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EP4577858A2 - Appareil, systèmes et procédés d'imagerie par fluorescence sur des capteurs plans - Google Patents

Appareil, systèmes et procédés d'imagerie par fluorescence sur des capteurs plans

Info

Publication number
EP4577858A2
EP4577858A2 EP23873538.5A EP23873538A EP4577858A2 EP 4577858 A2 EP4577858 A2 EP 4577858A2 EP 23873538 A EP23873538 A EP 23873538A EP 4577858 A2 EP4577858 A2 EP 4577858A2
Authority
EP
European Patent Office
Prior art keywords
imaging
tissue
collimator
filter
upr
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
EP23873538.5A
Other languages
German (de)
English (en)
Inventor
Moshiur M. Anwar
Micah ROSCHELLE
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.)
University of California
University of California San Diego UCSD
Original Assignee
University of California
University of California San Diego UCSD
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 University of California, University of California San Diego UCSD filed Critical University of California
Publication of EP4577858A2 publication Critical patent/EP4577858A2/fr
Pending legal-status Critical Current

Links

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
    • G01N21/62—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • 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

Definitions

  • the disclosed technology relates generally to lens-less (e.g., lens free) and chip-based fluorescence imagers, including examples of the devices, methods, and design principles for an intraoperative fluorescence imaging system for visualizing microscopic disease.
  • Examples also include lab-on-chip sensors, planar imagers and imaging arrays, DNA, RNA, protein microarrays, ELISA, and PCR-based diagnostics.
  • Examples include implantable imagers and sensors.
  • Examples also include high throughput cell imagers. This has broad implications in the implementation of multi-color (multi-target or multiplexed) and high-performance miniaturized fluorescence imaging systems.
  • PSMs Positive surgical margins
  • current laparoscopic fluorescence imagers are only capable of macroscopic (millimeter plus scale) visualization and have seen limited application in multiplexed imaging.
  • a system for fluorescence imaging includes an optical front-end including a first layer of an optical filter and a second layer of a collimator.
  • the optical front-end comprises a plurality of layers of alternating layers of materials with different refractive indices to create a long pass, short pass, band-pass, notch, or multiband pass interference filter, which may include an SF-2023-054-3-PCT-0-UPR absorption filter.
  • the optical front-end is affixed to an imaging sensor comprising an array of pixels and wherein the system further comprises a visualizations system in electrical communication with the imaging chip.
  • the imaging sensor may be a CMOS sensor.
  • the front-end may be of a thickness of less than or equal to 5mm, 2.5mm, 1.25mm, 500 ⁇ m, 250 ⁇ m, and/or 150 ⁇ m.
  • the collimator can be angle- selective, where the collimator has 2, 3, 4, 5, or 6 orders of magnitude or greater rejection at 30 degrees or greater off axis.
  • the collimator can be a parallel-hole collimator, a fiber optic plate, an absorptive material surrounding an array of optical fibers, an absorptive material surrounding one or more holes filled with a transparent material, or an absorptive material surrounding holes that are either filled with air or in a vacuum.
  • the angle-selective device including but not limited to a parallel-hole collimator or fiber optic plate, may be microfabricated in the layers comprising the image sensor design utilizing metal or active silicon layers as well as specialized post- processing steps.
  • the imaging chip is operationally integrated with a surgical tool, which can be a periscopic probe or a laparoscopic robotic instrument.
  • the imaging chip can include one or more of the following: at least one LED or at least one laser diode light source, is operationally integrated with an implantable imager, or is operationally integrated with a lab-on a chip.
  • Labs-on a chip can be a microarray of DNA, RNA, proteins, cells, tissue, or any biological sample or a diagnostic assay, including a PCR test, ELISA, or lateral flow assay.
  • the imaging chip can also be lens-free and/or use machine learning to improve image quality.
  • a sample being imaged can be one or more of diseased tissue and cancerous tissue.
  • a labeling agent being used can be an antibody, an antibody mimetic, nanobody, a peptide, a peptoid, an aptamer, or a small molecule ligand that selectively binds to the cellular, protein, DNA, RNA, molecular or chemical marker of interest.
  • a cellular marker of interest can be one or more of a tumor-specific antigen, a tumor-associated antigen, an immune-cell-specific antigen, and an immune activation marker.
  • a sample can be imaged with at least two different fluorophore conjugates, where each fluorophore conjugate includes a different fluorophore that emits fluorescent light at a different emission wavelength, and where each fluorophore conjugate comprises a different binding agent that selectively binds to a different marker.
  • SF-2023-054-3-PCT-0-UPR Additional embodiments include a method for imaging a biological sample, including applying a marker to a tissue, and obtaining an image of the tissue using a system, such as described above.
  • Further embodiments include resecting the tissue to remove diseased tissue from the marked tissue and/or illuminating the tissue with a light source.
  • obtaining an image of the tissue includes contacting the optical front-end to the tissue.
  • the marker can be a fluorescent dye selected from SYBR green, SYBR gold, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 570, Quasar 670, Quasar 705, fluorescein, fluorescein isothiocyanate, hexachlorofluorescein, 4′,6-diamidino-2-phenylindole, Hoechst, rhodamine, carboxy-X-rhodamine, and combinations thereof, or the marker is a fluorescent probe comprising a binding agent and a fluorophore.
  • the fluorophore can be selected from Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 784, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, and the binding moiety can be selected from a carbohydrate, a lipid, a peptide, a nanobody, a nucleic acid, a protein, and a small molecule.
  • Figure 1 illustrates a conceptual diagram of an example composite filter, in accordance with various embodiments.
  • Figures 2A-2D illustrate aspects of composite filters, in accordance with various embodiments.
  • Figures 3A-3B provide conceptual diagrams of versatile image sensors for intraoperative navigation, in accordance with various embodiments.
  • SF-2023-054-3-PCT-0-UPR Figure 4 illustrates an example of an implantable imaging device, in accordance with various embodiments.
  • Figures 5A-5C illustrate conceptual diagrams of various composite filters, in accordance with various embodiments.
  • Figures 6A-6C illustrate exemplary data regarding various composite filters, in accordance with various embodiments.
  • Figures 7A-7D provide graphs illustrating example characterizations of an optical front-end designed, in accordance with various embodiments.
  • Figures 8A-8E provide illustrations of example resolution measurements of an optical front-end designed, in accordance with various embodiments.
  • Figure 9 is an image illustrating various fascia on and near a prostate, in accordance with various embodiments.
  • Figures 10A-10C provide examples of ex vivo imaging of resected prostate tissue using an optical front-end designed, in accordance with various embodiments.
  • Figure 11 provides a conceptual diagram of an example interference filter directly coated on a fiber optic plate (FOP), in accordance with various embodiments.
  • Figure 12 provides an example of imaging of PC3-PIP cell cultures, in accordance with the various embodiments.
  • Figure 13 provides a graph illustrating exemplary data of signal noise ratios vs. cell cluster sizes, in accordance with the various embodiments.
  • Figure 14 provides a diagram of 2D cross-section of geometry used for deriving 2D PSF of a general lens-less imager, in accordance with the various embodiments.
  • Figure 15 provides an exemplary model of target and background tissue as a 2D plane with z-axis symmetry, in accordance with various embodiments.
  • Figures 16A-16D provide exemplary data comparing disclosed embodiments with a15 ⁇ m-thick amorphous silicon (a-Si) filter.
  • DETAILED DESCRIPTION Fluorescence contact imagers are provided. Certain embodiments utilize a composite emission filter. Such devices can be lens-less or lens-free, as in they do not utilize lenses. Without lenses, certain instances are smaller than conventional imaging SF-2023-054-3-PCT-0-UPR systems while maintaining high resolution. Certain embodiments are used in contact imaging to deliver wide-field of view microscopy while maintaining a thin and planar form factor. Some devices can be used for multiplexed imaging.
  • the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.”
  • the term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. [0034] It should be noted that many of the terms used herein are relative terms.
  • the terms “upper” and “lower” are relative to each other in location, i.e., an upper SF-2023-054-3-PCT-0-UPR component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the component is flipped.
  • the terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows through the outlet out of the structure.
  • the terms “horizontal” and “vertical” are used to indicate direction relative to an absolute reference, i.e., ground level.
  • top and bottom are used to refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth.
  • upwards and downwards are also relative to an absolute reference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.
  • ranges such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 11 ⁇ 2, SF-2023-054-3-PCT-0-UPR and 43 ⁇ 4.
  • ranges that include the language “less than” or “greater than” should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values from the value given to the extreme.
  • FOP fiber optic plate
  • interference filter refers to an optical filter designed to provide near- total transmittance of one spectral band with strong rejection of adjacent bands.
  • interference filters can be composed of a stack of alternating layers of materials with different refractive indices. By controlling the thickness of the alternating layers, specific wavelengths of light can either constructively or destructively interfere at each interface.
  • Interference filters can have long-pass, short-pass, band-pass, or notch characteristic or any combination of multiple long-pass, short-pass, band-pass, or notch characteristics in different spectral regions.
  • angle of incidence refers to an angle between a ray incident on a surface and the line perpendicular (at 90° angle) to the surface at the point of incidence, called the normal.
  • the ray can be formed by any waves, such as optical, acoustic, microwave, and X-ray.
  • the angle of incidence at which light is first totally internally reflected is known as the critical angle.
  • the angle of reflection and angle of refraction are other angles related to beams.
  • Surfaces can include (but are not limited to) functional items, such as a FOP, composite filter, in interference filter, a substrate, a sensor (e.g., an imaging sensor), and/or any other functional surface, in addition to non- functional surfaces.
  • peptide oligopeptide
  • polypeptide protein
  • amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, phosphorylation, glycosylation, acetylation, hydroxylation, oxidation, and the like as well as chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones.
  • the terms also include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
  • the terms include polypeptides including one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.
  • isolated is meant, when referring to a protein, polypeptide, or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type.
  • isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • substantially purified generally refers to isolation of a substance (compound, protein, nucleic acid, nanoparticles) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, or more preferably 90-95% of the sample.
  • Techniques for purifying substances of interest include, for example, ion- exchange chromatography, affinity chromatography and sedimentation according to density.
  • tumor refers to a cell or population of cells whose growth, proliferation or survival is greater than SF-2023-054-3-PCT-0-UPR growth, proliferation or survival of a normal counterpart cell, e.g. a cell proliferative, hyperproliferative or differentiative disorder. Typically, the growth is uncontrolled.
  • malignancy refers to invasion of nearby tissue.
  • Neoplasia or a secondary, recurring or recurrent tumor, cancer or neoplasia refers to spread or dissemination of a tumor, cancer or neoplasia to other sites, locations or regions within the subject, in which the sites, locations or regions are distinct from the primary tumor or cancer.
  • Neoplasia, tumors and cancers include benign, malignant, metastatic and non- metastatic types, and include any stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.) of neoplasia, tumor, or cancer, or a neoplasia, tumor, cancer or metastasis that is progressing, worsening, stabilized or in remission.
  • carcinomas such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma
  • cancers such as, but are not limited to, pancreatic cancer, lung cancer (non-small cell lung cancer, small cell lung cancer), gastric cancer, ovarian cancer, endometrial cancer, colorectal cancer, oral cancer, skin cancer, cholangiocarcinoma, head and neck cancer, breast cancer, ovarian cancer, melanoma, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophage
  • carcinomas such as squamous cell carcinoma, adenocarcinoma, a
  • a "ligand” or “binding agent” is any molecule that can be used to target a fluorophore to a cell or other target.
  • the ligand is a molecule that selectively binds to a target analyte of interest (e.g., cellular marker) with high binding affinity.
  • a target analyte of interest e.g., cellular marker
  • high binding affinity is meant a binding affinity of at least about 10-4 M, usually at least about 10-6 M or higher, e.g., 10-9 M or higher.
  • the ligand may be any of a variety of different types of molecules, as long as it exhibits the requisite binding affinity for the target analyte when conjugated to a fluorophore.
  • the ligand has medium or even low affinity for its target analyte, e.g., less than about 10-4 M.
  • the ligand may be a small molecule or large molecule ligand.
  • small molecule ligand is meant a ligand having a size of less than 10,000 daltons, usually ranging in size from about 50 to about 5,000 daltons, and more usually from about 100 to about 1000 daltons in molecular weight.
  • large molecule is meant a ligand having a size of more than 10,000 daltons in molecular weight.
  • a small molecule ligand may be any molecule, as well as binding portion or fragment thereof, that is capable of binding with the requisite affinity to the target analyte of interest (e.g., cellular marker).
  • the small molecule is a small organic molecule that is capable of binding to the target analyte of interest.
  • the small molecule will include one or more functional groups necessary for structural interaction with the target analyte, e.g., groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions.
  • the drug moiety will include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and will typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the small molecule will also comprise a region that may be modified and/or participate in conjugation to a fluorophore, without substantially adversely affecting the small molecule's ability to bind to its target analyte.
  • Small molecule ligands may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Small molecule ligands may also include organic compounds comprising alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing
  • Small molecules may include structures found among biomolecules, including peptides, carbohydrates, fatty acids, vitamins, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • the small molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Small molecules may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the small molecule may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e., a compound diversity combinatorial library. When obtained from such libraries, the small molecule employed will have demonstrated some desirable affinity for the protein target in a convenient binding affinity assay.
  • Combinatorial libraries, as well as methods for the production and screening are described in: U.S. Pat. Nos.
  • Small molecule ligands may also include drugs that selectively bind to receptors on cells, including, without limitation, growth factor receptors, receptor tyrosine kinases, receptor protein serine/threonine kinases, G-protein coupled receptors, cytokine receptors, lectin receptors, and folate receptors.
  • drugs that selectively bind to receptors on cells including, without limitation, growth factor receptors, receptor tyrosine kinases, receptor protein serine/threonine kinases, G-protein coupled receptors, cytokine receptors, lectin receptors, and folate receptors.
  • anti-cancer drugs that bind to such cellular receptors may be used as ligands to target fluorophores to cancer cells.
  • Exemplary drugs that may be used as ligands to target cancer cells include, without limitation, Acitinib, Afatinib, Axitinib, Erlotinib, Cabozantinib, Crizotinib, Gefitinib, Imatinib, Ibrutinib, Lapatinib, Neovastat, Nilotinib, Pazopanib, Perifosine, Ponatinib, Regorafenib, Sorafenib, Sunitinib, Trametinib, and Vandetenib.
  • the ligand can also be a large molecule.
  • antibody encompasses monoclonal antibodies as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies.
  • the term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat.
  • F(ab′)2 and F(ab) fragments Fv molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single- chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al.
  • Fv is an antibody fragment which contains an antigen-recognition and - binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH- VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444- 6448.
  • the term "affibody molecule” refers to a molecule that consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa.
  • a monoclonal antibody, for comparison, is 150 kDa, and a single-domain antibody, the smallest type of antigen- binding antibody fragment, 12-15 kDa.
  • the phrase "specifically (or selectively) binds" with reference to binding of an antibody or other binding agent to an antigen or analyte refers to a binding reaction that is determinative of the presence of the antigen or analyte in a heterogeneous population of proteins and other biologics.
  • an antigen or analyte e.g., cellular marker such as a tumor-marker or immune activation marker
  • the specified antibodies or other binding agents bind to a particular antigen or analyte at at least two times the background and do not substantially bind in a significant amount to other molecules present in the sample.
  • Specific binding to an antigen or analyte under such conditions may require an antibody or other binding agent that is selected for its specificity for a particular antigen or analyte.
  • antibodies raised to an antigen from specific species such as rat, mouse, or human can be selected to obtain only those antibodies that are specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane.
  • conjugated refers to the joining by covalent or noncovalent means of two compounds or agents (e.g., binding agent specific for a tumor marker or immune activation marker conjugated to a fluorophore).
  • subject refers to a vertebrate, preferably a mammal.
  • vertebrate any member of the subphylum chordata, including, without limitation, humans and other primates, SF-2023-054-3-PCT-0-UPR including nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
  • Imaging Chip Many embodiments describe a composite emission filter and can provide multiplexed, high-resolution imaging. Such filters can be used in contact imaging, including fluorescence contact imaging.
  • FIG. 1 is a conceptual diagram of an example composite filter, in accordance with various embodiments described herein.
  • multi-layer interference filters are used for this purpose. These filters can be engineered for extremely high performance at any visible and NIR wavelength and can even be made to have multiple passbands for multiplexed imaging. However, the performance degrades rapidly for obliquely incident light. As a result, previous work in lens-less fluorescence imaging has focused on using absorption filters. Here a certain material is chosen that selectively absorbs the excitation light while passing the emissions. These filters are inherently angle-insensitive but suffer from weak performance due to material imperfections.
  • FIG. 2A illustrates an exemplary schematic of a composite filter with an imaging sensor in accordance with many embodiments.
  • a fiber optic plate (FOP) and interference filter form a composite emission filter for an imaging sensor.
  • an interference filter can include a substrate, which may provide structure to the interference filter.
  • Such substrates can be any suitable material to provide structure and/or optical throughput.
  • Such substrates include (but are not limited to) fused silica.
  • the substrate can be of any relevant thickness to provide the proper structure, rigidity, and/or any other property provided by the substrate.
  • an interference filter is disposed or deposited directly on a FOP, such as illustrated in Figure 1.
  • the interference filter maybe coated or deposited directly on the image sensor or on any another planar surfaces of the system.
  • the filter may be microfabricated with integrated circuit technology as part of the image sensor design and/or created through post-processing of the image sensor.
  • the imaging sensor can be any relevant sensor.
  • the imaging sensor is a Complementary Metal Oxide Semiconductor (CMOS) imaging sensor. Sensors can be an array of pixels with any relevant a number of pixels, size of pixels, and/or pitch of pixels to provide a desired form factor.
  • CMOS Complementary Metal Oxide Semiconductor
  • the array may have dimensions ranging from approximately 24X24 pixels up to approximately 96X96 pixels, and the dimensions do not need to be equal.
  • SF-2023-054-3-PCT-0-UPR sensors can have pixels of 24X24 pixels, 24X28 pixels, 24X32 pixels, 24X36 pixels, 24X40 pixels, 24X44 pixels, 24X48 pixels, 24X52 pixels, 24X56 pixels, 24X60 pixels, 24X64 pixels, 24X68 pixels, 24X72 pixels, 36X36 pixels, 36X40 pixels, 36X44 pixels, 36X48 pixels, 36X52 pixels, 36X56 pixels, 36X60 pixels, 36X64 pixels, 36X68 pixels, 36X72 pixels, 36X76 pixels, 36X80 pixels, 36X84 pixels, 36X88 pixels, 36X92 pixels, 36X96 pixels, 48X48 pixels, 48X52 pixels, 48X56 pixels, 48X60 pixels, 48X64 pixels, 48X68 pixels, 48X72 pixels, 48X76 pixels, 48X80 pixels, 48X84 pixels, 48X92 pixels, 36
  • the foregoing ranges can be oriented in a longer horizontal or vertical direction, such that an embodiment that is 36X80 pixels is considered equivalent to an array of 80X36 pixels.
  • Pixel size can further be selected from an appropriate size.
  • the pixels are 28 ⁇ m X 28 ⁇ m, 32 ⁇ m X 32 ⁇ m, 36 ⁇ m X 36 ⁇ m, 40 ⁇ m X 40 ⁇ m, 44 ⁇ m X 44 ⁇ m, 48 ⁇ m X 48 ⁇ m, 52 ⁇ m X 52 ⁇ m, 56 ⁇ m X 56 ⁇ m, or 60 ⁇ m X 60 ⁇ m.
  • the pitch of pixels can further be adjusted based on manufacturing ability and/or size constraints.
  • Pitch can be selected from approximately 30 ⁇ m to approximately 75 ⁇ m. It should be noted that pitch cannot be less than a dimension of a pixel, thus pitch can be approximately 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, or 75 ⁇ m. It should be noted that a specific embodiment can comprise a pixel array of 80X36 pixels, where each pixel is 44 ⁇ m X 44 ⁇ m with a pitch of 55 ⁇ m.
  • FIG. 2B illustrates an exemplary interface between layers of an interference filter of various embodiments.
  • Interference filters can be fabricated from periodic layers of materials with different refractive indices and precisely tuned thicknesses to cause constructive or destructive interference at each interface for specific wavelengths.
  • SF-2023-054-3-PCT-0-UPR As an angle of incidence (AOI) increases, the optical path length difference between each layer (OP2-OP1) decreases, altering the spectral interference of the filter and causing the overall filter response to shift to shorter wavelengths.
  • AOI angle of incidence
  • This material may be organic (such as a dye in a polymer), colored glass, or a semiconductor material that acts as a bandpass, short pass, or long pass filter.
  • a semiconductor material that acts as a bandpass, short pass, or long pass filter.
  • amorphous silicon which acts as a long pass filter in the near infrared wavelength.
  • Other examples include gallium phosphide, cadmium sulfide, gallium arsenide, indium phosphide, and crystalline silicon.
  • certain embodiments include an angle-selective collimator. Certain embodiments can use a parallel hole collimator to achieve angle-selectivity, while other embodiments use a FOP to achieve angle-selectivity.
  • FOPs of various embodiments include fibers contain a cladding (outer) SF-2023-054-3-PCT-0-UPR and core (inner) region with differing refractive indices.
  • the refractive index of the cladding may be chosen to be lower than that of the core such that optical propagation through the fiber is governed by total internal reflection.
  • FOPs allow for large aspect ratios to be achieved without limiting the transmittance of light close to normal incidence. Light incident on the FOP with angles less than the critical angle of the fibers is transmitted through the fibers, while light incidence at angles larger than the critical angle passes through the fibers and into the surrounding absorptive media.
  • the FOP may be composed of fibers combining any materials with a significant refractive index difference surrounded by an absorptive media including but not limited to any of the aforementioned materials.
  • Certain embodiments utilize a low numerical aperture (NA) FOP to provide the angle selectivity.
  • NA numerical aperture
  • a low-NA FOP allows light transmission at small AOIs while absorbing or otherwise rejecting light with a high AOI.
  • Figure 2C provides an illustration of an exemplary FOP, where the FOP comprises a matrix of thin optical fibers embedded in a dark, extra-mural absorbing (EMA) glass.
  • EMA extra-mural absorbing
  • the light is guided by total internal reflection through the fiber matrix.
  • the AOI increases beyond the NA of the fibers, the light is passed through EMA glass experiencing significant attenuation that increases with AOI as the optical pathlength increases.
  • Using optical fibers as the transmissive medium in this way allows for high aspect ratios necessary for adequate absorption at large AOIs, without significantly reducing the transmittance for AOIs close to normal incidence.
  • the numerical aperture of the fibers may be (but are not limited to) the range of 0.001-1, such as 0.001, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and/or 1.
  • 0.001-1 such as 0.001, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and/or 1.
  • aspect ratio is defined as the ratio of the length of the fibers (also referred to as height or depth and refers to a dimension that is perpendicular to the plane of the image) to the diameter of the fibers.
  • a larger aspect ratio implying that the fibers are significantly longer than their diameter, will result in increased angle selectivity, as angled light will pass through more absorptive material before exiting the collimator.
  • the fibers can be any SF-2023-054-3-PCT-0-UPR shape, including asymmetric, that has a longer length than width.
  • Another parameter is the normal incidence transmittance of the collimator which is determined in part by the fill factor (percentage of surface area that is covered by transmissive fibers). Higher normal incidence transmittance is desired to minimize the insertion loss due to the collimator. However, a smaller fill factor and, hence, lower normal incidence transmittance can increase absorption of angled emissions.
  • Fiber size can be between approximately 5 ⁇ m and 25 ⁇ m, such as 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 ⁇ m, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, 20 ⁇ m, 21 ⁇ m, 22 ⁇ m, 23 ⁇ m, 24 ⁇ m, or 25 ⁇ m.
  • packing density and/or pitch are related parameters to demonstrate a number of fibers in a given distance or area—pitch being distance between equivalent points on consecutive fibers, while packing density is number of fibers in a given area.
  • Pitch can be approximately 6 ⁇ m and 30 ⁇ m, depending on fiber size, manufacturing capabilities, and/or desired pitch.
  • pitch can be 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 ⁇ m, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, 20 ⁇ m, 21 ⁇ m, 22 ⁇ m, 23 ⁇ m, 24 ⁇ m, 25 ⁇ m, 26 ⁇ m, 27 ⁇ m, 28 ⁇ m, 29 ⁇ m, or 30 ⁇ m.
  • individual fibers possess a diameter of approximately 9 ⁇ m and a pitch of approximately 12 ⁇ m.
  • thickness is less than 2 cm, others ⁇ 1 cm, others ⁇ 0.5 cm, and others ⁇ 0.2 cm. In other embodiments, thickness can be between approximately 50 ⁇ m and 500 ⁇ m, such as 50 ⁇ m, 75 ⁇ m, 100 ⁇ m, 125 ⁇ m, 150 ⁇ m, 175 ⁇ m, 200 ⁇ m, 225 ⁇ m, 250 ⁇ m, 275 ⁇ m, 300 ⁇ m, 325 ⁇ m, 350 ⁇ m, 375 ⁇ m, 400 ⁇ m, 425 ⁇ m, 450 ⁇ m, 475 ⁇ m, or 500 ⁇ m. Select embodiments utilize a FOP having a thickness of 250 ⁇ m.
  • a FOP can also act as a collimator, such as schematically illustrated in Figure 2D. Collimators restrict the angle of view of each pixel by blocking light incident at oblique angles, deblurring the image. By acting as a collimator, FOPs can improve imaging resolution by attenuating divergent light that contributes to blur, effectively restricting the field of view (FOV) of each pixel to a smaller area. In lens-less fluorescence contact imaging, for maximum resolution the fluorescent signal from each cell may be isolated to the pixel directly opposite it. However, fluorophores can emit light isotopically.
  • FOPs can be fabricated from a combination of any transmissive (core) and absorptive media (surrounding or cladding).
  • the transmissive hole array can be constructed simply as air gaps within the material or can be filled with a media that is optically transmissive in the spectral region of interest including but not limited to epoxy, glass, silicon (for example for NIR), semiconductors transparent to the wavelength of interest, or PDMS.
  • FOPs may even be fabricated on-chip by patterning semiconductor materials with adequate absorption in the visible region (as cladding material) and that are compatible with common microfabrication techniques.
  • the collimator may also be fabricated by creating pillars of transmissive material with no cladding around them (such SF-2023-054-3-PCT-0-UPR that ‘air’ is the cladding) providing a lower index of refraction surrounding area to trap light within the transmissive pillars.
  • Certain collimators may also utilize optical phenomena such as refraction or interference to achieve similar properties. Such implementations are not limited to but may take the form of micro-lens arrays or diffraction gratings with the characteristics being that they are planar and have a thickness of less than 5mm and can sufficiently collimate the incident light. In some instances, the thickness can be less than 1 cm.
  • a suitable collimator e.g., FOP
  • FOP fluorescence emission
  • the fluorescence emissions are often 4 to 6 orders of magnitude weaker than the excitation background.
  • fluorescence filters in those applications may be capable of providing 4 to 6 orders of magnitude excitation rejection.
  • this threshold may be lowered to 2 orders of magnitude excitation rejection. These, for example, can be applications with a high fluorescence signal. Additionally, alternative applications are also conceivable in which this threshold may be raised to 10 orders of magnitude excitation rejection.
  • Interference filters are capable of providing this level of rejection at normal incidence, but as described previously with increasing incident angle off the axis perpendicular to the surface of the filter, the filter characteristic shifts to lower wavelengths increasing excitation bleed-through.
  • the precise angle at which the excitation bleed-through exceeds the threshold determined by the level of rejection may be dependent on the proximity of the emission spectra of the excitation source in relation to the cut-off wavelength of the filter and can be determined through measurement. The closer that the excitation wavelength is to the cut-off wavelength of the filter, the lower the angle will be at which rejection provided by the interference filter starts to decay. For angles beyond this angle, the planar collimator may add additional rejection to preserve filtering performance above the determined threshold.
  • the optical front-end should provide this.
  • the rejection of the interference filter decays to 2 orders of magnitude (from 4-6 at normal incidence, where normal is defined as perpendicular to the plane of incidence)
  • the planar SF-2023-054-3-PCT-0-UPR collimator provides at least an additional 3 orders of magnitude rejection to reach the threshold.
  • the aspect ratio may influence the system. The higher the aspect ratio, the more selective the collimator plate is (e.g., by allowing only a smaller set of angles of light to pass through).
  • Higher aspect ratios can therefore be made by having a taller substrate, but this increases the form-factor of the device.
  • Thinner collimator plates i.e. reduced height
  • too small a width begins to reduce the pixel fill factor, as an increased amount of blocking or absorptive material is placed over the photosensitive element (i.e. photodiode) of the imager, blocking more light and reducing signal. Therefore, the system may utilize an adequate fill factor, which sets the lower bound of the width, and an appropriate angle selection, which then defines the height of the layer.
  • the height (thickness) of the collimator layer may increase the bulk of the overall device.
  • the system may utilize 2 orders of magnitude or greater rejection at 30 degrees or greater off axis. In some instances, the system may utilize 3 orders of magnitude or greater rejection at 30 degrees or greater off axis. In some instances, the system may utilize 4 orders of magnitude or greater rejection at 30 degrees or greater off axis. In some instances, the system may utilize 5 orders of magnitude or greater rejection at 30 degrees or greater off axis. In some instances, the system may utilize 6 orders of magnitude or greater rejection at 30 degrees or greater off axis. In some instances, the system may utilize the total optical front-end to be less than 1 cm thick. In some instances, the system may utilize the total optical front-end to be less than 0.5 cm thick.
  • the system may utilize the total optical front-end to be less than 0.25 cm thick. In some instances, the system may utilize the total optical front-end to be less than 1 mm thick. In some instances, the system may utilize the total optical front-end to be less than 0.5 mm thick. In some instances, the system may utilize the total optical front-end to be less than 250 microns thick. In some instances, the system may utilize the total optical front-end to be less than 150 microns thick. In certain examples, the thickness of the layer of optical filter material is less than 100 microns.
  • the thickness of the layer of filter material may range from 1 micron to 100 microns, including any thickness within this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, SF-2023-054-3-PCT-0-UPR 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns in thickness.
  • the system may utilize the fill factor over the photosensitive element to be greater than 95%. In some cases, the system may utilize the fill factor over the photosensitive element to be greater than 75%.
  • the system may utilize the fill factor over the photosensitive element to be greater than 50%. In some cases, the system may utilize the fill factor over the photosensitive element to be greater than 25%. In some cases, the system may utilize the fill factor over the photosensitive element to be greater than 15%. In some instances, the system may utilize the aspect ratio of the holes or fibers (height divided by width (or diameter)) to be greater than or equal to 30. In some instances, the system may utilize the aspect ratio of the holes or fibers (height divided by width (or diameter)) to be greater than or equal to 20. In some instances, the system may utilize the aspect ratio of the holes or fibers (height divided by width (or diameter)) to be greater than or equal to 10.
  • the system may utilize the aspect ratio of the holes or fibers (height divided by width (or diameter)) to be greater than or equal to 5. In some instances, the system may utilize the aspect ratio of the holes or fibers (height divided by width (or diameter)) to be greater than or equal to 3.
  • the disclosed optical front-end can be fabricated by separately manufacturing each of the layers including but not limited to a planar collimator and any number of interference filters and then binding them together with optically transparent binding agent including but not limited to epoxy.
  • Each of the components can be fabricated on a mechanical substrate, such as glass, that has transmission properties compatible with fluorescence imaging (i.e.
  • the disclosed optical front-end has the advantage that it may be fabricated entirely from commercially available parts, allowing for quick and easy implementation.
  • the interference filters can be coated directly on the planar collimator providing that the planar collimator has a smooth surface and is composed of a material compatible with available material deposition techniques including but not limited to vacuum thermal evaporation, sol-gel technique, chemical bath deposition, spray pyrolysis technique, plating, electroplating, electroless deposition, chemical vapor SF-2023-054-3-PCT-0-UPR deposition, low pressure chemical vapor deposition, plasma enhanced chemical vapor deposition, atomic layer deposition, radio frequency sputtering, direct current sputtering, ion plating evaporation, molecular beam epitaxy, arc evaporation, laser beam evaporation, and electron beam evaporation.
  • the interference filter may be coated on either or both sides of the planar collimator.
  • the disclosed optical front-end has applications in any fluorescence imaging or sensing system which do not primarily rely on lenses. In many instances, lenses are not used. However, micro lenses, phase masks, amplitude masks, or micro lenses arrays of various sizes and arrangements can also be integrated on the optical front-end. These applications include but are by not limited to implantable fluorescence imagers or sensors for in vivo imaging; multiplexed sensing arrays for DNA analysis, protein analysis, PCR, flow cytometry, cell counting, fluorescent bead counting; or small form-factor fluorescence cameras or microscopes for pathological analysis, live cell imaging, or intraoperative imaging.
  • interference filters are used for this purpose as they can be engineered to have high out-of-band rejection, sharp cut-off transitions, and near-total passband transmittance for any visible or near-IR spectral band.
  • interference filters are innately angle- sensitive; for progressively oblique incident angles, the filter passband shifts to shorter wavelengths, increasing excitation bleed-through. Consequently, this class of filters has included focusing optics to ensure captured excitation (illumination) and emission light is incident perpendicular to the filter surface.
  • the excitation light may be introduced obliquely and there are no collimating lenses to guarantee that all light is normally incident on the filter, precluding the use of interference filters.
  • PSMs positive surgical margins
  • SF-2023-054-3-PCT-0-UPR incomplete resection have been found to occur in more than 20% of prostatectomies, the highest rate for all cancers among men.
  • PSMs significantly increase a risk of recurrence and mortality and require adjuvant treatment, incurring additional costs and burden to the patient.
  • Fluorophores can be conjugated to biological probes, such as antibodies or small molecules, targeted towards specific cellular markers, generating high-contrast fluorescence signals with cellular-level specificity.
  • FIGS. 3A-3B provide conceptual diagrams of an example versatile image sensor for intraoperative navigation, in accordance with various techniques described herein.
  • the techniques described herein include an intraoperative imaging chip.
  • this system all of the bulky optical components are removed and images are captured with just the image sensor itself.
  • systems can maintain adequate resolution and high sensitivity by capturing fluorescence emission before they diverge.
  • this technology is capable of microscopic detection.
  • taking advantage of its small and inherently planar form factor it is highly maneuverable and can be integrated on existing surgical tools.
  • Two challenges are in maintaining resolution SF-2023-054-3-PCT-0-UPR without lenses and integrating the optical front-end necessary for separating the fluorescence signal from the excitation background.
  • various embodiments include a light source to provide an excitation wavelength.
  • the light source can be placed such that it illuminates a target tissue at an oblique angle.
  • the contact sensor of various imaging chips can then intercept normally incident light emitted from markers or dyes on the tissue.
  • Additional embodiments can include a visualization system in electrical communication with an imaging chip as described herein.
  • the visualization system can be used to observe any visible or fluorescent signal emitted from a surgical device or scope incorporating an imaging chip, as described herein.
  • Such visualization systems can include a monitor (or other display), a fluorometer, a luminometer, and/or any other device for measuring a signal emitted from a sample or tissue.
  • the visualization system is connected to an imaging chip via electronic connections (e.g., wires, cords, etc.) or via a wireless interface, such as ultrasound, RF, low frequency EM, magnetic, etc.
  • a wireless interface such as ultrasound, RF, low frequency EM, magnetic, etc.
  • implantable sensors for in vivo imaging whereby the entire image sensor must fit within the body, or within a tumor, lesion, organ, or portion of tissue. This can include imaging within tissue, or in vivo flow cytometry (for tagged cells, molecules, or nanoparticles), or imaging of genetically engineered cells, such as with CAR-T cells.
  • Certain embodiments are directed to implantable imagines, such as illustrated in Figure 4. The smaller form factor of a lens-less design may be better for implantation.
  • a FOP and filter can be mounted to an imaging sensor (e.g., a CMOS sensor) and an application specific integrated circuit (ASIC) and printed circuit board (PCB).
  • Additional embodiments include a wireless interface to transmit images to an external device.
  • the wireless interface can be any wireless modality of power and data transfer, including ultrasound, RF, low frequency EM, magnetic.
  • Power to the implantable device can be provided via capacitors, batteries (e.g., Li-Ion, alkaline, NIMH, and/or other battery type), or other electrical storage device. Such batteries can be charged via any applicable means, such as via motion or pressure (e.g., piezo electric device), wireless (e.g., Qi protocol).
  • a light source such as a light emitting SF-2023-054-3-PCT-0-UPR diode, laser diode, and/or any other applicable light source.
  • Such light sources can be wavelength specific based on inherent emission or by using filters. Such light can be tuned or keyed to an excitation wavelength of a fluorescent marker, such as described herein.
  • Another notable application is lab-on-chip sensors utilizing fluorescence imaging and sensing. These systems can span from DNA- or RNA-based microarrays to protein microarrays, to cellular or tissue microarrays. This can include point-of-care sensors, and point-of-care antigen- or PCR-based diagnostic assays.
  • Such light source can be a white light source or a source specific to a particular wavelength, spectrum, and/or range of wavelengths.
  • Wavelengths can be generated by using a specific light source or diode that illuminates a specific wavelength or range of wavelengths. Additional embodiments can utilize one or more filters to provide a wavelength of light. Certain implementations may have a system that allows to change a wavelength or range of wavelengths emitted from the light source and/or utilize multiple light sources, where each light source is capable of providing a single wavelength or range of wavelengths. [0098] Wavelengths or ranges of wavelengths can range from ultraviolet (UV) light to infrared (IR) wavelengths of light.
  • UV ultraviolet
  • IR infrared
  • Exemplary ranges include near-UV ( ⁇ 300nm to ⁇ 380nm), violet ( ⁇ 380nm to ⁇ 440nm), blue ( ⁇ 440nm to ⁇ 485nm), cyan ( ⁇ 485nm to ⁇ 510nm), green ( ⁇ 510nm to ⁇ 565nm), yellow ( ⁇ 565nm to ⁇ 590nm), orange ( ⁇ 590nm to ⁇ 625nm), red ( ⁇ 625nm to ⁇ 740nm), or near-IR ( ⁇ 740nm to ⁇ 850nm).
  • Additional systems can include a computing device for image analysis, feature recognition, and/or any other purpose.
  • imaging chips can be used in vivo and/or ex vivo.
  • In vivo imaging includes imaging live tissue within a subject (e.g., animal, mammal, non- human mammal, human, primate, simian, monkey, ape, rodent, mouse, ungulate, etc.), while ex vivo can include imaging tissue samples, biopsies, and/or any other excised tissue sample.
  • Ex vivo samples can be placed imaged on a slide (e.g., microscope slide) or directly from the excised tissue itself.
  • a marker e.g., dye or probe
  • a probe can be applied to a tissue, whether in vivo or ex vivo.
  • Such dye or probe can be colorimetric and/or fluorescent.
  • a probe can include a fluorophore or other chromatographic moiety for imaging a cell or tissue type.
  • the chromatographic moiety can be conjugated to an agent that targets a molecule of interest—such agents may be referred to as a binding agent, targeting agent, binding moiety, and/or binding moiety.
  • binding agents can include (but are not limited to) a carbohydrate, a lipid, a peptide, a nucleic acid (e.g., RNA, DNA, etc.), a protein, and/or other large or small molecule of interest.
  • a carbohydrate e.g., a lipid, a peptide, a nucleic acid (e.g., RNA, DNA, etc.), a protein, and/or other large or small molecule of interest.
  • Exemplary fluorescent moieties and dyes can include (but are not limited to) SYBR green, SYBR gold, a CAL Fluor dye such as CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, and CAL Fluor Red 635, a Quasar dye such as Quasar 570, Quasar 670, and Quasar 705, an Alexa Fluor such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647,and Alexa Fluor 784, a cyanine dye such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, fluorescein, 2', 4', 5', 7'-tetrachloro-4-7-dichlorofluorescein (TET), carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), 6-carboxy-4',5'- dichlor
  • Dyes can further include near-IR dyes or fluorophores, such as IRDye dyes (e.g., IRDye 800CW, IRDye 680RD, IRDye 700, IRDye 750, and IRDye 800RS), CF dyes (e.g., CF680, CF680R, CF750, CF770, and CF790), Tracy dyes (e.g., Tracy 645 and Tracy 652), Alexa dyes (e.g., Alexa Fluor® 660 dye, Alexa Fluor® 700 dye, Alexa Fluor® 750 dye, and Alexa Fluor® 790), cyanine dyes (e.g., Cy7 and Cy7.5), thienothiadiazole dyes, phthalocyanine dyes, squaraine dyes, rhodamine dyes and analogues (e.g., Si ⁇ pyronine, Si ⁇ rhodamine, Te ⁇ rhodamine, and Changsha),
  • Antibodies that can be used include (but are not limited to) monoclonal antibodies, polyclonal antibodies, as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies.
  • Antibodies may include hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2 and F(ab) fragments; Fv molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al.
  • the ligand will include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and will typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, or preferably at least two of the functional chemical groups.
  • the small molecule may also comprise a region that may be modified and/or participate in conjugation to a fluorophore, without substantially adversely affecting the small molecule's ability to bind to its target analyte.
  • Small molecule ligands are also found among biomolecules including peptides, carbohydrates, SF-2023-054-3-PCT-0-UPR fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • the small molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Small molecules may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the small molecule may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e., a compound diversity combinatorial library. When obtained from such libraries, the small molecule employed will have demonstrated some desirable affinity for the protein target in a convenient binding affinity assay.
  • Combinatorial libraries, as well as methods for the production and screening are described in: U.S. Pat. Nos.
  • the binding agent comprises a membrane-targeted cleavable probe that becomes activated when it encounters a protease.
  • probes comprise a synthetic peptide substrate comprising a protease cleavage site coupled to a fluorophore and a membrane targeting domain. Upon cleavage by a protease, the fluorophore is deposited in cell membranes.
  • protease-activated peptide probes see, e.g., Page et al. (2015) Nature Communications 6 (8448), Backes et al. (2000) Nat. Biotechnol.18:187-193; herein incorporated by reference.
  • Fluorophores may be conjugated to binding agents by any suitable method.
  • the fluorophore and binding agent may be directly linked, e.g., via a single bond, or indirectly linked e.g., through the use of a suitable linker, e.g., a polymer linker, a chemical linker, or one or more linking molecules or moieties.
  • a suitable linker e.g., a polymer linker, a chemical linker, or one or more linking molecules or moieties.
  • attachment of the fluorophore and binding agent may be by way of one or more covalent interactions.
  • the fluorophore or binding agent may be functionalized, e.g., by addition or creation of a reactive functional group.
  • a functional linker refers to any suitable linker that has one or more functional groups for the attachment of one molecule to another.
  • the functional linker comprises an amino functional group, a thiol functional group, a hydroxyl functional group, an imidazolyl functional group, a guanidinyl functional group, an alkyne functional group, an azide functional group, or a strained alkyne functional group.
  • Such a small distance can range from less than 1 mm up to approximately 10 mm, such as approximately 0.1 mm, 0.25 mm, 0.5 mm, 0.75 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm.
  • Imaging can include illuminating tissue with white light and/or a subset of light (e.g., specific wavelengths and/or ranges of wavelengths). For example, illumination can occur with light that overlaps with a fluorophore’s excitation spectrum or excitation maximum. Imaging can utilize a single exposure or multiple exposures which can be combined or averaged.
  • probes SF-2023-054-3-PCT-0-UPR or dyes mark diseased tissues, so marked tissue is excised. Additional instances use a first dye that is specific for healthy tissue and a second dye that is specific for diseased tissue; in these situations, the tissue marked as diseased is removed.
  • tissue samples (such as described above) can be imaged by a composite filter and imaging sensor. Such imaging can reveal presence or absence of certain targets, such as target cells or target tissues. Such targets can include diseased tissues, such as cancerous, neoplastic, infected, and/or any other form of diseased tissue.
  • certain embodiments perform in vivo imaging.
  • Such computer-readable storage media can comprise RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage, or other magnetic storage devices, flash memory, or any other medium that can be used to store desired program code in the form of instructions or data structures and that can be accessed by a computer. Also, any connection is properly termed a computer-readable medium.
  • coaxial cable, fiber optic cable, twisted pair, digital subscriber line (DSL), or wireless technologies such as infrared, radio, and microwave are included in the definition of medium.
  • DSL digital subscriber line
  • computer-readable storage media and data storage media do not include connections, carrier waves, signals, or other transitory media, but are instead directed to non-transitory, tangible storage media.
  • Disk and disc includes compact disc (CD), laser disc, optical disc, digital versatile disc (DVD), floppy disk and SF-2023-054-3-PCT-0-UPR Blu-ray disc, where disks usually reproduce data magnetically, while discs reproduce data optically with lasers. Combinations of the above should also be included within the scope of computer-readable media.
  • processors such as one or more digital signal processors (DSPs), general purpose microprocessors, application specific integrated circuits (ASICs), field programmable logic arrays (FPGAs), or other equivalent integrated or discrete logic circuitry.
  • DSPs digital signal processors
  • ASICs application specific integrated circuits
  • FPGAs field programmable logic arrays
  • processor may refer to any of the foregoing structure or any other structure suitable for implementation of the techniques described herein.
  • functionality described herein may be provided within dedicated hardware and/or software modules configured for encoding and decoding or incorporated in a combined codec.
  • the techniques could be fully implemented in one or more circuits or logic elements.
  • the techniques of this disclosure may be implemented in a wide variety of devices or apparatuses, including a wireless handset, an integrated circuit (IC) or a set of ICs (e.g., a chip set).
  • IC integrated circuit
  • a set of ICs e.g., a chip set.
  • Various components, modules, or units are described in this disclosure to emphasize functional aspects of devices configured to perform the disclosed techniques, but do not necessarily require realization by different hardware units.
  • the FOP When excitation light is incident on the FOP with AOIs larger than the NA of the fibers, the light will pass through the EMA sidewalls and experience significant attenuation. However, a small fraction of the incident light scatters through the apertures, escaping the absorptive side walls, and exiting the FOP at near-normal incidence to the sensor. Consequently, it is preferred to place the interference filter below the FOP to block the excitation that is scattered. If the interference filter is placed above the FOP, the excitation rejection will be limited by the scattering effects and not the absorption performance of the FOP. Thus, for obliquely incident excitation the FOP is responsible for providing excitation rejection and should be placed on top of the interference filter.
  • the optical front-end best blocks excitation near normal incidence when the interference filter is on top and shows the highest performance for oblique excitation with the FOP on top.
  • the difference in performance between the two orientations at the extremes of both regimes is between 1-2 orders of magnitude.
  • the measurements also show that the angle-sensitivity of the front-end can be completely rectified by placing the same interference on both sides of the FOP. With this modification the filter shows superior performance for both normal incident and oblique excitation.
  • This design comes at the cost of added fabrication complexity and is only necessary when the AOI of the excitation light is not known.
  • the excitation may be introduced obliquely, so it is sufficient to have a single interference filter on the bottom of the FOP.
  • FIGS 7A-7D are graphs illustrating example characterizations of an optical front-end designed in accordance with the techniques described herein.
  • Figure 7A a comparison of angular selectivity of FOP, on-chip collimators made of an angle-selective grating (ASG), and a lens-less imager with no collimators is shown.
  • the FOP compared to the ASGs reduces the FWHM by almost 3x to 12.65°.
  • the performance of the FOP, interference filter, and final filter design was measured and characterized at different AOIs on an optical breadboard setup.
  • Figure 7A shows the measured angular transmittance of the FOP in air at 488nm compared to the on-chip angle-selective gratings (ASGs) and the theoretical angular sensitivity of a lens-less imager without collimators.
  • FWHM full width at half maximum
  • This improvement over ASGs is due to the fact that on-chip collimators may be constructed with reflective metal as opposed to optically absorbing material and have aspect ratios limited by the thickness of on-chip metal layers and design rules of the process.
  • FIG. 7C a spectra of multi-bandpass interference filter are shown.
  • the filter provides more than 6 orders of rejection of 488nm and 633nm excitation while passing a majority of emitted fluorescence for various filters.
  • the spectral design for the imager including the transmittance spectra of the interference filter and the emission spectra for two particular fluorophores (AlexaFluor488 and IRDye680LT), with corresponding excitation laser lines at 488nm and 633nm—is shown in Figure 7C.
  • the interference filter provides sufficient bandwidth to capture emitted fluorescence while providing 6 orders of magnitude of excitation rejection.
  • the filter maintains adequate excitation rejection, but near 20° for 488nm and 35° for 633nm, the transmittance sharply increases as the filter passband shifts over the excitation wavelengths, becoming practically transparent at 33° and 48°, respectively.
  • the 488nm excitation shifts into the filter passband at a smaller AOI as it is closer to the filter band- edge than 633nm.
  • the composite filter maintains more than 5 orders of magnitude rejection at 488nm and more than 6 orders of magnitude rejection at 633nm.
  • Figures 8A-8E are example resolution measurements of an optical front-end designed in accordance with the techniques described herein.
  • Figure 8A an experimental setup for resolution measurements with USAF 1951 test target is shown.
  • the test target is used to pattern excitation light onto a uniform layer of Cy5.5 dye contained with a coverslip on the surface of the target.
  • the target assembly is placed directly on an imaging chip described herein.
  • the system imaged a standard negative United States Air Force 1951 (USAF-1951) resolution test target.
  • USAF target is used to pattern excitation light onto a uniform layer of Cy5.5 dye, which is placed directly on the imaging chip ( Figure 8A).
  • Figure 8B a reference image of test target taken at 2.5x using a benchtop fluorescence microscope is shown.
  • Figure 8C an image taken with the imaging chip of group 2 on the test target is shown, which lies near the resolution limit of the sensor.
  • the reference image of Figure 8B can be compared with the chip image of the elements in group 2 ( Figure 8C), which lay at the resolution limit of the sensor.
  • FIG. 9 is an image illustrating various fascia on and near a prostate.
  • a goal in tumor resection surgeries is the complete removal of all gross and microscopic disease with minimal damage to neighboring healthy tissue.
  • surgeries are primarily guided through visual examination, touch, or white light imaging. Because these techniques lack adequate contrast, many surgeries fall short of this goal.
  • positive margins where cancer cells are detected along the margin of the tumor cavity indicating an incomplete resection, are common across many cancer types.
  • the rate of occurrence is estimated to be as large as a quarter of all patients. This number is greater than 20% for prostate cancer patients. Overall, what this means, is that effected patients have a two-times increased risk of recurrence.
  • Figures 10A–10C are examples of ex vivo imaging of resected prostate tissue using an optical front-end designed in accordance with the techniques described herein.
  • an imaging chip prototype notated as VISION
  • the system imaged banked, resected patient tissue with tumor and nerves.
  • the tissue samples are paraffin embedded, sectioned at 4 ⁇ m thickness, and mounted onto glass slides.
  • the samples were fluorescently stained for both prostate cancer with an anti-PSMA antibody conjugated to red and nerves with an anti-S100 antibody conjugated to green.
  • slides are scanned with a fluorescence microscope and compared against full-slide H&E scans by a trained pathologist. Relevant areas of each slide are then imaged with VISION.
  • Dual- SF-2023-054-3-PCT-0-UPR color imaging of both fluorescence channels is achieved with a single chip by taking a separate exposure with each excitation wavelength.
  • multiple exposures are captured and then averaged for each channel. For the prostate channel, 100 exposures at 75ms are used for each image, while for nerves, 100 exposures at 50ms are used.
  • FIG 10A images of resected prostate tumor immuno-fluorescently (IF) stained for prostate cancer with red and nerves green are shown. Comparison with full slide microscope scans of IF (iv) and H&E staining (iv) shows that VISION can clearly identify tumor (ii) and nerves (v). Simultaneous detection of both tumors and nerves with the same imager (i) is possible by overlaying separate exposures of each channel. (vi) VISION is highly sensitive and is able to detect a microscopic foci ( ⁇ 100 cells) along the inked margin.
  • FIG 10A shows a side-by-side comparison of images of a resected prostate tumor captured with VISION and a benchtop fluorescence microscope alongside H&E images.
  • VISION clearly identifies tumor foci and nerves, including a microscopic tumor foci of less than 100 cells along the inked margin of sample.
  • Figure 10B images of extra-prostatic extension (EPE) into fibroadipose tissue are shown.
  • EPE extra-prostatic extension
  • VISION (iv) is able to clearly identify most tumor and nerves identified in microscope IF (i, iii) and histological (ii, v) references.
  • Figure 10C images of metastatic tumor foci in lymph node are shown.
  • VISION detects sight of metastatic tumor (iv) visible in microscope IF (i, iii) and histological (ii, v) references.
  • FIGs 10B-10C two other clinically relevant examples were imaged using VISION to visualize extra- prostatic extension (EPE) of tumor into fibroadipose tissue interspersed with nerves and to identify a metastatic lymph node.
  • EPE extra- prostatic extension
  • FIG 11 is a conceptual diagram of an example interference filter directly coated on a fiber optic plate (FOP), in accordance with the techniques described herein.
  • the techniques described herein fabricate the optical front-end by directly coating a multi- SF-2023-054-3-PCT-0-UPR bandpass interference filter on top of a Low-NA fiber optic plate.
  • the filter has two bands: one in the green region and another in the NIR band, with good spectral characteristics which excite at 488nm and 633nm.
  • the interference filter shows significant excitation bleed through at large angles of incidence.
  • the FOP is highly absorptive at large angles of incidence, compensating for this deficiency.
  • FIG. 12 is an example of imaging of PC3-PIP cell cultures, in accordance with the techniques described herein.
  • a system as described herein imaged PC3-PIP cell cultures stained with anti-PSMA with dyes in both channels.
  • the system imaged FoVs containing cell clusters of different sizes with a single 50ms exposure time and measured the highest at pixel signal to noise ratio.
  • the two clusters labeled A and B contain cell clusters of 100 and 50 cells and are imaged with SNRS of 22 and 10, respectively.
  • Example 7 Signal Noise Ratios
  • Figure 13 is a graph illustrating signal noise ratios vs. cell cluster sizes, in accordance with the techniques described herein.
  • Figure 13 shows that a system described herein is capable of imaging clusters as small as 100 cells with an SNR of 10dB, meaning that these signals are more than 10x above our noise level. And the NIR channel 100 cell clusters are visible at SNRs of 6dB.
  • the SNR can be significantly improved using longer exposure times, higher laser powers, and the use of better CMOS technologies, which can move toward single cell detection.
  • Example 8 Signal Noise Ratios [0146]
  • An imaging system is typically characterized by its point spread function (PSF).
  • the PSF is equivalent to the impulse response function of a linear shift invariant (LSI) system and can be determined by finding the image produced by the system when imaging an ideal point source.
  • LSI linear shift invariant
  • the PSF acts as a blurring kernel, spreading out each point source in the sample plane. This can be quantified by measuring full width at half maximum (FWHM) of the PSF.
  • FWHM full width at half maximum
  • $"% ⁇ is found by placing a point source on the sample plane located at angle ⁇ off the z axis from the pixel at origin on the imager plane and determining fraction of total light emitted by the point source that is incident on the pixel for each ⁇ , ⁇ 3 ⁇ $"% ⁇ ⁇ light incident on pixel ⁇ & G&'H IJK&&IL HKMh& [0149]
  • the point source emits isotopically with its total surface flux, ⁇ , at radial distance U inversely proportional the area of the sphere defined by U, 1 ⁇ 5 ⁇ ⁇ 4 ⁇
  • the pixel subtends a cone of this sphere defined by the perimeter of the pixel.
  • the pixel pitch ( ⁇ 5-55 ⁇ m) is often much smaller than the separation distance (0.5-1.5mm) due to the filter thickness and spacer necessary for epi- SF-2023-054-3-PCT-0-UPR illumination of the sample.
  • the area of the spherical patch at the base of the cone is equivalent to the effective area of the pixel.
  • the above model only considers a single 2D sample plane.
  • the image of each 2D slice is super-imposed on the image plane.
  • scattering and absorption effects may be taken into account for each layer requiring a more complex model.
  • the imaging depth will be limited to a couple of mm as there is significant attenuation with increasing depth due to the absorption and scattering of tissue, which are high for visible and NIR wavelengths, and quadratic spreading loss of the fluorescence emissions.
  • a collimator can be used to further restrict the pixel angle of view (AoV).
  • Typical lens-less collimators are Parallel-hole collimators, which are composed of an array of holes within an absorptive media. Light at AOIs close to normal incidence see a direct path through the collimator, while at oblique AOIs the light may pass through the sidewalls of the collimator and is attenuated.
  • the angle-selectivity of a Parallel-hole collimator is determined directly by the aspect ratio (channel length/hole diameter) of the holes. Larger aspect ratios provide sharper angle selectivity. [0156]
  • the relation derived above for the PSF of a lens-less imager can be modified to incorporate the effect of additional angle selectivity provided by the collimator.
  • the maximum collection efficiency is 50% due to the fact that a planar sensor with infinite area only collects light from one side of the point source.
  • a custom PCB is used to supply all power and biasing for the imaging chip as well as to read off and digitize each captured image frame. All timing and control signals necessary for image acquisition are generated by an FPGA, which is also used as digital interface between the imaging chip and a computer. A custom software GUI is used to visualize and capture image data on a laptop.
  • Resolution measurements are performed using a negative USAF-1951 resolution test target. The test target is coated one with Cy5.5 dye dissolved in DMSO and is sealed with a quartz coverslip. To verify even distribution of dye, a reference image of the target is taken on a benchtop microscope at 2.5x with an integration time of 1s. The target is then placed dye-coated-side down directly on the imaging chip.
  • Excitation light is provided by the aforementioned fiber-coupled and collimated 633nm laser operated at approximately 1mW. To minimize background, all imaging is performed in an optically isolated box. Each element is centered on the imager to minimize illumination variation and is imaged. 10050ms frames are captured for each imaging area and averaged to produce the final image. The contrast for each element is measured in ImageJ (NIH). Contrast is defined as ⁇ (I ⁇ _max-I_min) ⁇ /(I_max+ I_min-2I_background), where I_max is the maximum pixel value in the bright bars on the target, I_min is the minimum pixel value in the dark bars on the target, and I_background is the average pixel value when the excitation source is off.
  • the paraffin-embedded tissue blocks for each sample are sectioned at 4 ⁇ m and mounted onto glass slides.
  • One representative slide from each block is stained with hemoxylin and eosin (H&E) for histological analysis.
  • the remaining slides are used for immunofluorescence staining.
  • H&E hemoxylin and eosin
  • To label prostate tumor an anti-PSMA rabbit primary antibody is used with an IRDye 680LT goat anti-rabbit secondary antibody (LI-COR).
  • an anti-S100 mouse primary antibody is used with an Alexa Fluor 488 anti-mouse secondary antibody (Invitrogen).
  • the imaging chip is then inverted and placed on the coverslip such that it is imaging the same sample area as the microscope.
  • an excitation wavelength in the passband of the chip is shined through a high-magnification objective and onto the chip.
  • the chip position is adjusted such that the excitation light is centered within the FOV of the imager.
  • the aforementioned collimated, fiber-coupled lasers are used to provide fluorescence excitation at 488nm and 633nm and are both operated at 15mW.
  • the excitation light is introduced at the back of the slide and is incident on the chip at approximately 70° with the beam parallel to the long dimension of the chip. For each region of interest on the sample, separate microscope and chip images are acquired with each excitation wavelength.
  • a system for fluorescence imaging comprising: an optical front-end comprising a first layer of an optical filter and a second layer of a collimator.
  • Aspect 7 The system of any one or more of Aspects 1–5, wherein a thickness of the optical front-end is less than or equal to 5mm.
  • Aspect 7. The system of any one or more of Aspects 1–6, wherein the thickness of the optical front-end is less than or equal to 2.5mm.
  • Aspect 8. The system of any one or more of Aspects 1–7, wherein the thickness of the optical front-end is less than or equal to 1.25mm.
  • Aspect 9 The system of any one or more of Aspects 1–8, wherein the thickness of the optical front-end is less than or equal to 500 ⁇ m.
  • Aspect 10. The system of any one or more of Aspects 1–9, wherein the thickness of the optical front-end is less than or equal to 250 ⁇ m.
  • Aspect 13 The system of any one or more of Aspects 1–10, wherein the thickness of the optical front-end is less than or equal to 150 ⁇ m SF-2023-054-3-PCT-0-UPR Aspect 12.
  • Aspect 13 The system of Aspect 12, wherein the collimator further has one of: 2 orders of magnitude rejection at 30 degrees or greater off axis, 3 orders of magnitude rejection at 30 degrees or greater off axis, 4 orders of magnitude rejection at 30 degrees or greater off axis, 5 orders of magnitude rejection at 30 degrees or greater off axis, or 6 orders of magnitude or greater rejection at 30 degrees or greater off axis.
  • Aspect 14 The system of any one or more of Aspects 1–10, wherein the thickness of the optical front-end is less than or equal to 150 ⁇ m SF-2023-054-3-PCT-0-UPR Aspect 12.
  • Aspect 15 The system of any one or more of 3 1–112, wherein the collimator is a parallel-hole collimator.
  • Aspect 15 The system of any one or more of Aspects 1–14, wherein the collimator is a fiber optic plate.
  • Aspect 16 The system of any one or more of Aspects 1–15, wherein the collimator comprises an absorptive material surrounding an array of optical fibers.
  • Aspect 17. The system of any one or more of Aspects 1–15, wherein the collimator is an absorptive material surrounding one or more holes filled with a transparent material.
  • Aspect 18 The system of any one or more of Aspects 1–15, wherein the collimator is an absorptive material surrounding holes that are either filled with air or in a vacuum.
  • Aspect 19 The system of any one or more of Aspects 1–18, wherein the imager is operationally integrated with a surgical tool.
  • Aspect 20 The system of Aspect 19, wherein the surgical tool is a periscopic probe or SF-2023-054-3-PCT-0-UPR laparoscopic robotic instrument.
  • Aspect 21 The system of any one or more of Aspects 1–20, wherein the imager comprises at least one LED or at least one laser diode light source.
  • Aspect 22 The system of any one or more of Aspects 1–18 or 21, wherein the imager is operationally integrated with an implantable imager.
  • Aspect 23 The system of any one or more of Aspects 1–18 or 21, wherein the imager is operationally integrated with a lab-on a chip.
  • Aspect 24 The system of Aspect 23, wherein the lab on chip is a microarray of DNA, RNA, proteins, cells, tissue, or any biological sample.
  • Aspect 25 The system of Aspect 23, wherein the lab on chip is a diagnostic assay, including a PCR test, ELISA, or lateral flow assay.
  • Aspect 26 The system of any one or more of Aspects 23–25, wherein the imager comprises at least one LED or at least one laser diode light source.
  • Aspect 27 The system of any one or more of Aspects 1–26, wherein the system is lens-free.
  • Aspect 28 The system of any one or more of Aspects 1–27, wherein the system utilizes machine learning to improve image quality.
  • Aspect 29 The system of any one or more of Aspects 1–27, wherein the system utilizes machine learning to improve image quality.
  • Aspect 30 The system of any one or more of Aspects 1–28, wherein a sample being imaged by the system comprises one or more of diseased tissue and cancerous tissue.
  • Aspect 30 The system of any one or more of Aspects 1–29, wherein a labeling agent SF-2023-054-3-PCT-0-UPR being imaged comprises one or more of an antibody, an antibody mimetic, a peptide, a peptoid, an aptamer, or a small molecule ligand that selectively binds to the cellular, protein, DNA, RNA, molecular or chemical marker of interest.
  • Aspect 31 Aspect 31.
  • Aspect 30 wherein the cellular marker of interest comprises one or more of a tumor-specific antigen, a tumor-associated antigen, and an immune activation marker.
  • Aspect 32 The system of any one or more of Aspects 1–31, wherein a sample is imaged with at least two different fluorophore conjugates, wherein each fluorophore conjugate comprises a different fluorophore that emits fluorescent light at a different emission wavelength, and wherein each fluorophore conjugate comprises a different binding agent that selectively binds to a different marker.
  • Aspect 33 A method for imaging a biological sample, comprising: applying a marker to a tissue; and obtaining an image of the tissue using a system as described in any of Aspects 1-31.
  • Aspect 34 The method of Aspect 33, further comprising resecting the tissue to remove diseased tissue from the marked tissue.
  • Aspect 35 The method of any of Aspects 33-34, wherein the marker is a fluorescent dye selected from SYBR green, SYBR gold, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 570, Quasar 670, Quasar 705, fluorescein, fluorescein isothiocyanate, hexachlorofluorescein, 4′,6-diamidino-2-phenylindole, Hoechst, rhodamine, carboxy-X-rhodamine, and combinations thereof.
  • the marker is a fluorescent dye selected from SYBR green, SYBR gold, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL
  • Aspect 36 The method of any of Aspects 33-34, wherein the marker is a fluorescent probe comprising a binding agent and a fluorophore.
  • the marker is a fluorescent probe comprising a binding agent and a fluorophore.
  • Aspect 38 The method of Aspect 36, wherein the binding moiety is selected from a carbohydrate, a lipid, a peptide, a nucleic acid, a protein, and a small molecule.
  • Aspect 40 The method of any of Aspects 33-38 further comprising illuminating the tissue with a light source.
  • Aspect 40 The method of any of Aspects 33-39, wherein obtaining an image of the tissue comprises contacting the optical front-end to the tissue.
  • DOCTRINE OF EQUIVALENTS [0185] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, a number of well- known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.

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Abstract

La divulgation concerne un système et des procédés d'imagerie de tissu. De tels systèmes peuvent être utilisés pour l'imagerie in vivo ou ex vivo. L'imagerie in vivo peut impliquer un imageur et un élément frontal optique. De tels éléments frontaux avant peuvent comprendre un collimateur et un filtre. La combinaison peut être utilisée pour réduire les interférences ou le bruit provoqués par une lumière oblique. Des systèmes supplémentaires peuvent être utilisés pour une imagerie laparoscopique et/ou une chirurgie guidée par image, telle qu'une résection de tumeur.
EP23873538.5A 2022-09-27 2023-09-26 Appareil, systèmes et procédés d'imagerie par fluorescence sur des capteurs plans Pending EP4577858A2 (fr)

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WO2015042529A2 (fr) * 2013-09-20 2015-03-26 The Regents Of The University Of California Procédés, systèmes et dispositifs permettant d'imager des tumeurs microscopiques
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EP3885801A1 (fr) * 2020-03-25 2021-09-29 Ams Ag Filtre d'interférence, dispositif optique et procédé de fabrication d'un filtre d'interférence
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