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WO2025212881A1 - Démélange spectral de fluorophores imagés - Google Patents

Démélange spectral de fluorophores imagés

Info

Publication number
WO2025212881A1
WO2025212881A1 PCT/US2025/022946 US2025022946W WO2025212881A1 WO 2025212881 A1 WO2025212881 A1 WO 2025212881A1 US 2025022946 W US2025022946 W US 2025022946W WO 2025212881 A1 WO2025212881 A1 WO 2025212881A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
calibration
spectrum
image
beads
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/022946
Other languages
English (en)
Inventor
Anastasia KURNIKOVA
Chrisgen Vonnegut
Aron BEEKMAN
Martin DE QUINCEY
Ognjen Golub
Ronald Kuhn
Audrey Nicole MILLIGAN
Meike PEDERSEN
Adam YORK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FEI Deutschland GmbH
Life Technologies Corp
Original Assignee
FEI Deutschland GmbH
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by FEI Deutschland GmbH, Life Technologies Corp filed Critical FEI Deutschland GmbH
Publication of WO2025212881A1 publication Critical patent/WO2025212881A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G01N21/278—Constitution of standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • Microscopy of sectioned tissue samples stained with fluorescent dyes and/or immuno staining provides valuable histological, cellular and biomarker information.
  • fluorescent dyes and/or immuno staining e.g., direct or indirect staining with primary or secondary antibodies conjugated to fluorophores
  • detection of fluorescence from neighboring fluorescent channels in the channel of interest can hamper identification of actual targets.
  • One workaround would be to limit the number of targets by staining samples with a few spectrally separate fluorophores.
  • multiplexing the detection is often required since the availability of tissue samples can be limited, and the tissue and the detection reagents can be expensive.
  • Another example provides a method of calibrating an imaging device.
  • the method includes obtaining a first image of an unstained version of a sample, acquiring an unstained spectral profile of the sample using the first image, obtaining, for each of a plurality of stained versions of the sample, a second image, and extracting a plurality of spectral profiles associated with the plurality of stained images, each spectral profile associated a fluorophorc of the plurality of fluorophores.
  • a portion of the sample is stained with a different fluorophore of a plurality of fluorophores.
  • the method includes generating an unmixing matrix based on the unstained spectral profile and the plurality of spectral profiles.
  • a calibration slide for use in the calibration of an imaging device.
  • the calibration slide comprises a microscope slide, and a plurality of detectable calibration beads disposed in a mountant within a defined area on the surface of the microscope slide.
  • the mountant has a refractive index that at least matches the refractive index of the microscope slide.
  • the calibration slide contains between 1,300,000 to about 2,600,000 beads within a 10 mm 2 area on the slide surface.
  • the beads have an average diameter ranging from about 0.1 pm to about 6.0 pm, with a preferred average diameter of about 4 pm to about 5 pm.
  • the coefficient of variation (CV) of the bead diameter ranges from about 2% to 5%, with an optimal CV of around 2%.
  • Fluorophores may include cyanine-based dyes, hemi-cyanine -based dyes, rhodamine-based dyes, coumarin-based dyes, pyrene-based dyes, indacene-based dyes, or indole-based dyes. These beads encapsulate the fluorophores, and the slide maintains a shelf life of 1 year or more when stored at 2°C to 30°C, protected from light. [0010]
  • a method for preparing the calibration slide is disclosed. The method involves dispersing fluorescent beads in a dispersal solution, spreading the solution onto a microscope slide, drying the beads, and then mounting them using a mounting solution.
  • the beads are typically polystyrene based, with a diameter of about 4.7 pm, and the dispersal solution contains about 0.2% w/v beads, including a non-ionic detergent.
  • the bead solution is spread onto the slide at a thickness of about 10 pm to about 15 pm, resulting in a bead density of about 4000 to about 8000 beads per 20x field of view.
  • the mounting solution which is preferably glycerol-based, has a refractive index that matches that of the microscope slide.
  • the method includes staining the beads with a plurality of fluorescent dyes, which emit light across different wavelengths (385 nm - 860 nm), optimizing the stain concentration for visibility with less than 100 msec exposure time.
  • an imaging system in another aspect, includes the calibration slide and an imaging device calibrated with the calibration slide.
  • FIGS. 1A-1B show an example of spatial transcriptomics.
  • FIG. 2 illustrates a process diagram of linear unmixing of a 9-plex sample.
  • FIG. 3A represents a block diagram of an example fluorescence microscope.
  • FIG. 3B represents a block diagram of an example controller of the fluorescence microscope of FIG. 3A.
  • FIG. 8 shows a block diagram of a method for obtaining a single-color control sample spectrum.
  • FIG. 10 illustrates examples metrics regarding the calibration of a fluorescence microscope.
  • FIG. 13 shows an illustration of a calibration slide.
  • the term “a,” “an,” “the” and similar terms used in the context of the disclosure are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
  • “a,” “an,” or “the” means “one or more” unless otherwise specified.
  • the term “or” can be conjunctive or disjunctive.
  • the term “and/or” refers to both the conjunctive and disjunctive.
  • the term “substantially” means to a great or significant extent, but not completely.
  • the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value.
  • the symbol means “about” or “approximately.”
  • ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range.
  • a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ⁇ 10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”
  • active ingredient or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.
  • control As used herein, the terms “control,” or “reference” are used herein interchangeably.
  • a “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result.
  • Control also refers to control experiments or control cells.
  • Antibody as used herein means an immunoglobulin or a fragment thereof and encompasses any polypeptide comprising an antigen-binding site regardless of the source, method of production, and other characteristics.
  • An “analyte” or “antigen” as used herein refers to any substance recognized by an antibody, or another means of detection.
  • a “detectable label” as used herein refers to any molecule which may be detected directly or indirectly to reveal the presence of a target in the sample.
  • a direct detectable label may be used.
  • Direct detectable labels may be detected without the need for additional molecules. Examples include fluorescent dyes, radioactive substances, and metal particles.
  • Indirect detectable labels may be used, which require the employment of one or more additional molecules. Examples include enzymes that affect a color change in a suitable substrate, as well as any molecule that may be specifically recognized by another substance carrying a label or react with a substance carrying a label.
  • Other examples of indirect detectable labels thus include antibodies, antigens, nucleic acids and nucleic acid analogs, ligands, substrates, and haptens.
  • More than one type of color may be used, for instance, by attaching distinguishable labels to a single detection unit or by using more than one detection unit, each carrying a different and distinguishable label.
  • detectable labels that can be used in spatial biology workflows, either alone or in combination with the methods described herein include gold or other metal particles, heavy atoms, spin labels, radioisotopes, and quantum dots.
  • Detectable labels can be attached (e.g., conjugated) to a variety of different substances, including, without limitation, haptens, antigens, nucleic acids or nucleic acid analogues, proteins, such as receptors, peptide ligands, enzymes, enzyme substrates, or antibodies (including antibody fragments).
  • detectable labels and substances that can be conjugated to detectable labels include polymers, polymer particles, bead or other solid surfaces and substrates.
  • Fluorophore as used herein is a molecule that emits detectable electro-magnetic radiation upon excitation with electro-magnetic radiation at one or more wavelengths.
  • fluorophores A large variety of fluorophores are known in the ail and arc developed by chemists for use as detectable molecular labels and can be conjugated to affinity molecules described herein.
  • fluorophorc and “fluorescent dye” may be used interchangeably herein.
  • Chrophor refers to a chemical compound that can, by chemical or other means, be converted into a chromophore.
  • exemplary chromogens include, but are not limited to, naphthols, aryl diazonium salts, and 1,3-diketones.
  • chromophore refers to an aromatic compound including a chemical grouping that gives color to the compound by causing displacement of, or appearance of, absorbent bands in the visible spectrum.
  • the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • a probe may react with a target, or directly bind to a target, or indirectly react with or bind to a target by directly binding to another substance that in turn directly binds to or reacts with a target.
  • the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or archaea, among other life forms. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.
  • primates e.g., humans, male or female; infant, adolescent, or adult
  • non-human primates e.g., rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or archaea, among other
  • multiplex detection refers typically refers to detection of more than five molecular markers or data types within the same biological sample (e.g., tissue section or cell), while preserving spatial information.
  • Multiplexing also refers to techniques for measuring the expression or presence of multiple genes, proteins, or other molecules in the same tissue sample, while maintaining their spatial location.
  • 1A shows the spectral emission profiles of 9 fluorophores illustrating the spectral overlap of different dyes (DAPI, eFluor 506, Alexa FluorTM 488, Alexa FluorTM 514, Alexa FluorTM 555, eFluor 615, Alexa FluorTM 647, Alexa FluorTM 700, Alexa FluorTM ' 750).
  • DAPI eFluor 506, Alexa FluorTM 488, Alexa FluorTM 514, Alexa FluorTM 555, eFluor 615, Alexa FluorTM 647, Alexa FluorTM 700, Alexa FluorTM ' 750.
  • the spectral profile of DAPI ranges from approximately 375 nm to approximately 650 nm.
  • DAPI has a peak intensity at approximately 450 nm.
  • Each fluorophore has a unique spectral range and peak intensity.
  • FIG. IB shows example spectrally mixed (left) and unmixed (right) composite images of human tonsil tissue sample stained with 9 fluorophores. While the mixed image includes all fluorescent channels, the unmixed image includes only the desired fluorescent channels.
  • FIG. 2 illustrates a process diagram of linear unmixing of a 9-plex sample.
  • a multiplex (c.g., 9-plcx) tissue sample is received.
  • ten control samples arc obtained, where the multiplex tissue sample is imaged as an unstained sample and for nine fluorescent dyes.
  • ten single color control slides are prepared (e.g., ten images are captured, one for each control sample).
  • the spectral profiles 208 of each fluorophore and tissue autofluorescence are obtained to produce an unmixing matrix.
  • a mixed 9-plex image of the sample stained with each of the nine fluorescent dyes is captured.
  • the unmixing matrix 212 is employed to calculate the relative contribution from each fluorophore for every pixel of the mixed 9-plex image 210.
  • an unmixed image is obtained that includes only the desired fluorescent channels.
  • a 9-plex is a representative example of a multiplex tissue sample and that unmixing other multiplex tissue samples such as but not limited to 12-plex and 15-plex are possible.
  • FIG. 3A shows a block diagram of an example fluorescence microscope 300.
  • the fluorescence microscope 300 includes a controller component 302, a light source 304 (e.g., a laser light source), and fluorescence detectors 306.
  • the light source may include, for example, one or more light emitting diodes (LEDs), one or more laser diodes, one or more photomultiplier tubes (PMTs), a single-photon avalanche diode (SPAD) array, dichroic filters, emission filters, and combinations thereof.
  • the light source 304 projects light, such as laser light, onto the sample.
  • the controller component 302 may include an electronic processor 310, data storage device(s) 312, and an input/output (TO) interface 314.
  • the controller component 302 is suitable for the application and setting, and can include, for example, multiple electronic processors, multiple I/O interfaces, multiple data storage devices, or combinations thereof.
  • some or all of the components included in the controller component 302 may be attached to one or more mother boards and enclosed in a housing (e.g., including plastic, metal and/or other materials).
  • some of these components may be fabricated onto a single system-on-a-chip or SoC (e.g., an SoC may include one or more processing devices and one or more storage devices).
  • SoC system-on-a-chip
  • one or more of these components may be situated in a separate housing.
  • the electronic processor 310 may be situated in a first housing, while the data storage device(s) 312 are situated in a second housing communicatively coupled to the first housing.
  • processors or “electronic processor” refers to any device(s) or portion(s) of a device that process electronic data from registers and/or memory to transform that electronic data that may be stored in registers and/or memory.
  • the electronic processor 310 may include one or more digital signal processors (DSPs), application-specific integrated circuits (ASICs), central processing units (CPUs), graphics processing units (GPUs), cryptoprocessors (specialized processors that execute cryptographic algorithms within hardware), server processors, or any other suitable processing devices.
  • DSPs digital signal processors
  • ASICs application-specific integrated circuits
  • CPUs central processing units
  • GPUs graphics processing units
  • cryptoprocessors specialized processors that execute cryptographic algorithms within hardware
  • server processors or any other suitable processing devices.
  • the data storage device 312 may include one or more local or remote memory devices such as random-access memory (RAM) devices (e.g., static RAM (SRAM) devices, magnetic RAM (MRAM) devices, dynamic RAM (DRAM) devices, resistive RAM (RRAM) devices, or conductive-bridging RAM (CBRAM) devices), hard drive-based memory devices, solid-state memory devices, networked drives, cloud drives, or any combination of memory devices.
  • RAM random-access memory
  • MRAM magnetic RAM
  • DRAM dynamic RAM
  • RRAM resistive RAM
  • CBRAM conductive-bridging RAM
  • the data storage device 312 may include memory that shares a die with a processor.
  • the memory may be used as a cache memory and may include embedded dynamic random-access memory (eDRAM) or spin transfer torque magnetic random-access memory (STT-MRAM), for example.
  • eDRAM embedded dynamic random-access memory
  • STT-MRAM spin transfer torque magnetic random-access memory
  • the data storage device 312 may include non-transitory computer readable media having instructions thereon that, when executed by one or more processors (e.g., the electronic processor 310), causes the controller component 302 to store various applications and data for performing one or more of the methods described herein or portions described herein. It should be understood that each method described herein may be implemented via one application or multiple applications and, in some examples, the data storage device 312 stores additional data in various configurations.
  • the I/O interface 314 of controller component 302 may include one or more communication chips, connectors, and/or other hardware and software to govern communications between the controller component 302 and other components.
  • the I/O interface 314 may include circuitry for managing wireless communications for the transfer of data to and from the controller component 302.
  • the I/O interface 314 may include one or more antennas (c.g., one or more antenna arrays) for receipt and/or transmission of wire communications.
  • the fluorescence microscope 300 includes one or more output devices, such as a display screen, which are connected to the electronic processor 310 via the I/O interface 314.
  • Example systems and methods described herein provide for calibrating a fluorescence microscope for capturing multiple sample images without requiring the need for repeated control samples.
  • the fluorescence microscope 300 may acquire data from biological samples labeled with multiple overlapping fluorophores. The images of tissue samples are then separated into individual fluorophore channels. For example, unmixing of these spectra is performed to produce individual images. Methods and operations described herein provide an unmixing algorithm performed by the fluorescence microscope 300 that provides maximal interpretability of the results and provides a guided calibration for a user of the fluorescence microscope 300 to extract spectra from calibration slides.
  • FIG. 4 illustrates a block diagram of an example method 400 for performing spectral unmixing of an image including multiple overlapping fluorophores.
  • the method 400 is described herein as being performed by the fluorescence microscope 300. However, it should be understood that the method 400 (or portions thereof) may be performed by one or more electronic controllers located within the fluorescence microscope 300 (such as the controller component 302), separate or remote from the fluorescence microscope 300, or a combination thereof. Additionally, in various instances, various blocks illustrated in the example method 400 may be removed, added, combined, or modified without departing from the spirit of the present disclosure. It further should be understood that, while steps are illustrated as occurring in series, certain steps could be performed simultaneously in parallel.
  • the fluorescence microscope 300 receives a sample.
  • a tissue sample is provided in a module received by the fluorescence microscope 300 for imaging.
  • the sample is a 9-plex sample that includes nine fluorescent dyes.
  • the controller component 302 captures a raw image (e.g., a mixed image) of the sample.
  • a raw image e.g., a mixed image
  • the controller component 302 controls the light source 304 to inteiTogate the sample, causing the sample to fluoresce.
  • the fluorescence from the sample is captured as an image by the fluorescence detectors 306.
  • the raw image includes all fluorescent light emitted by the sample.
  • the controller component 302 applies linear unmixing to the raw image to generate an unmixed image.
  • an unmixing matrix is applied to the raw image to separate each fluorophore for every pixel of the raw image, thereby producing unmixed images.
  • the unmixing matrix may be re-scaled according to the selected channels at step 406.
  • the unmixing matrix may be altered by the controller component 302 such that the unmixed image only includes the selected channels at step 406.
  • the controller component 302 provides the unmixed image.
  • the method 500 is described herein as being performed by the fluorescence microscope 300. However, it should be understood that the method 500 (or portions thereof) may be performed by one or more electronic controllers located within the fluorescence microscope 300 (such as the controller component 302), separate or remote from the fluorescence microscope 300, or a combination thereof. Additionally, in various instances, various blocks illustrated in the example method 500 may be removed, added, combined, or modified without departing from the spirit of the present disclosure. It further should be understood that, while steps are illustrated as occurring in series, certain steps could be performed simultaneously in parallel.
  • the controller component 302 receives a raw unstained image.
  • a raw unstained image For example, an unstained sample is provided for imaging by the fluorescence microscope 300.
  • the controller component 302 controls the light source 304 to interrogate the sample, causing the sample to fluoresce.
  • the fluorescence detectors 306 capture the fluorescent light from the sample, and the controller component 302 processes signals provided by the fluorescence detectors 306 to generate the raw unstained image.
  • the controller component 302 receives a selected region of interest of the sample.
  • a user of the fluorescence microscope 300 may select a region of interest (or field of view) within the SCC image by interfacing with the I/O interface 314.
  • the controller component 302 clips the SCC image to the region of interest such that only the region of interest is shown to the user.
  • the controller component 302 averages all pixels within the selected region of interest in the SCC image to obtain an unstained spectrum.
  • the unstained spectrum represents the values of all pixels within the selected region of interest for the SCC.
  • the unstained spectrum represents the natural spectrum of the sample with no dyes applied to the sample.
  • the controller component 302 provides the unstained spectrum.
  • the unstained spectrum may be output via an output device of the fluorescence microscope 300 (e.g., a device, such as the display, connected to the I/O interface 314).
  • the unstained spectrum may be stored in the data storage device(s) 312.
  • foreground and background florescent channels may be identified for each dye to assist with the unmixing operation.
  • the foreground channel may be the channel with the highest available fluorescence signal for a given fluorophore.
  • the background channel may be the closest available channel (relative to the foreground channel) with no fluorescence for a given fluorophore.
  • the fluorophore may be expected to have autofluorescence.
  • FIG. 6 illustrates a block diagram of an example method 600 for identifying foreground and background fluorescent channels for selected dyes (e.g., selected fluorophores). The method 600 is described herein as being performed by the fluorescence microscope 300.
  • the method 600 may be performed by one or more electronic controllers located within the fluorescence microscope 300 (such as the controller component 302), separate or remote from the fluorescence microscope 300, or a combination thereof. Additionally, in various instances, various blocks illustrated in the example method 600 may be removed, added, combined, or modified without departing from the spirit of the present disclosure. It further should be understood that, while steps are illustrated as occurring in series, certain steps could be performed simultaneously in parallel.
  • the controller component 302 receives a selected dye.
  • a dye may be selected by a user of the fluorescence microscope 300 for calibration of the fluorescence microscope 300.
  • the dye may be a fluorescent dye as described with respect to FIGS. 1A-1B.
  • the controller component 302 loads a reference spectrum for the selected dye.
  • the data storage device(s) 312 stores a plurality of reference spectrums, each reference spectrum associated with a dye that may be selected by the user.
  • the electronic processor 312 may retrieve the reference spectrum from the data storage device(s) 312.
  • the reference spectrum may be an SCC spectrum obtained by the controller component 302 for a particular dye (for example, as described with respect to method 800 of FIG.
  • the controller component 302 identifies a maximum intensity channel of the reference spectrum. For example, the controller component 302 determines a fluorescence channel within the reference spectrum that has the greatest average intensity, such as an intensity peak of the reference spectrum.
  • the controller component 302 sorts the selected florescence channels by LEDs and filters. For example, the fluorescence channels are sorted according to their wavelength proximity to the identified foreground channel for the given fluorophore. The fluorescence channels may be sorted first according to excitation wavelength (e.g., the LEDs), and then according to emission wavelength (e.g., the filters) as a heuristic for channel similarity.
  • the controller component 302 identifies the closest non-fluorescent channel relative to the foreground channel based on the unstained spectrum. For example, the closest non-fluorescent channel to the foreground channel is determined according to emission and excitation wavelengths. A heuristic may be defined by which the difference in emission wavelengths is weighted as a less important factor than the distance in excitation wavelengths. The “closest” non-fluorescent channel minimizes the distance between the excitation wavelengths.
  • the controller component 302 identifies the background channel based on the non-fluorescent channel. For example, the non-fluorescent channel identified at step 612 is selected as the background channel.
  • the controller component 302 receives a plurality of selected dyes. For example, multiple dyes may be selected by a user of the fluorescence microscope 300.
  • the selected dyes are dyes that are applied to a sampled to be imaged.
  • the controller component 302 selects multiple channels for capturing a fluorescent image of a sample stained with the dyes. In some instances, the controller component 302 determines which fluorescent channels will best fluoresce from the sample based on the plurality of selected dyes. For example, the controller component 302 calculates available fluorescent channels based on a spectrum associated with the selected dyes. In another example, the controller component 302 compares the selected dyes to a lookup table to identify fluorescent channels associated with the selected dyes. In other instances, a user selects desired channels to be unmixed from a captured mixed image of the stained sample.
  • the controller component 302 adjusts channel settings of the fluorescent microscope 300 based on the selected channels and selected field of view. For example, exposure time of the sample may be adjusted based on the selected channels.
  • the controller component 302 performs a SCC operation to generate a matrix of the spectra.
  • An example of the SCC operation is described below with respect to method 800 (shown in FIG. 8).
  • the SCC operation is repeated for each dye of the plurality of selected dyes.
  • the controller component 302 calculates an unmixing matrix based on the matrix of the spectra. For example, the controller component 302 computes the unmixing matrix by performing a pseudoinverse operation on the matrix of the spectra.
  • the controller component 302 provides the unmixing matrix.
  • the unmixing matrix may be output via an output device of the fluorescence microscope 300 (e.g., a device, such as the display, connected to the VO interface 314).
  • the unmixing matrix may be stored in the data storage device(s) 312.
  • FIG. 8 illustrates a block diagram of an example method 800 for obtaining a SCC spectrum.
  • the method 800 is described herein as being performed by the fluorescence microscope 300. However, it should be understood that the method 800 (or portions thereof) may be performed by one or more electronic controllers located within the fluorescence microscope 300 (such as the controller component 302), separate or remote from the fluorescence microscope 300, or a combination thereof. Additionally, in various instances, various blocks illustrated in the example method 800 may be removed, added, combined, or modified without departing from the spirit of the present disclosure. It further should be understood that, while steps are illustrated as occurring in series, certain steps could be performed simultaneously in parallel.
  • the fluorescence microscope 300 receives a sample.
  • the sample may be, for example, stained with a single lluorophore.
  • the controller component 302 acquires selected channels for imaging of the sample. For example, the controller component 302 determines which fluorescent channels will best fluoresce from the sample based on the selected fluorophore. In some instances, a user of the fluorescence microscope 300 selects channels for imaging of the sample.
  • the controller component 302 captures a raw SCC image of the sample.
  • the controller component 302 controls the light source 304 to interrogate the sample, causing the sample to fluoresce.
  • the fluorescence detectors 306 capture the fluorescent light from the sample, and the controller component 302 processes signals provided by the fluorescence detectors 306 to generate the raw SCC image.
  • the controller component 302 performs a spectral extraction operation on the raw SCC image to obtain a SCC spectrum.
  • An example of the spectral extraction operation is described below with respect to method 900 (shown in FIG. 9).
  • the controller component 302 may be configured to select a particular combination of LEDs in the light source 304, dichroic filters, and emission filters that are associated with the selected channels for imaging the sample.
  • the controller component 302 normalizes the SCC spectrum.
  • the SCC spectrum may be normalized to account for an exposure time of the sample to the laser from the light source 304. For example, each SCC spectrum may be divided by the exposure time of the sample to acquire a normalized SCC spectrum.
  • the controller component 302 provides the normalized SCC spectrum.
  • the normalized SCC spectrum may be output via an output device of the fluorescence microscope 300 (e.g., a device, such as the display, connected to the I/O interface 314).
  • the normalized SCC spectrum may be stored in the data storage device(s) 312.
  • the controller component 302 repeats the method 800 to capture all SCC images, and therefore SCC spectrums, which arc associated with a particular combination of fluorophores.
  • the controller component 302 conditions the foreground channel and the background channel for processing.
  • the controller component 302 may apply intensity normalization to the foreground channel and/or the background channel.
  • the controller component 302 may apply a scaling function to the foreground channel and/or the background channel.
  • the controller component 302 determines a difference between the foreground channel and the background channel. For example, the controller component 302 may subtract the background channel from the foreground channel or may subtract the foreground channel from the background channel, thereby generating a difference image.
  • the controller component 302 clips bottom intensity values of the difference image. For example, any negative values of the difference image may be clipped to a value of zero.
  • the controller component 302 applies a thresholding operation to the clipped difference image to generate a signal mask image (c.g., a pixel mask). For example, an Otsu’s method is performed on the clipped difference image to perform automatic image thresholding.
  • the controller component 302 clips foreground and background channel intensities to pre-defined thresholds to generate a clipped image.
  • the controller component 302 calculates a correlation coefficient between the signal mask image and the clipped image.
  • the controller component 302 averages non-zero foreground pixels in the signal mask image to obtain a first spectrum.
  • the controller component 302 subtracts the unstained spectrum from the first spectrum to generate a second spectrum.
  • the unstained image, the SCC images, the foreground channel information, and the background channel information are used to extract a mask of the foreground pixels, in which the tissue in the sample is labeled with a fluorescent marker.
  • This is achieved by subtracting the foreground and background channels, followed by an Otsu threshold operation. This operation returns a single intensity threshold that separates pixels into foreground pixels and background pixels.
  • the signal in the resulting pixels is averaged across images and the unstained spectrum is subtracted to obtain a best estimate of the signal in each channel.
  • additional processing is performed to filter the extracted signal.
  • the correlation of the pixel mask and the clipped image is calculated, and the extracted spectrum values a e set to zero for any channels where this correlation metric is below a pre-determined factor.
  • the fluorescence microscope 300 provides recommendations to a user for the best channels to be retained during unmixing. This enables users to achieve maximum acquisition speed by imaging only the most critical channels. For example, for each spectrum extracted across the full channel set, the core channel is always retained. Next, the least impactful channel (measured by the unmixing sensitivity) is removed. This provides a list of channels that may be dropped in the order of their importance to the unmixing output.
  • the controller component 302 loads spectral profiles associated with all selected dyes and associated with the unstained image.
  • An unmixing matrix is generated for the spectral profiles, and a sum of squares operation is performed across the fluorescence channels included in the unmixing matrix. This value is calculated for each unmixing matrix with one channel dropped, and the channel with maximum of the sum of squares after removal is removed such that only recommended channels remain. The procedure is repeated the first time to find core channels by reducing the subset of channels to the number of dyes, then repeated again to find recommended channels.
  • a signal mask for each unmixed multiplex channel is calculated by a thresholding algorithm such as an otsu threshold.
  • the 95 th percentile signal intensity in the selected pixels may be calculated from the calculated signal mask in the raw channels of the multiplex samples.
  • a scaling factor for each fluorophore is calculated as the ratio of the multiplex channel intensity to calibration sample intensity for that fluorophore.
  • each row of the signal matrix is scaled by the scaling factor.
  • fraction bleedthrough and EDR are calculated as before from the scaled signal matrix.
  • a user may inspect a cosine similarity matrix, unmixing sensitivity, and a complexity index for the selected dye panel to evaluate the collinearity and identify dye pairs that are difficult to unmix.
  • users may also inspect the signal mask identified from each of the SCCs to validate that the signal selected is correctly extracted. Additionally, a similarity angle maps may provide a visualization for users to inspect the similarity of their extracted spectrum to each pixel in the image. Finally, post-unmixing the SCCs, the projected dynamic range, dye bleedthrough, signal to unstained intensity, and IOU relative to max channels may be displayed to users.
  • Table 1 shows an example dataset including a nine-fluorophore panel.
  • the table indicates each applied fluorophore, an associated antibody, and a dilution value of the fluorophore within the sample.
  • the methods described herein may be used in an immunohistochemistry assay, an immunocytochemistry assay, an in-situ hybridization (ISH) assay, enzyme immuno-assays (EIA), enzyme linked immuno-assays (ELISA), blotting methods (e.g., Western, Southern, and Northern), labeling inside electrophoresis systems or on surfaces or arrays, or other general detection assays known in the art.
  • ISH in-situ hybridization
  • EIA enzyme immuno-assays
  • ELISA enzyme linked immuno-assays
  • blotting methods e.g., Western, Southern, and Northern
  • labeling inside electrophoresis systems or on surfaces or arrays or other general detection assays known in the art.
  • Samples may comprise a solid, for example, containing targets in a tissue slice from an organ.
  • Samples may be derived from living matter taken from any living organism, such as an animal, such as mammals (e.g., humans), plants, fungi, archaea, or bacteria.
  • samples may comprise eukaryotic cells, archaeal cells, or prokaryotic cells.
  • the tissue sample may be from a subject selected from humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.
  • tissue or cell samples may be prepared by a variety of methods known to those of ordinary skill in the art, depending on the type of sample and the assay format.
  • tissue or cell samples may be fresh or preserved, and may be, for example, flash-frozen, smeared, dried, embedded, or fixed on slides or other supports.
  • Samples may be prepared and stained using a free-floating technique.
  • a tissue section may be brought into contact with different reagents and wash buffers in suspension or freely floating in appropriate containers, for example microcentrifuge tubes, before being mounted on slides for further treatment and examination by the methods described herein.
  • a tissue section may be mounted on a slide or other substrate after an incubation with immuno-specific reagents. The remains of the staining process may then be conducted after mounting.
  • samples may be comprised in a tissue section mounted on a suitable solid substrate.
  • sections comprising samples may be mounted on a glass slide or other planar substrate, to highlight by selective staining certain morphological indicators of disease states or detection of detectable targets.
  • the substrate may be a glass or plastic microscope slide (e.g., 26 x 75 x 1 mm or 1 x 3 x 0.04 in).
  • FIG. 14 discloses an exemplary method 1400 describing the steps of preparing a calibration slide for a spectral imaging application.
  • polystyrene-based beads having a nominal diameter of about 4.7 pm are formulated at a concentration of 0.2% w/v. These beads are suspended in a solution containing 1 % of a non-ionic detergent, such as a PluronicTM surfactant (BASF) to help with dispersal of the beads.
  • a non-ionic detergent such as a PluronicTM surfactant (BASF) to help with dispersal of the beads.
  • the solution e.g., about 10- 15 pL
  • the beads are allowed to dry at elevated temperatures.
  • the bead density can range from about 4000-8000 beads per 20x field of view.
  • IHC samples may include, for instance: preparations comprising un-fixed fresh tissues and/or cells or solution samples; fixed and embedded tissue specimens, such as archived material; and frozen tissues or cells.
  • An IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibody, washing, applying secondary antibody-enzyme conjugate, washing, applying a tertiary antibody conjugated to a polymer and linked with an enzyme, applying a chromogen substrate, washing, counter staining, applying a cover slip, and microscopic examination using a fluorescence microscope and processed using imaging and unmixing software.
  • FFPE paraffin embedding
  • Any suitable fixing agent may be used. Examples include ethanol, acetic acid, picric acid, 2-propanol, 3,3'- diaminobenzidine tetrahydrochloride dihydrate, acetoin (mixture of monomer) and dimer, acrolein, crotonaldehyde (cis + trans), formaldehyde, glutaraldehyde, glyoxal, potassium dichromate, potassium permanganate, osmium tetroxide, paraformaldehyde, mercuric chloride, tolylene-2,4-diisocyanate, trichloroacetic acid, and tungstic acid.
  • Fresh biopsy specimens, cytological preparations (including touch preparations and blood smears), frozen sections, and tissues for IHC analysis may be fixed in organic solvents, including ethanol, acetic acid, methanol and/or acetone.
  • antigen retrieval it may be useful to pre-treat the samples to increase the reactivity or accessibility of a detectable target and to reduce nonspecific interactions.
  • the target is an antigen
  • a process called “antigen retrieval” may be used (and which is also known in the ail as target retrieval, epitope retrieval, target unmasking, or antigen unmasking). See, e.g., Shi et al., J. Histochem. Cytochem. 45(3): 327-343 (1997).
  • Antigen retrieval encompasses a variety of methods including enzymatic digestion with proteolytic enzymes, such as proteinase, pronase, pepsin, papain, trypsin, or neuraminidase.
  • Signal-to-noise ratio may be increased by different physical methods, including application of vacuum, ultrasound, or freezing and thawing tissue samples before or during incubation of the reagents.
  • Treatments may be performed to reduce nonspecific binding.
  • carrier proteins, carrier nucleic acid molecules, salts, or detergents may reduce or prevent non-specific binding.
  • Non-specific binding sites may be blocked in some embodiments with inert proteins like, HSA, BSA, ovalbumin, with fetal calf serum or other sera, or with detergents like polyoxyethylene sorbitan monolaurate (TWEEN®20), octylphenoxypolyethoxyethanol (Nonidet P-40), /-octylphenoxypolyethoxyethanol (TRITONTM X-100), triterpene glycosides (Saponin), nonionic polyoxyethylene surfactants (BRIJ®-35), or nonionic triblock copolymers (PLURONICS®).
  • fluorescein or its derivatives such as fluorescein-5 -isothiocyanate (FITC), 5- (and 6)-carboxyfluorescein, 5- or 6-earboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, xanthene and its derivatives; rhodamine and its derivatives, such as tetramethylrhodamine and tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC); cyanine and its derivatives,; coumarin and its derivatives, such as (diethyl- amino)coumarin or 7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester (AMCA), as well as BODIPY dyes, pyrene-based dyes, anthracene-based dye
  • Fluorophores for use in imaging application can include reactive functional groups (e.g., amine, carboxylic acid, azide, alkyne, succinimidyl ester, sulfonyl chloride, maleimide, and the like) that are capable of reaction with reactive group in or on a substance to provide a fluorescently-labeled substance.
  • reactive functional groups e.g., amine, carboxylic acid, azide, alkyne, succinimidyl ester, sulfonyl chloride, maleimide, and the like
  • fluorophores useful herein include, but are not limited to, fluorescent proteins such as green fluorescent protein (GFP) and its analogues or derivatives, fluorescent amino acids such as tyrosine and tryptophan and their analogues, and fluorescent nucleosides.
  • fluorescent proteins such as green fluorescent protein (GFP) and its analogues or derivatives
  • fluorescent amino acids such as tyrosine and tryptophan and their analogues
  • fluorescent nucleosides include, but are not limited to, fluorescent proteins such as green fluorescent protein (GFP) and its analogues or derivatives
  • fluorescent amino acids such as tyrosine and tryptophan and their analogues
  • nucleosides fluorescent nucleosides
  • Fluorescent molecules for use as detectable labels include commercially available compounds or their reactive counterparts, such as, for example, cyanine dyes such as Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7 from Cytiva (Marlborough, MA); cyanine and rhodamine-based DY dyes from Dyomics GmbH (Germany); and dyes that are commercially available from Sigma- Aldrich Co. (St.
  • Thermo Fisher Scientific including, e.g., eFluorTM dyes, Alexa FluorTM dyes (e.g., Alexa FluorTM 350, 488, 555, 568, 594, 647, 680 and 750), Alexa FluorTM Plus dyes, NovaFluorTM dyes, Oregon Green 488, Pacific Blue (3-carboxy- 6, 8-difluoro-7-hydroxy coumarin), and Rhodamine Green.
  • Alexa FluorTM dyes e.g., Alexa FluorTM 350, 488, 555, 568, 594, 647, 680 and 750
  • Alexa FluorTM Plus dyes e.g., Alexa FluorTM Plus dyes
  • NovaFluorTM dyes Oregon Green 488, Pacific Blue (3-carboxy- 6, 8-difluoro-7-hydroxy coumarin
  • Rhodamine Green eFluorTM dyes
  • Alexa FluorTM dyes e.g., Alexa
  • fhiorophores and reactive versions thereof suitable for spatial imaging techniques include the iFluor and mFluor reagents, as well as PE-Cy5 and APC-Cy7 tandem fluorescent probes from AAT Bioquest and ATTO fluorescent labels from Atto-Tec GmbH (Siegen, Germany).
  • a target or analyte may comprise one or more of lipids; glyco-lipids; carbohydrates; polysaccharides; salts; ions; or a variety of other organic and inorganic substances.
  • a target or analyte may be expressed on the surface of the sample, such as on a membrane or interface.
  • a target or analyte may be contained in the interior of the sample.
  • an interior target or analyte may comprise a target or analyte located within the cell membrane, periplasmic space, cytoplasm, or nucleus, or within an intracellular compartment or organelle.
  • One embodiment described herein is a method for calibrating an imaging device and analyzing a sample for a plurality of analytes comprising incubating a solution comprising one or more affinity molecules that labels one or more analytes in the sample and detecting a signal from each affinity molecule that is bound to the plurality of analytes, thereby detecting the presence or amount of each analyte in the plurality of analyte.
  • the affinity molecule is conjugated to a fluorophore, conjugated to an enzyme, bound by another affinity molecule that is conjugated to a fluorophore, or bound by another affinity molecule that is conjugated to an enzyme.
  • the detecting is performed using an imaging device selected from a light or fluorescence microscope, a charge coupled device (CCD) camera or imager, a phosphorimager, or a combination thereof.
  • the method further comprises calibrating an imaging device by obtaining a first image of an unstained version of a sample; acquiring an unstained spectral profile of the sample using the first image; obtaining, for each of a plurality of stained versions of the sample, a second image, wherein, in each of the stained images, a portion of the sample is stained with a different fluorophore of a plurality of fluorophores; acquiring a plurality of spectral profiles associated with the plurality of stained images, each spectral profile associated a fluorophore of the plurality of fluorophores; generating an unmixing matrix based on the unstained spectral profile and the plurality of spectral profiles; obtaining a mixed image of a second sample, wherein, in the mixed image, the second sample is stained with each fluorophor

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Abstract

L'invention concerne des systèmes et des procédés d'étalonnage d'un imageur pour le démélange d'images avec de multiples fluorophores. Un procédé donné à titre d'exemple mis en œuvre par un dispositif informatique consiste à recevoir une ou plusieurs images témoin monochromes, un canal de premier plan et un canal d'arrière-plan. Les images de contrôle monochromes sont des images de l'échantillon ayant un fluorophore unique appliqué à l'échantillon, et le canal de premier plan et le canal d'arrière-plan sont associés au fluorophore. Le procédé consiste à déterminer une différence entre le canal de premier plan et le canal d'arrière-plan, à acquérir un masque de pixels de premier plan à partir de la différence, à faire la moyenne d'un ensemble de pixels de premier plan dans le masque de pixels de premier plan pour générer un premier spectre et à soustraire un spectre non coloré du premier spectre pour générer un second spectre. Le second spectre définit un profil spectral de l'échantillon. L'invention concerne également une lame d'étalonnage et un procédé de préparation d'une lame d'étalonnage.
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