WO2022009104A2

WO2022009104A2 - Focusing a microscope using fluorescent images

Info

Publication number: WO2022009104A2
Application number: PCT/IB2021/056073
Authority: WO
Inventors: Amir ZAIT; Arnon Houri Yafin; Roi PADAN; Dan Gluck; Ofir Rimer; Sar GROSS; Lior OFER; Yochay Shlomo ESHEL; Sarah LEVY; Joseph Joel POLLAK
Original assignee: S.D. Sight Diagnostics Ltd
Priority date: 2020-07-07
Filing date: 2021-07-07
Publication date: 2022-01-13
Also published as: WO2022009104A3

Abstract

Apparatus and methods are described for use with a microscope (24) that acquires images of a bodily sample disposed in a sample carrier (22). A fluorescent, microscopic image of a given x-y region is acquired, while a focal plane of the microscope (24) is set at an estimated optimum focal plane. A focusing region-of-interest is identified within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest. A set of fluorescent focusing images of the region-of-interest are acquired with the microscope (24) focused at respective focusing levels. An optimum focal plane at which to acquire a microscopic image of the region is determined, at least partially based upon a relationship between a contrast-indicative parameter of each of the images and the focusing level at which the microscope (24) was focused when the image was acquired. Other applications are also described.

Description

FOCUSING A MICROSCOPE USING FLUORESCENT IMAGES

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. Provisional Patent Application No. 63/048,702 to Zait et al., filed July 07, 2020, entitled "Focusing a microscope using fluorescent images," which is incorporated herein by reference.

FIELD OF EMBODIMENTS OF THE INVENTION

Some applications of the presently disclosed subject matter relate generally to analysis of bodily samples, and, in particular, to optical density and microscopic measurements that are performed upon blood samples.

BACKGROUND

In some optics-based methods (e.g., diagnostic, and/or analytic methods), a property of a biological sample, such as a blood sample, is determined by performing an optical measurement. For example, the density of a component (e.g., a count of the component per unit volume) may be determined by counting the component within a microscopic image. Similarly, the concentration and/or density of a component may be measured by performing optical absorption, transmittance, fluorescence, and/or luminescence measurements upon the sample. Typically, 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 measurements that are performed upon the portion of the sample that is contained within the chamber of the sample carrier are analyzed in order to determine a property of the sample.

Observation of a sample with a microscope generally requires three dimensional adjustment of focus. In order to image a portion of the sample disposed at a given 3-dimensional location within the sample carrier, the imaging field of the microscope is typically aligned with the portion of the sample in the plane that is perpendicular to the optical axis of the microscope (also known as x-y positioning), and the focal plane of the microscope is made to coincide with the portion of the sample along the optical axis of the microscope (also referred to as z- positioning). Typically, x-y positioning is performed by sequentially moving the microscope and the sample carrier with respect to each other within the x-y plane, such that microscopic images of imaging fields of the sample are captured at respective x-y locations, and the z-positioning is performed automatically using an autofocus system cooperating with the microscope. SUMMARY OF EMBODIMENTS

Typically, before using a microscope to acquire a diagnostic image of a given image field of a bodily sample (such as a blood sample), the microscope is focused, i.e., the optimum focal plane at which to focus the microscope in order to acquire the diagnostic image is determined, and the focal plane of the microscope is set accordingly. (A diagnostic image should be interpreted herein to mean an image that is analyzed in order to determine parameters of the sample, and/or components thereof.) Typically, a plurality of images that (partially or fully) overlap with each other in the x-y plane are acquired, with the focal plane of the microscope set at respective focusing levels along the z-axis. (In the present application, the term z-axis is used to refer to the optical axis of the optical system, and the x-y plane is used to denote the plane that is perpendicular to the optical axis, as is common in the art. The terms "z-axis" and "optical axis" are used interchangeably.) The images are analyzed in order to determine the optimum focal plane at which to focus the microscope in order to acquire the diagnostic image. For some applications, in order to acquire the diagnostic image, the focal plane of the microscope is set to a focusing level that is derived from the optimum focal plane, as described in further detail hereinbelow.

In some cases, in order to determine the optimum focal plane at which to focus the microscope in order to acquire a diagnostic fluorescent image, a proxy focusing target is used. For example, brightfield images of the sample may be acquired, and the optimum focal plane may be determined by analyzing the brightfield images, for example, using techniques described in US 10,176,565 to Greenfield, which is incorporated herein by reference. For some applications, it is desirable to determine the optimum focal plane for a diagnostic fluorescent image using the target fluorescent entities themselves, rather than using a proxy for determining the optimum focal plane at which to acquire a diagnostic image of the target entities.

It is typically the case that fluorescing entities that are visible in fluorescent images are relatively sparsely distributed throughout the sample. This is a particular issue for certain types of samples, such as fine needle aspirate samples. Typically, the relative sparsity of the fluorescent entities in the images renders it somewhat challenging to focus the microscope using such entities. Moreover, for some applications of the present invention, the microscope is focused relatively frequently, during the acquisition of microscopic issues of a sample. For example, the microscope may be focused for each and every image field, or every x image fields, where x may be an integer of between 2 and 10. A further challenge when focusing a microscope using fluorescent entities is that the fluorescent entities typically undergo photobleaching. It is typically necessary to determine the correct focal plane for the microscope before the fluorescent entities have undergone significant photobleaching, such that the fluorescent entities are still fluorescing in the diagnostic image that is ultimately acquired at the determined optimum focal plane. For some applications, the above challenges are overcome by performing a focusing procedure as described herein.

There is therefore provided, in accordance with some applications of the present invention, a method for use with a microscope that is configured to acquire images of a bodily sample disposed in a sample carrier, including: acquiring a fluorescent, microscopic image of a given x-y region within the sample carrier, while a focal plane of the microscope is set at an estimated optimum focal plane for the x-y region; identifying a focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest; acquiring a set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis; for the set of fluorescent focusing images, determining a relationship between a contrast- indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired; identifying an optimum focal plane at which to acquire a microscopic image of the given x-y region, at least partially based upon said relationship; and acquiring a diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focusing level that is based upon the identified optimum focal plane.

In some applications, acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane includes acquiring a diagnostic image of the x-y region of the sample carrier in a different imaging modality than an imaging modality in which the set of fluorescent focusing images are acquired.

In some applications, acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane includes acquiring a diagnostic image of the x-y region of the sample carrier in a similar imaging modality to an imaging modality in which the set of fluorescent focusing images are acquired.

In some applications, acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane includes acquiring a diagnostic image of the x-y region of the sample carrier with the focal plane of the microscope set to the identified optimum focal plane.

In some applications, acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane includes acquiring a diagnostic image of the x-y region of the sample carrier with the focal plane of the microscope set to a focal plane that is offset by a predetermined amount with respect to the identified optimum focal plane.

In some applications, identifying the optimum focal plane at which to acquire the microscopic image of the given x-y region includes identifying, as the optimum focal plane, a focusing level at which the contrast-indicative parameter indicates that image contrast is at a maximum.

In some applications, determining the relationship between a contrast-indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired further includes interpolating the relationship such as to estimate the contrast-indicative parameter for interpolated focusing levels, and identifying the optimum focal plane at which to acquire the microscopic image of the given x-y region includes identifying one of the interpolated focusing levels as the optimum focal plane.

In some applications, the method is for use with a blood sample disposed within the sample carrier, and identifying the focusing region-of-interest within the acquired image, includes identifying the focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: platelets leukocytes, pathogens, and any combination thereof.

In some applications, the method is for use with a fine needle aspirate sample disposed within the sample carrier, and identifying the focusing region-of-interest within the acquired image comprises identifying the focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, parasites, and any combination thereof.

In some applications, acquiring the set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis, includes acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region.

In some applications, acquiring the set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region includes acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis, such that subsequent to acquiring the set of fluorescent focusing images of the region-of-interest, an intensity of fluorescence of entities within the given x-y region is more than 90 percent of an intensity of fluorescence of the entities when the entities first begin to fluoresce.

In some applications, acquiring the set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region includes selecting a wavelength at which to excite the sample in order to acquire the set of fluorescent focusing images, such as to prevent causing substantial photobleaching of entities within the given x-y region.

In some applications, acquiring the set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region includes intermittently exciting the sample with excitation light during the acquisition of the set of fluorescent focusing images.

In some applications, acquiring the set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis includes acquiring a set of focusing images along a range of focusing levels that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane.

In some applications, the method further includes, in response to identifying an initially- determined optimum focal plane that is toward an edge of said range of focusing levels, acquiring a new set of fluorescent focusing images of the region-of-interest along a range of focusing levels that are centered around the initially -determined optimum focal plane and that extend from the given distance above the initially -determined optimum focal plane to a given distance below the initially-determined optimum focal plane. In some applications, the method further includes, in response to being unable to detect an optimum focal plane within said range of focusing levels, acquiring focusing images along an extended range of distances above the estimated optimum focal plane and below the estimated optimum focal plane.

There is further provided, in accordance with some applications of the present invention, apparatus for use a bodily sample disposed in a sample carrier, the apparatus including: a microscope configured to acquire microscopic images of the bodily sample disposed in the sample carrier; and at least one computer processor configured to: drive the microscope to acquire a fluorescent, microscopic image of a given x-y region within the sample carrier, while a focal plane of the microscope is set at an estimated optimum focal plane for the x-y region, identify a focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, drive the microscope to acquire a set of fluorescent focusing images of the region- of-interest with the microscope focused at respective focusing levels along the optical axis, for the set of fluorescent focusing images, determine a relationship between a contrast-indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired, identify an optimum focal plane at which to acquire a microscopic image of the given x-y region, at least partially based upon said relationship, and drive the microscope to acquire a diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focusing level that is based upon the identified optimum focal plane.

In some applications, the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier in a different imaging modality than an imaging modality in which the set of fluorescent focusing images are acquired.

In some applications, the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier in a similar imaging modality to an imaging modality in which the set of fluorescent focusing images are acquired. In some applications, the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the identified optimum focal plane.

In some applications, the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focal plane that is offset by a predetermined amount with respect to the identified optimum focal plane.

In some applications, the computer processor is configured to identify the optimum focal plane at which to acquire the microscopic image of the given x-y region by identifying as the optimum focal plane, a focusing level at which the contrast-indicative parameter indicates that image contrast is at a maximum.

In some applications, the computer processor is configured to determine the relationship between the contrast-indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired by interpolating the relationship such as to estimate the contrast-indicative parameter for interpolated focusing levels, and the computer processor is configured to identify one of the interpolated focusing levels as the optimum focal plane.

In some applications, the apparatus is for use with a blood sample disposed within the sample carrier, and the computer processor is configured to identify the focusing region-of-interest within the acquired image based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: platelets leukocytes, pathogens, and any combination thereof.

In some applications, the apparatus is for use with fine needle aspirate sample disposed within the sample carrier, and the computer processor is configured to identify the focusing region- of-interest within the acquired image based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, parasites, and any combination thereof.

In some applications, the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region. In some applications, the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis, such that subsequent to the microscope acquiring the set of fluorescent focusing images of the region-of-interest, an intensity of fluorescence of entities within the given x-y region is more than 90 percent of an intensity of fluorescence of the entities when the entities first begin to fluoresce.

In some applications, the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region by selecting a wavelength at which to excite the sample in order to acquire the set of fluorescent focusing images, such as to prevent causing substantial photobleaching of entities within the given x-y region.

In some applications, the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region by intermittently exciting the sample with excitation light during the acquisition of the set of fluorescent focusing images.

In some applications, the computer processor is configured to drive the microscope to acquire the set of focusing images along a range of focusing levels that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane.

In some applications, in response to identifying an initially -determined optimum focal plane that is toward an edge of said range of focusing levels, the computer processor is configured to drive the microscope to acquire a new set of fluorescent focusing images of the region-of- interest along a range of focusing levels that are centered around the initially-determined optimum focal plane and that extend from the given distance above the initially -determined optimum focal plane to a given distance below the initially-determined optimum focal plane.

In some applications, in response to being unable to detect an optimum focal plane within said range of focusing levels, the computer processor is configured to drive the microscope to acquire focusing images along an extended range of distances above the estimated optimum focal plane and below the estimated optimum focal plane.

There is further provided, in accordance with some applications of the present invention, a computer software product, for use with a microscope, and a bodily sample disposed within a sample carrier, the computer software product including a non-transitory computer-readable medium in which program instructions are stored, which instructions, when read by a computer cause the computer to perform the steps of: drive the microscope to acquire a fluorescent, microscopic image of a given x-y region within the sample carrier, while a focal plane of the microscope is set at an estimated optimum focal plane for the x-y region, identify a focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, drive the microscope to acquire a set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis, for the set of fluorescent focusing images, determine a relationship between a contrast- indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired, identify an optimum focal plane at which to acquire a microscopic image of the given x-y region, at least partially based upon said relationship, and drive the microscope to acquire a diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focusing level that is based upon the identified optimum focal plane.

The present invention will be more fully understood from the following detailed description of embodiments thereof, taken together with the drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a block diagram showing components of a biological sample analysis system, in accordance 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. 3 A, 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;

Figs. 4A and 4B are fluorescent, microscopic images of a portion of a blood sample, after staining the sample with Acridine Orange and with a Hoechst reagent and exciting the sample such that the platelet fluoresces, by illuminating the sample with UV light and blue light, respectively, in accordance with some applications of the present invention;

Fig. 5 is a graph showing variance of a fluorescent, microscopic images that are acquired at a given x-y location, as a function of the locations of the focal planes of the microscopic images along the optical axis of the microscope, in accordance with some applications of the present invention; and

Fig. 6 is a flowchart showing steps of a focusing procedure that are performed in accordance with some applications of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Reference is now made to Fig. 1A, which is block diagram showing components of a biological sample analysis system 20, in accordance with some applications of the present invention. Typically, a biological sample (e.g., a blood sample) is placed into 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. For example, the optical measurement devices may include a microscope (e.g., a digital microscope), a spectrophotometer, a photometer, a spectrometer, a camera, a spectral camera, a hyperspectral camera, a fluorometer, a spectrofluorometer, and/or a photodetector (such as a photodiode, a photoresistor, and/or a phototransistor). For some applications, 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) sends instructions to the computer processor via a user interface 32. For some applications, 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. Typically, the computer processor generates an output via an output device 34. Further typically, the output device includes a display, such as a monitor, and the output includes an output that is displayed on the display. For some applications, 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. For some applications, user interface 32 acts as both an input interface and an output interface, i.e., it acts as an input/output interface. For some applications, 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.

Reference is now made to Figs. 2A, 2B, and 2C, which 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, while 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. For some applications, one or more optical measurement devices 24 (and/or computer processor 28 and memory 30) is housed inside optical measurement unit 31. In order to perform the optical measurements upon the sample, sample carrier 22 is placed inside the optical measurement unit. For example, the optical measurement unit may define a slot 36, via which the sample carrier is inserted into the optical measurement unit. Typically, the optical measurement unit includes a stage 64, which is configured to support sample carrier 22 within the optical measurement unit. For some applications, 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) functions as user interface 32 and/or output device 34.

Typically, the optical measurement unit includes microscope system 37 (shown in Figs. 2B-C) configured to perform microscopic imaging of a portion of the sample. For some applications, the microscope system includes a set of light sources 65 (which typically include a set of brightfield light sources (e.g. light emitting diodes) that are configured to be used for brightfield imaging of the sample, a set of fluorescent light sources (e.g. light emitting diodes) that are configured to be used for fluorescent imaging of the sample), and a camera (e.g., a CCD camera, or a CMOS camera) configured to image the sample. Typically, 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. For some applications, the optical-density-measurement 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. For some applications, each of the aforementioned sets of light sources (i.e., the set of brightfield light sources, the set of fluorescent light sources, and the set optical-density-measurement light sources) includes a plurality of light sources (e.g. a plurality of light emitting diodes), each of which is configured to emit light at a respective wavelength or at a respective band of wavelengths.

Reference is now made to Figs. 3 A and 3B, which 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). Typically, 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. Typically, the sample chambers of the sample carrier are filled with a bodily sample, such as blood via sample inlet holes 38. For some applications, the sample chambers define one or more outlet holes 40. The outlet holes are configured to facilitate filling of the sample chambers with the bodily sample, by allowing air that is present in the sample chambers to be released from the sample chambers. Typically, as shown, 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.

Reference is made to Fig. 3C, which shows an exploded view of sample carrier 22, in accordance with some applications of the present invention. For some applications, 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. For example, as shown, 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 bodily sample, by allowing air to flow to the outlet holes, and/or by allowing the bodily sample to flow around the central portion of the sample chamber.

For some applications, a sample carrier as shown in Figs. 3A-C is used when performing a complete blood count on a blood sample. For some such applications, the sample carrier is used with optical measurement unit 31 configured as generally shown and described with reference to Figs. 2A-C. For some applications, 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. 2C)). For some applications, first set 52 of sample chambers includes a plurality of sample chambers, while second set 54 of sample chambers includes only a single sample chamber, as shown. However, 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. For example, the diluent may contain pH buffers, stains, fluorescent stains, antibodies, sphering agents, lysing agents, etc. Typically, the second portion of the blood sample, which is placed inside second set 54 of sample chambers is a natural, undiluted blood sample. Alternatively or additionally, 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.

For some applications, 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. For example, the staining substance may be configured to stain DNA with preference over staining of other cellular components. Alternatively, the staining substance may be configured to stain all cellular nucleic acids with preference over staining of other cellular components. For example, the sample may be stained with Acridine Orange reagent, Hoechst reagent, and/or any other staining substance that is configured to preferentially stain DNA and/or RNA within the blood sample. Optionally, 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). Typically, the first portion of the sample is stained with Acridine Orange and with a Hoechst reagent. For example, the first (diluted) portion of the blood sample may be prepared using techniques as described in US 9,329,129 to Poliak, 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. For some applications, 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. For example, the first portion of the sample may be stained with methylene blue and/or Romanowsky stains. For some applications, 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.

Referring again to Figs. 2B-C, typically, sample carrier 22 is supported within the optical measurement unit by stage 64. Further typically, 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. For some applications, 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 an objective lens 66 of a microscope unit of the optical measurement unit is disposed below the glass layer of the sample carrier. Typically, at least some light sources 65 that are used during microscopic measurements that are performed upon the sample (for example, light sources that are used during brightfield imaging) illuminate the sample carrier from above the molded component. Further typically, at least some additional light sources (not shown) illuminate the sample carrier from below the sample carrier (e.g., via the objective lens). For example, light sources that are used to excite the sample during fluorescent microscopy may illuminate the sample carrier from below the sample carrier (e.g., via the objective lens).

Typically, prior to being imaged microscopically, 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 Poliak, which is incorporated herein by reference. For some applications, 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). Typically, 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. Subsequent to the cells having been left to settle on the base surface of the sample chamber (e.g., by having been left to settle for a predefined time interval), at least one microscopic image of at least a portion of the monolayer of cells is typically acquired. Typically, 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. Typically, 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. For some applications, respective imaging fields have different optimum depth levels from each other.

It is noted that, in the context of the present application, 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"). Within the monolayer there may be some overlap of cells, such that within certain areas there are two or more overlapping layers of cells. For example, 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.

For some applications, the microscopic analysis of the first portion of the blood sample is performed with respect to the monolayer of cells. Typically, the first portion of the blood sample is imaged under brightfield imaging, i.e., under illumination from one or more light sources (e.g., one or more light emitting diodes, which typically emit light at respective spectral bands). Further typically, the first portion of the blood sample is additionally imaged under fluorescent imaging. Typically, 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. Typically, for the fluorescent imaging, a separate set of light sources (e.g., one or more light emitting diodes) is used to illuminate the sample at the known excitation wavelengths. As described hereinabove, for some applications, the sample is stained with Acridine Orange reagent and Hoechst reagent. For some such applications, 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.

As described with reference to US 2019/0302099 to Poliak, which is incorporated herein by reference, for some applications, sample chambers belonging to set 52 (which is used for microscopy measurements) 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. For example, if a blood sample, and/or a monolayer formed by the sample, has a relatively low density of red blood cells, then 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. Conversely, if a blood sample, and/or a monolayer formed by the sample, has a relatively high density of red blood cells, then such measurements may be performed upon a sample chamber of the sample carrier having a relatively low height, for example, such that there is a sufficient sparsity of cells, and/or such that there is a sufficient sparsity of cells within the monolayer of cells formed by the sample, that the cells can be identified within microscopic images. For some applications, such methods are performed even without the variation in height between the sample chambers belonging to set 52 being precisely known.

For some applications, based upon the measurand that is being measured, the sample chamber within the sample carrier upon which to perform optical measurements is selected. For example, 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. Conversely, in order to determine mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red blood cell distribution width (RDW), red blood cell morphologic features, and/or red blood cell abnormalities, 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. Similarly, in order to count platelets, classify platelets, and/or extract any other attributes (such as volume) of platelets, 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.

In accordance with the above-described examples, it is preferable to use 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. Therefore, for some applications, 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. For some applications, 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.

Typically, in order to perform optical density measurements upon the sample, it is desirable to know the optical path length, the volume, and/or the thickness of the portion of the sample upon which the optical measurements were performed, as precisely as possible. Typically, 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). For example, the concentration and/or density of a component may be measured by performing optical absorption, transmittance, fluorescence, and/or luminescence measurements upon the sample.

Referring again to Fig. 3B, for some applications, sample chambers belonging to set 54 (which is used for optical density measurements), 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 Poliak, 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 Poliak, which is incorporated herein by reference. For some applications, 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. For some applications, 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.

As described hereinabove, while the sample is disposed in the sample carrier, optical measurements are performed upon the sample using one or more optical measurement devices 24. Typically, 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. Typically, the sample carrier is inserted into optical measurement unit 31, which houses the optical measurement device while the optical measurements are performed. Typically, the optical measurement unit houses the sample carrier such that the molded layer is disposed above the glass layer, and such that the optical measurement unit is disposed below the glass layer of the sample carrier and is able to perform optical measurements upon the sample via the glass layer. The sample carrier is formed by adhering the glass layer to the molded component. For example, the glass layer and the molded component may be bonded to each other during manufacture or assembly (e.g. using thermal bonding, solvent-assisted bonding, ultrasonic welding, laser welding, heat staking, adhesive, mechanical clamping and/or additional substrates). For some applications, the glass layer and the molded component are bonded to each other during manufacture or assembly using adhesive layer 46.

For some applications, the apparatus and methods described herein are applied to a fine needle aspirate sample. For some such applications, 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.

Reference is now made to Figs. 4A and 4B, which are fluorescent, microscopic images of a portion of a blood sample, after staining the sample with Acridine Orange and with a Hoechst reagent and exciting the sample such that an entity 70 fluoresces, by illuminating the sample, respectively, with UV light (Fig. 4A) and blue light (Fig. 4B), in accordance with some applications of the present invention. For some applications, stained entities that emit fluorescent light are used as targets for focusing the microscope, by an autofocus system of the microscope. For some such applications, the stained entities are stained with stains that are configured to stain DNA, RNA, and/or or other cellular organelles. For example, the stained entities may be stained with Acridine Orange and/or with a Hoechst reagent. Typically, by staining the blood sample with stains that are configured to stain DNA and RNA, the entities that fluoresce are cells that have a nucleus or cytoplasm. Alternatively or additionally, other fluorescent stains may be used to stain the bodily sample. For example, antibody-bound stains may be used. In the example shown in Figs. 4A and 4B, the stained entity is a platelet. However, the scope of the present invention includes staining other entities, such as leukocytes, and/or pathogens. For some applications, the apparatus and methods described herein are applied to a fine needle aspirate sample. For some such applications, 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.

Typically, before acquiring a diagnostic image of a given image field of the sample (i.e., an image that is analyzed in order to determine parameters of the sample, and/or components thereof), the microscope is focused, i.e., the optimum focal plane at which to focus the microscope in order to acquire the diagnostic image is determined, and the focal plane of the microscope is set accordingly. Typically, a plurality of images that (partially or fully) overlap with each other in the x-y plane are acquired, with the focal plane of the microscope set at respective focusing levels along the z-axis. (In the present application, the term z-axis is used to refer to the optical axis of the optical system, and the x-y plane is used to denote the plane that is perpendicular to the optical axis, as is common in the art. The terms "z-axis" and "optical axis" are used interchangeably.) The images are analyzed in order to determine the optimum focal plane at which to focus the microscope in order to acquire the diagnostic image. (For some applications, in order to acquire the diagnostic image, the focal plane of the microscope is set to a focusing level that is derived from the optimum focal plane, as described in further detail hereinbelow.)

In some cases, in order to determine the optimum focal plane at which to focus the microscope in order to acquire a diagnostic fluorescent image, a proxy focusing target is used. For example, brightfield images of the sample may be acquired, and the optimum focal plane may be determined by analyzing the brightfield images, for example, using techniques described in US 10,176,565 to Greenfield, which is incorporated herein by reference. For some applications, it is desirable to determine the optimum focal plane using the target fluorescent entities themselves, rather than using a proxy for determining the optimum focal plane at which to acquire a diagnostic image of the target entities.

As indicated in Figs. 4A and 4B, it is typically the case that fluorescing entities that are visible in fluorescent images are relatively sparsely distributed throughout the sample. This is a particular issue for certain types of samples, such as fine needle aspirate samples. Typically, the relative sparsity of the fluorescent entities in the images renders it somewhat challenging to focus the microscope using such entities. Moreover, for some applications of the present invention, the microscope is focused relatively frequently, during the acquisition of microscopic issues of a sample. For example, the microscope may be focused for each and every image field, or every x image fields, where x may be an integer of between 2 and 10. A further challenge when focusing a microscope using fluorescent entities is that the fluorescent entities typically undergo photobleaching. It is typically necessary to determine the correct focal plane for the microscope before the fluorescent entities have undergone significant photobleaching, such that the fluorescent entities are still fluorescing in the diagnostic image that is ultimately acquired at the determined optimum focal plane. For some applications, the above challenges are overcome by performing a focusing procedure as described herein.

An estimated optimum focal plane estimate is calculated. Typically, the estimated optimum focal plane is calculated based upon factors such as mechanical measurements of the system, focal planes of neighboring cells and/or in neighboring imaging fields, features of the sample carrier, a focal plane that has been established for a brightfield image of a partially- overlapping or fully-overlapping image field, etc. For some applications, the estimated optimum focal plane is calculated by analyzing entities within one or more preliminary images, and based on the sizes of the entities in the preliminary images, calculating the estimated optimum focal plane. (It is typically the case that entities will appear bigger in images that are less well focused upon the entities.) In the next step, the microscope typically acquires an image at the estimated optimum focal plane. In the acquired image, a region-of-interest that is suitable for remaining steps of the focusing procedure is identified. For example, a vertical strip of predetermined width may be identified as a suitable region-of-interest based on this strip containing more nuclei than other, similar vertical strips of the predetermined width, and/or based on this strip having higher fluorescence intensity levels than any other, similar vertical strips of the predetermined width.

It is noted that, in fluorescent images, fluorescing entities typically provide a relatively high level of contrast with respect to their surroundings (as indicated in Figs. 4A and 4B). Therefore, it is typically relatively easy to identify such entities in the acquired image, and to thereby determine parameters such as the region in which the most nuclei are visible, and/or the region having the highest fluorescence levels. Thus, such entities (as well as parameters relating to such entities) are typically identifiable even in a low cell density solution. Typically, once the region-of-interest has been identified, the procedure for determining the optimum focal plane proceeds as follows.

Typically, a plurality of focusing images of the region-of-interest are acquired at respective focusing levels along the optical axis, within a relatively short amount of time. For example, between 35 and 65 focusing images may be acquired within less than 130 ms, e.g., between 70 and 130 ms. Typically, the focusing images are acquired at a rate of between 1.5 ms per image and 2.5 ms per image, e.g., approximately 2 ms per image. Typically, the set of focusing images are centered around the estimated optimum focal plane. For example, the set of focusing images may be acquired along a range of focusing levels along the optical axis that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane. Typically, the overall time that it takes to complete the focusing of the microscope with respect to a given image field is constrained so as to avoid significant photobleaching effect to the imaged field and/or neighboring fields during the focusing procedure. Further typically, the duration of the focusing procedure with respect to a given image field is such that by the end of the focusing with respect to the image field, the intensity of fluorescence of the entities within the image field or within neighboring image fields is more than 90 percent, more than 95 percent, or more than 99 percent of the intensity of fluorescence of the entities when the entities first begin to fluoresce (i.e., before the entities have undergone any photobleaching). On the other hand, over the course of the focusing procedure, focusing images need to be acquired at a sufficient number of focusing levels for the optimum focal plane may be determined relatively accurately. Typically, factors such as the image acquisition rate, the speed at which the focal plane of the microscope is adjusted, and/or the portion of the image field that is imaged, are constrained by the aforementioned factors.

For some applications, in each of the focusing images, only a portion of the image field within the x-y plane is acquired, in order to speed up the image-acquisition rate. Typically, the focusing images are acquired while the microscope focal plane is moved along the optical axis with respect to the sample carrier at the maximum possible speed. (In accordance with respective applications, the microscope focal plane is moved with respect to the sample carrier, by (a) moving the sample carrier with respect to the microscope, (b) moving the microscope with respect to the sample carrier, and/or (c) by modifying the focal length of the microscope.) Further typically, the focusing images are acquired at the greatest image-acquisition rate that the imaging sensor (e.g., the CCD) of the microscope unit will allow. For some applications, the image-acquisition rate of the focusing images, and/or the speed of movement of the microscope focal plane along the optical axis with respect to the sample carrier varies over the course of the focusing procedure, e.g., in accordance with techniques described in US 2016/0246046 to Yorav Raphael, which is incorporated herein by reference. For some applications, pixels belonging to the focusing images are binned during the image-acquisition process, in order to allow for faster image-acquisition and image-processing times.

For some applications, during the focusing procedure, the illumination of the sample with the excitation light is performed intermittently (e.g., using a blinking LED), in order to reduce the total exposure time of the sample to the excitation light. For some applications, a combination of fluorescent images and brightfield images are used to determine the optimum focal plane, while reducing the exposure of the sample to the excitation light, relative to if only fluorescent images were to be used for the focusing procedure. For example, techniques described in US 10,176,565 to Greenfield, which is incorporated herein by reference, may be combined with the techniques described herein.

Reference is now made to Fig. 5, which is a graph showing variance of fluorescent, microscopic images that are acquired at a given x-y location, as a function of the locations of the focal planes of the microscopic images along the optical axis of the microscope, in accordance with some applications of the present invention. Fig. 5 shows an example of a typical plot for a plurality of focusing images that are acquired along the optical axis for a given image field, with each focusing image being represented by a small circle. As shown, the plot of variance against z-axis value is typically interpolated between the circles representing the focusing images.

Typically, each focusing image is analyzed to provide one or more parameters that are indicative of contrast, such as variance or coefficient of variance. By way of example, Fig. 5 shows variance measurements plotted against locations of respective focusing levels along the optical axis. The focusing level (or an interpolated focusing level) having the maximal variance is the level at which edges of the fluorescent entities are the clearest. Therefore, this focusing level typically corresponds to the optimum focal plane. For some applications, a similar procedure is performed with a different contrast-indicative parameter, such as, coefficient of variance, standard deviation, and/or sum of absolute-value of derivatives. For some applications, the optimum focal plane corresponds to one of the focusing levels at which an image was acquired during the focusing procedure. Alternatively, interpolation of the variances of the acquired focusing images is performed, and an optimum focal plane may be determined to correspond to interpolated focusing level that lies between the focusing levels of two of the acquired focusing images.

For some applications (not shown), a variance curve of the type shown in Fig. 5 defines a plurality of local maxima. For some applications, one of the maxima is selected as the optimum focal plane based upon criteria, such as, the overall maximum coefficient of variance, FWHM of the respective maxima (with the narrowest maximum typically being selected), or distinctness of the maximum from its local background (e.g., the difference between the maximum and the background normalized in units of the background standard deviations.) For some applications, a plurality of optimum focal planes are selected to correspond to each of a plurality of local maxima of the variance curve. For such applications, respective diagnostic images are typically acquired with the focal plane of the microscope set at each of the optimum focal planes (and/or derivatives thereof). For some applications, in response to identifying a plurality of local maxima, or if the local maximum does not satisfy certain criteria (e.g., maximum coefficient of variance, FWHM of the maximum, or distinctness of the maximum from its local background), the focusing procedure is repeated using a different region-of-interest (e.g., a square-shaped region-of-interest instead of a strip). Alternatively, in response to identifying a plurality of local maxima, or if the local maximum does not satisfy certain criteria (e.g., maximum coefficient of variance, FWHM of the maximum, or distinctness of the maximum from its local background), the computer processor determines that there is no optimum focal plane for a given region within the x-y plane (e.g., for a given image field). It is noted that operations that are described in the present paragraph with reference to a variance curve are typically performed by the computer processor as a series of algorithmic steps, without the computer processor actually generating a variance curve.

For some applications, once the optimum focal plane is determined, a diagnostic image of the sample is acquired with the focal plane of the microscope set based upon the determined optimum focal plane. In accordance with respective applications, the aforementioned diagnostic images may be acquired in various modalities. For example, the diagnostic images may be fluorescent, microscopic images that are acquired using the same parameters (e.g., the same excitation wavelength band and the same imaging wavelength band) as those used for the focusing images, fluorescent, microscopic images that are acquired using the different parameters from those used for the focusing images, and/or brightfield images. For some applications, once the optimum focal plane is determined, a diagnostic image of the sample is acquired with the focal plane of the microscope set to the determined optimum focal plane. Alternatively or additionally, one or more diagnostic images are acquired with the focal plane of the microscope set to a focal plane that is derived from the optimum focal plane, e.g., offset from the optimum focal plane by a given distance or by a given number of focal depths. For some applications, such off-focus images are acquired in order to extract certain parameters that cannot be extracted from on-focus images alone. Alternatively or additionally, the focal plane for images that are acquired in certain imaging modalities may be offset with respect to the determined optimum focal plane, in order to account for phenomena, such as axial chromatic aberrations.

Reference is now made to Fig. 6, which is a flowchart summarizing steps of a focusing procedure that is performed in accordance with some applications of the present invention.

In a first step 80, an estimated optimum focal plane for a given x-y region (e.g., a given image field) is calculated and a fluorescent, microscopic image of the x-y region is acquired with the focal plane of the microscope set to the estimated optimum focal plane.

Subsequently, in step 82, a focusing region-of-interest is identified within the acquired image, e.g., based on the number of nuclei identified within the region-of-interest, and/or based on the fluorescent intensity levels within the region-of-interest. It is noted that step 82 is optional, and for some applications, step 84 is performed with respect to the entire x-y region.

Typically, in the next step 84, a set of focusing images of the region-of-interest are acquired at respective focusing levels along the optical axis, within a relatively short amount of time.

In step 86, for each of the focusing images, a parameter that is indicative of a level of contrast of the image (e.g., variance, coefficient of variance, etc.) is determined, and a relationship between the parameter and location of the focusing level along the z-axis is determined.

In step 88, the optimum focal plane is determined based upon the above-mentioned relationship. Typically, the optimum focal plane is selected to correspond to the focusing level at which the parameter that is indicative of a level of contrast of the image (e.g., variance, coefficient of variance, etc.) is at a maximum. As described hereinabove, in response to detecting a plurality of local maxima, and/or in response to the one or more maxima not satisfying certain criteria, steps 82-86 may be repeated using a different region-of-interest, and/or the computer processor may determine that an optimum focal plane cannot be found for this x-y region.

It is noted that, as described with reference to step 84, typically, a set of focusing images of the region-of-interest are acquired at respective focusing levels along the optical axis. Typically, the set of focusing images are centered around the estimated optimum focal plane. For example, the set of focusing images may be acquired along a range of focusing levels that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane. For some applications, in response to determining that the optimum focal plane (as initially identified) lies towards the edge of the aforementioned range of focusing levels (e.g., within a given percentage of the edge, within a given number of focusing levels from the edge, or within a given absolute distance from the edge), a new set of focusing images of the region-of-interest are acquired at respective focusing levels along the optical axis. Typically, this new set of focusing images is acquired over a similar range of focusing levels along the optical axis, but with the range being centered around the optimum focal plane as found in the previous step.

As described in the above paragraph, for some applications, the set of focusing images are acquired along a range of focusing levels that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane. For some applications, in response to being unable to detect an optimum focal plane within the aforementioned range of focusing levels, focusing images are acquired along an extended range of distances above the estimated optimum focal plane and below the estimated optimum focal plane. For example, if initially, a set of focusing images are acquired along a range of focusing levels extending from a distance D above the estimated optimum focal plane to a distance -D below the estimated optimum focal plane, the range of the focusing images may be extended such that focusing images are acquired along a range of focusing levels extending from a distance of between 2D and 3D above the estimated optimum focal plane to a distance of -2D and -3D below the estimated optimum focal plane. For some applications, the steps described in the present paragraph and/or the previous paragraph are repeated iteratively, until an optimum focal plane is found.

In step 90, once the optimum focal plane is determined, a diagnostic image of the sample is acquired with the focal plane of the microscope set based upon the determined optimum focal plane. As described hereinabove, the aforementioned diagnostic images may be acquired in various modalities. For some applications, once the optimum focal plane is determined, a diagnostic image of the sample is acquired with the focal plane of the microscope set to the determined optimum focal plane. Alternatively or additionally, one or more diagnostic images are acquired with the focal plane of the microscope set to a focal plane that is derived from the optimum focal plane, e.g., offset from the optimum focal plane by a given distance or by a given number of focal depths.

For some applications, the sample as described herein is a sample that includes blood or components thereof (e.g., a diluted or non-diluted whole blood sample, a sample including predominantly red blood cells, or a diluted sample including predominantly red blood cells), and parameters are determined relating to components in the blood such as platelets, white blood cells, anomalous white blood cells, circulating tumor cells, red blood cells, reticulocytes, Howell-Jolly bodies, sickle cells, tear-drop cells, etc. For some applications, the sample includes a fine needle aspirate sample. For some such applications, parameters are determined relating to components in the sample such as: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, and/or parasites.

Applications of the invention described herein can take the form of a computer program product accessible from a computer-usable or computer-readable medium (e.g., a non-transitory computer-readable medium) providing program code for use by or in connection with a computer or any instruction execution system, such as computer processor 28. For the purposes of this description, a computer-usable or computer readable medium can be any apparatus that can comprise, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. The medium can be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system (or apparatus or device) or a propagation medium. Typically, the computer-usable or computer readable medium is a non- transitory computer-usable or computer readable medium.

Examples of a computer-readable medium include a semiconductor or solid state memory, magnetic tape, a removable computer diskette, a random-access memory (RAM), a read-only memory (ROM), a rigid magnetic disk and an optical disk. Current examples of optical disks include compact disk-read only memory (CD-ROM), compact disk-read/write (CD-R/W) and DVD.

A data processing system suitable for storing and/or executing program code will include at least one processor (e.g., computer processor 28) coupled directly or indirectly to memory elements (e.g., memory 30) through a system bus. The memory elements can include local memory employed during actual execution of the program code, bulk storage, and cache memories which provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution. The system can read the inventive instructions on the program storage devices and follow these instructions to execute the methodology of the embodiments of the invention.

Network adapters may be coupled to the processor to enable the processor to become coupled to other processors or remote printers or storage devices through intervening private or public networks. Modems, cable modem and Ethernet cards are just a few of the currently available types of network adapters.

Computer program code for carrying out operations of the present invention may be written in any combination of one or more programming languages, including an object-oriented programming language such as Java, Smalltalk, C++ or the like and conventional procedural programming languages, such as the C programming language or similar programming languages.

It will be understood that algorithms described herein, can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general-purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer (e.g., computer processor 28) or other programmable data processing apparatus, create means for implementing the functions/acts specified in the algorithms described in the present application. These computer program instructions may also be stored in a computer-readable medium (e.g., a non-transitory computer-readable medium) that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable medium produce an article of manufacture including instruction means which implement the function/act specified in the flowchart blocks and algorithms. The computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide processes for implementing the functions/acts specified in the algorithms described in the present application.

Computer processor 28 is typically a hardware device programmed with computer program instructions to produce a special purpose computer. For example, when programmed to perform the algorithms described herein, computer processor 28 typically acts as a special purpose sample- analysis computer processor. Typically, the operations described herein that are performed by computer processor 28 transform the physical state of memory 30, which is a real physical article, to have a different magnetic polarity, electrical charge, or the like depending on the technology of the memory that is used.

The apparatus and methods described herein may be used in conjunction with apparatus and methods described in any one of the following patents or patent applications, all of which are incorporated herein by reference:

US 9,522,396 to Bachelet;

US 10,176,565 to Greenfield;

US 10,640,807 to Poliak; US 9,329,129 to Poliak;

US 10,093,957 to Poliak;

US 10,831,013 to Yorav Raphael;

US 10,843,190 to Bachelet;

US 10,482,595 to Yorav Raphael;

US 10,488,644 to Eshel;

WO 17/168411 to Eshel;

US 2019/0302099 to Poliak;

US 2019/0145963 to Zait;

WO 19/097387 to Yorav-Raphael;

WO 21/079305 to Pecker;

WO 21/079306 to Pecker;

WO 21/116955 to Yafin;

WO 21/116957 to Gluck;

WO 21/116959 to Eshel;

WO 21/116960 to Zait; and WO 21/116962 to Halperin.

It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. Rather, the scope of the present invention includes both combinations and subcombinations of the various features described hereinabove, as well as variations and modifications thereof that are not in the prior art, which would occur to persons skilled in the art upon reading the foregoing description.

Claims

1. A method for use with a microscope that is configured to acquire images of a bodily sample disposed in a sample carrier, comprising: acquiring a fluorescent, microscopic image of a given x-y region within the sample carrier, while a focal plane of the microscope is set at an estimated optimum focal plane for the x-y region; identifying a focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest; acquiring a set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis; for the set of fluorescent focusing images, determining a relationship between a contrast- indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired; identifying an optimum focal plane at which to acquire a microscopic image of the given x-y region, at least partially based upon said relationship; and acquiring a diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focusing level that is based upon the identified optimum focal plane.

2. The method according to claim 1, wherein acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane comprises acquiring a diagnostic image of the x-y region of the sample carrier in a different imaging modality than an imaging modality in which the set of fluorescent focusing images are acquired.

3. The method according to claim 1, wherein acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane comprises acquiring a diagnostic image of the x-y region of the sample carrier in a similar imaging modality to an imaging modality in which the set of fluorescent focusing images are acquired.

4. The method according to claim 1, wherein acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane comprises acquiring a diagnostic image of the x-y region of the sample carrier with the focal plane of the microscope set to the identified optimum focal plane.

5. The method according to claim 1, wherein acquiring the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the focusing level that is based upon the identified optimum focal plane comprises acquiring a diagnostic image of the x-y region of the sample carrier with the focal plane of the microscope set to a focal plane that is offset by a predetermined amount with respect to the identified optimum focal plane.

6. The method according to claim 1, wherein identifying the optimum focal plane at which to acquire the microscopic image of the given x-y region comprises identifying, as the optimum focal plane, a focusing level at which the contrast-indicative parameter indicates that image contrast is at a maximum.

7. The method according to claim 1, wherein determining the relationship between a contrast- indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired further comprises interpolating the relationship such as to estimate the contrast-indicative parameter for interpolated focusing levels, and wherein identifying the optimum focal plane at which to acquire the microscopic image of the given x-y region comprises identifying one of the interpolated focusing levels as the optimum focal plane.

8. The method according to claim 1, wherein the method is for use with a blood sample disposed within the sample carrier, and wherein identifying the focusing region-of-interest within the acquired image comprises identifying the focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: platelets leukocytes, pathogens, and any combination thereof.

9. The method according to claim 1, wherein the method is for use with a fine needle aspirate sample disposed within the sample carrier, and wherein identifying the focusing region-of-interest within the acquired image comprises identifying the focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, parasites, and any combination thereof.

10. The method according to any one of claims 1-9, wherein acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis comprises acquiring the set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region.

11. The method according to claim 10, wherein acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region comprises acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis, such that subsequent to acquiring the set of fluorescent focusing images of the region-of-interest, an intensity of fluorescence of entities within the given x-y region is more than 90 percent of an intensity of fluorescence of the entities when the entities first begin to fluoresce.

12. The method according to claim 10, wherein acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region comprises selecting a wavelength at which to excite the sample in order to acquire the set of fluorescent focusing images, such as to prevent causing substantial photobleaching of entities within the given x-y region.

13. The method according to claim 10, wherein acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region comprises intermittently exciting the sample with excitation light during the acquisition of the set of fluorescent focusing images.

14. The method according to any one of claims 1-9, wherein acquiring the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis comprises acquiring a set of focusing images along a range of focusing levels that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane.

15. The method according to claim 14, further comprising, in response to identifying an initially-determined optimum focal plane that is toward an edge of said range of focusing levels, acquiring a new set of fluorescent focusing images of the region-of-interest along a range of focusing levels that are centered around the initially-determined optimum focal plane and that extend from the given distance above the initially-determined optimum focal plane to a given distance below the initially-determined optimum focal plane.

16. The method according to claim 14, further comprising in response to being unable to detect an optimum focal plane within said range of focusing levels, acquiring focusing images along an extended range of distances above the estimated optimum focal plane and below the estimated optimum focal plane.

17. Apparatus for use with a bodily sample disposed in a sample carrier, the apparatus comprising: a microscope configured to acquire microscopic images of the bodily sample disposed in the sample carrier; and at least one computer processor configured to: drive the microscope to acquire a fluorescent, microscopic image of a given x-y region within the sample carrier, while a focal plane of the microscope is set at an estimated optimum focal plane for the x-y region, identify a focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, drive the microscope to acquire a set of fluorescent focusing images of the region- of-interest with the microscope focused at respective focusing levels along the optical axis, for the set of fluorescent focusing images, determine a relationship between a contrast-indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired, identify an optimum focal plane at which to acquire a microscopic image of the given x-y region, at least partially based upon said relationship, and drive the microscope to acquire a diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focusing level that is based upon the identified optimum focal plane.

18. The apparatus according to claim 17, wherein the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier in a different imaging modality than an imaging modality in which the set of fluorescent focusing images are acquired.

19. The apparatus according to claim 17, wherein the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier in a similar imaging modality to an imaging modality in which the set of fluorescent focusing images are acquired.

20. The apparatus according to claim 17, wherein the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to the identified optimum focal plane.

21. The apparatus according to claim 17, wherein the computer processor is configured to drive the microscope to acquire the diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focal plane that is offset by a predetermined amount with respect to the identified optimum focal plane.

22. The apparatus according to claim 17, wherein the computer processor is configured to identify the optimum focal plane at which to acquire the microscopic image of the given x-y region by identifying, as the optimum focal plane, a focusing level at which the contrast-indicative parameter indicates that image contrast is at a maximum.

23. The apparatus according to claim 17, wherein the computer processor is configured to determine the relationship between the contrast- indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired by interpolating the relationship such as to estimate the contrast-indicative parameter for interpolated focusing levels, and wherein the computer processor is configured to identify one of the interpolated focusing levels as the optimum focal plane.

24. The apparatus according to claim 17, wherein the apparatus is for use with a blood sample disposed within the sample carrier, and wherein the computer processor is configured to identify the focusing region-of-interest within the acquired image based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: platelets leukocytes, pathogens, and any combination thereof.

25. The method according to claim 17, wherein the apparatus is for use with fine needle aspirate sample disposed within the sample carrier, and wherein the computer processor is configured to identify the focusing region-of-interest within the acquired image based on fluorescent entities that are disposed within the focusing region-of interest, the entities being selected from the group consisting of: macrophages, histiocytes, mast cells, plasma cells, melanocytes, epithelial cells, mesenchymal cells, mesothelial cells, bacteria, yeast, parasites, and any combination thereof.

26. The apparatus according to any one of claims 17-25, wherein the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region- of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region.

27. The apparatus according to claim 26, wherein the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis, such that subsequent to the microscope acquiring the set of fluorescent focusing images of the region-of-interest, an intensity of fluorescence of entities within the given x-y region is more than 90 percent of an intensity of fluorescence of the entities when the entities first begin to fluoresce.

28. The apparatus according to claim 26, wherein the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region by selecting a wavelength at which to excite the sample in order to acquire the set of fluorescent focusing images, such as to prevent causing substantial photobleaching of entities within the given x-y region.

29. The apparatus according to claim 26, wherein the computer processor is configured to drive the microscope to acquire the set of fluorescent focusing images of the region-of-interest with the microscope focused at respective focusing levels along the optical axis without causing substantial photobleaching of entities within the given x-y region by intermittently exciting the sample with excitation light during the acquisition of the set of fluorescent focusing images.

30. The apparatus according to any one of claims 17-25, wherein the computer processor is configured to drive the microscope to acquire the set of focusing images along a range of focusing levels that extend from a given distance above the estimated optimum focal plane to a given distance below the estimated optimum focal plane.

31. The apparatus according to claim 30, wherein, in response to identifying an initially- determined optimum focal plane that is toward an edge of said range of focusing levels, the computer processor is configured to drive the microscope to acquire a new set of fluorescent focusing images of the region-of-interest along a range of focusing levels that are centered around the initially-determined optimum focal plane and that extend from the given distance above the initially-determined optimum focal plane to a given distance below the initially-determined optimum focal plane.

32. The apparatus according to claim 30, wherein, in response to being unable to detect an optimum focal plane within said range of focusing levels, the computer processor is configured to drive the microscope to acquire focusing images along an extended range of distances above the estimated optimum focal plane and below the estimated optimum focal plane.

33. A computer software product, for use with a microscope, and a bodily sample disposed within a sample carrier, the computer software product comprising a non-transitory computer- readable medium in which program instructions are stored, which instructions, when read by a computer cause the computer to perform the steps of: drive the microscope to acquire a fluorescent, microscopic image of a given x-y region within the sample carrier, while a focal plane of the microscope is set at an estimated optimum focal plane for the x-y region, identify a focusing region-of-interest within the acquired image, based on fluorescent entities that are disposed within the focusing region-of interest, drive the microscope to acquire a set of fluorescent focusing images of the region-of- interest with the microscope focused at respective focusing levels along the optical axis, for the set of fluorescent focusing images, determine a relationship between a contrast- indicative parameter of each of the images and the focusing level along the optical axis at which the microscope was focused when the image was acquired, identify an optimum focal plane at which to acquire a microscopic image of the given x-y region, at least partially based upon said relationship, and drive the microscope to acquire a diagnostic image of the x-y region of the sample carrier, with the focal plane of the microscope set to a focusing level that is based upon the identified optimum focal plane.