor may thus alternate between different combinations of coherence-degree and wavelength, thereby capitalizing on the advantages of each combination.
  • multiple degrees of spatial coherence includes providing multiple degrees of spatial coherence using any suitable combination of features described above with reference to Figs. 5A-9B.
  • multiple degrees of spatial coherence may be provided by varying the number of active light sources (as in Figs. 4A-B) and also varying the size of aperture 84 (as in Figs. 5A-8B).
  • the illumination module is configured to emit light with a spatial coherence that varies with respect to different respective axes, typically by virtue of the range of angles from which the sample is illuminated varying with respect to the axes.
  • the emitted light may be incoherent with respect to a first axis and partially-coherent with respect to a second axis, which may be perpendicular to the first axis.
  • each image acquired by the imaging sensor provides the advantages of multiple degrees of spatial coherence.
  • the illumination module have only a single mode of operation.
  • Figs. 4A-B were described as corresponding to different respective modes of operation. However, Figs. 4A-B may also correspond to a single mode of operation, in which the number of light sources 68 that emit the light varies with respect to different respective axes.
  • Fig. 4A shows a cross-section through the illumination unit along one axis (the illumination with respect to this axis being incoherent, for example), while Fig. 4B shows another cross-section along another (e.g., perpendicular) axis (the illumination with respect to this axis being partially-coherent, for example).
  • each pair of figures in Figs. 5A-B and Figs. 6A-B may correspond to a single mode of operation in which stop aperture 90 is shaped asymmetrically with respect to the axes (e.g., is wider along one axis than along another, e.g., is elliptically shaped), such that the spatial coherence varies with respect to the axes by virtue of the light passing through the stop aperture.
  • Figs. 5A and 6A show a cross-section along one axis (the illumination with respect to this axis being incoherent, for example), while Figs. 5B and 6B show another cross-section along another (e.g., perpendicular) axis (the illumination with respect to this axis being partially-coherent, for example).
  • FIGs. 10A-B are schematic illustrations of illumination module 65 comprising a photodiode 112, in accordance with some embodiments of the present invention.
  • illumination module 65 further comprises at least one photodiode 112 configured to receive back reflection of the light emitted from light sources 68, such as back reflection from a condenser lens 110, which is typically uncoated, given that coatings tend to decrease back reflection.
  • the output from photodiode 112 is digitized, optionally amplified, and passed to processor 28 for analysis. Based on this analysis, the processor may adjust (e.g., in real-time) the voltage or current supplied to the light source that emitted the light, so as to correct the light intensity. Alternatively or additionally, the processor may output a message indicating the status of the light source.
  • each photodiode 112 is configured to receive a particular wavelength of light.
  • Fig. 10A shows a photodiode configured for blue light
  • Fig. 10B shows a photodiode configured for ultraviolet light.
  • Such use of back reflection for monitoring and/or correcting the intensity of the light sources may be implemented in any of the embodiments of illumination module 65 described herein, as shown in Fig. 5A for example, and may also be implemented for fluorescent microscopy and for non-imaging applications, such as discrete spectroscopy.

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Abstract

L'invention concerne un appareil et des procédés d'imagerie microscopique d'un échantillon. En commutant, par un processeur (28), entre de multiples modes de fonctionnement d'un module d'éclairage en champ clair (65), la lumière est émise par le module d'éclairage (65), à différents degrés respectifs de cohérence spatiale, de telle sorte que la lumière passe à travers l'échantillon et est ensuite focalisée, par un objectif (66), sur un capteur d'imagerie (67). De multiples images de l'échantillon sont délivrées, à partir du capteur d'imagerie (67), sur la base de la lumière focalisée sur le capteur d'imagerie (67), les images variant les unes des autres en conséquence des différents degrés de cohérence spatiale. L'invention concerne également d'autres applications.
PCT/IB2024/062064 2023-12-04 2024-12-01 Variation de cohérence spatiale dans un système de microscope Pending WO2025120470A1 (fr)

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