System and method for transferring markings - Patents.com

Abstract

Techniques disclosed herein relate to systems and methods for transferring markings. The system can include an imaging device for imaging stained and unstained slides, and a display for displaying one or more images of the stained and unstained slides. In various embodiments, the system can include a mechanical stage, such as a scaffold, for positioning or orienting the stained slide relative to the unstained slide. In various embodiments, the system can be coupled to a first application for aligning the images to enable digital marking of the unstained slide, and a second application for digitally marking one or more features and converting the digital markings into coordinates used for further processing, such as dissection.

Description

[Technical field]

[0001] RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 225,079, filed July 23, 2021, the entire contents of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates generally to the field of histology and / or pathobiology. More specifically, the present disclosure relates to the creation and transfer of pathologist markings for automated downstream analysis or processing of biological samples. [Background technology]

[0003] Various methods have been proposed to address the cumbersome nature of peeling procedures for preparation and / or processing of histological samples for downstream analysis or processing. Current tissue dissection processes are either fully manual or fully automated. In a fully manual process, a razor blade is used to peel and directly collect a region of interest (ROI) or "S" region from a substrate, such as a glass slide. For example, a conventional workflow for substrate submission involves a fully manual process, in which a user manually transfers a pathologist region of interest marking on a stained slide to an unstained slide using an off-the-shelf standard marking pen. The user then uses a razor blade or equivalent to peel and collect the region of interest (e.g., "S") on the marked unstained substrate into a container.

[0004] In the case of a fully automated process, the operator inserts the sample into the machine, which automatically retrieves the "S" region. However, the success rate of obtaining the desired "S" region is not satisfactory as it is not close to 100%. The fully manual process, although entirely dependent on the operator's hand / eye coordination, can achieve a success rate close to 100%, dramatically improving the consistency and accuracy of tissue stripping. However, the fully manual process also poses ergonomic / safety issues as the constant force applied to the glass surface may cause lacerations to the operator and ergonomic issues (e.g., carpal tunnel).

[0005] Current implementations of physical and digital pathology have improved many areas of histology / pathology workflow. However, there are still several areas of significant unmet need. One unmet need is the inability to digitally process some substrate submissions using commercially available digital pathology systems. A significant number of cases still require manual glass workflow processing. Thus, a method is needed to transform this process to achieve a fully digital workflow. Summary of the Invention

[0006] According to various embodiments disclosed herein, a system for transferring pathologist markings is provided that includes an imaging device configured to image a stained or unstained slide, a display for displaying one or more images of the stained or unstained slide, a mechanical stage / scaffolding for positioning or orienting the stained slide relative to the unstained slide to enable physical marking of the unstained slide, and an application used to digitally depict one or more features based on the physical markings and convert the physical markings to coordinates used for further processing.

[0007] According to various embodiments disclosed herein, a method for transferring a marking is provided that includes projecting an image of a stained slide with pathologist markings onto a display, placing an unstained slide on a scaffold above the display, aligning the unstained slide with the projected image of the stained slide by moving the scaffold, and physically transferring the pathologist markings onto the unstained slide.

[0008] According to various embodiments disclosed herein, a system for transferring pathologist markings is provided that includes an imaging device configured to image stained and unstained slides, a display for displaying one or more images of the stained and unstained slides, a first application used in aligning the one or more images of the stained or unstained slides, and a second application used in digitally marking one or more features and converting the digital markings of the one or more features into coordinates.

[0009] According to various embodiments disclosed herein, a method for digitally transferring a marking is provided that includes acquiring an image of a stained slide under an imager, projecting the image of the stained slide on a display, marking the image of the stained slide, transferring the marking of the stained slide to an unstained slide, and capturing the marked unstained slide with the marking transferred.

[0010] These and other aspects and embodiments are described in detail below. The preceding information and the following detailed description include illustrative examples of the various aspects and embodiments and provide an overview or framework for understanding the nature and character of the claimed aspects and embodiments. The drawings provide illustration and a further understanding of the various aspects and embodiments, and are incorporated in and constitute a part of this specification.

[0011] The accompanying drawings are not necessarily drawn to scale. Like reference numbers and designations in the various drawings refer to like elements. For purposes of clarity, not every component may be labeled in every drawing. The drawings are as follows: [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of an exemplary system for transferring a marking, according to various embodiments. [Diagram 2]1 illustrates an exemplary embodiment of a system for transferring a marking, according to various embodiments. [Diagram 3] 1 illustrates an exemplary embodiment of a system for transferring a marking, according to various embodiments. [Figure 4] 1 is a flowchart of an exemplary method for transferring a marking, according to various embodiments. [Diagram 5] 1 is a flowchart of an exemplary method for transferring a marking, according to various embodiments. [Figure 6] FIG. 1 is a block diagram illustrating a computer system in accordance with various embodiments. [Figure 7] FIG. 1 shows an example of a situation where the variation between successive tissue slides is too large to allow automated image registration methods. As shown in the figure, a first slice (shown in the light rectangle on the left) is expected to overlap with a successive slice (dark rectangle) with moderate variation between slices. Such expected variation is shown in the graph on the top right. However, when large variations occur between the first and second slices, image registration using algorithm-based methods becomes impossible. In contrast, the method described herein is advantageous in that such large variations between slices can be taken into account and appropriately processed to facilitate transfer or marking from one slide or image thereof to a second slide or image thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure and corresponding embodiments solve the known problem of processing biological specimens with a fully digital workflow using commercially available digital pathology systems. The disclosed systems, tools, methodologies, and / or approaches are suitable for use in transferring pathologist markings for histology and / or pathobiology, and provide improved reliability and consistency in creating, converting, and / or transferring markings for use during automated downstream analysis or processing of biological specimens.

[0014] It should be understood that the following disclosure provides many different embodiments or examples for implementing different features of the present disclosure. Specific embodiments or examples of components and arrangements are described below to simplify the present disclosure. It should be understood that these are merely examples and are not intended to be limiting. For example, the dimensions of elements are not limited to the disclosed ranges or values, but may depend on process conditions and / or desired characteristics of the apparatus. Furthermore, the formation of a first feature above or on a second feature in the following description may include embodiments in which the first and second features are formed in direct contact, and may include embodiments in which additional features may be formed between the first and second features such that the first and second features do not have to be in direct contact. Various features may be arbitrarily drawn to different scales for brevity and clarity.

[0015] Some of the terms used herein are defined as described in this section. Other terms are defined or exemplified elsewhere in this disclosure. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] Additionally, spatially relative terms such as "below," "below," "lower," "above," "above," and the like may be used herein to describe the relationship of one element or feature to another element(s) or feature(s) as depicted in the drawings for ease of description. The spatially relative terms are intended to encompass various orientations of the device during use or operation in addition to the orientation depicted in the drawings. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein may be interpreted accordingly. Additionally, the term "made from" may mean either "comprising" or "consisting of."

[0017] In this application, the use of "or" means "and / or" unless stated otherwise. In the context of multiple dependent claims, the use of "or" refers to more than one preceding independent or dependent claim in the alternative only.

[0018] The term "about" means a range of plus or minus 10% of the value, unless the context of the disclosure dictates otherwise or contradicts such an interpretation, e.g., "about 5" means 4.5 to 5.5, "about 100" means 90 to 100, and so on. For example, in a list of numerical values ​​such as "about 49, about 50, about 55," "about 50" means a range extending to less than half the interval(s) between the preceding and succeeding values, e.g., from greater than 49.5 to less than 52.5. Furthermore, the phrase "approximately below" or "approximately above" a value should be understood in light of the definition of the term "about" provided herein.

[0019] When a range of values ​​is provided in this disclosure, it is intended that each intervening value between the upper and lower limit of that range, as well as any other stated or intervening value in that stated range, be encompassed within the disclosure. For example, when a range of 1 μm to 8 μm is recited, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are also intended to be disclosed.

[0020] As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0021] As used herein, the term "detection" refers to the process of determining a value or set of values ​​associated with a sample by measuring one or more parameters in the sample, and may further include comparing a test sample to a reference sample. According to the present disclosure, detection of a tumor includes identifying, assaying, measuring and / or quantifying one or more markers.

[0022] As used herein, the term "likelihood" generally refers to a probability, relative probability, presence or absence, or degree.

[0023] As used herein, the terms "comprise" (or variations thereof), "contain" (or variations thereof), "have" (or variations thereof), or "include" (or variations thereof) are not intended to be limiting, are inclusive or open-ended, and do not exclude additional, unrecited additives, components, integers, elements, or method steps. For example, a process, method, system, composition, kit, or apparatus that includes a list of features is not necessarily limited to only those features and may include other features not expressly recited or inherent in such process, method, system, composition, kit, or apparatus.

[0024] The term "sample" as used herein refers to a composition obtained or derived from a subject of interest, including cells and / or other molecular entities that are characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. Preferably, the sample is a "biological sample", which means a sample from a living organism, such as, for example, cells, tissues, organs, etc. In some embodiments, the source of the tissue sample may be blood or any blood component; bodily fluids; solid tissue from fresh, frozen and / or preserved organs or tissue samples or specimens or aspirates; cells or plasma from any time point in the subject's pregnancy or development. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, fluids (e.g., lymph, amniotic fluid, milk, whole blood, urine, CSF, saliva, sputum, tears, sweat, mucus, tumor lysates, and cell culture media), homogenized tissues, tumor tissues, and cell extracts. Samples also include biological samples that have been manipulated, e.g., treated with reagents to solubilize or concentrate certain components, such as proteins or nucleic acids, or embedded in a semi-solid or solid matrix, for the purpose of making thin sections, e.g., thin sections of tissues or cells in a histological sample. Samples can include environmental components, e.g., water, soil, mud, air, resins, minerals, etc. Preferably, biological samples include DNA (e.g., gDNA, mtDNA), RNA (e.g., mRNA, tRNA), proteins, or combinations thereof, obtained from a subject (e.g., a human or other mammalian subject). In some embodiments, the term "sample" refers to cells, tissues, specimens, tissue samples, FFPE tissues, blood, or other biological materials attached using standard molecular biology methods.

[0025] As used herein, the term "cell" is used interchangeably with the term "biological cell." Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, etc., prokaryotic cells, bacterial cells, fungal cells, protozoan cells, etc., cells dissociated from tissues, such as muscle, cartilage, fat, skin, liver, lung, nervous tissue, etc., immune cells, such as T cells, B cells, natural killer cells, macrophages, etc., embryos (e.g., zygotes), oocytes, egg cells, sperm cells, hybrid cells, cultured cells, cells from cultured lines, cancer cells, infected cells, transfected and / or transformed cells, reporter cells, etc. Mammalian cells can be from, for example, humans, mice, rats, horses, goats, sheep, cows, primates, etc.

[0026] As used herein, the term "tumor" includes any cell or tissue that may have undergone transformation at the genetic, cellular, or physiological level compared to normal or wild-type cells. The term generally refers to a neoplastic growth that may be benign (e.g., a tumor that does not form tumor metastases and does not destroy adjacent normal tissue) or malignant / cancerous (e.g., a tumor that is usually capable of invading surrounding tissues and producing metastases, may recur after attempted removal, and is likely to result in the death of the host unless properly treated). See Steadman's Medical Dictionary, 28th Ed Williams & Wilkins, Baltimore, MD (2005).

[0027] The term "cancer" refers to abnormal cell proliferation, particularly carcinomas and cell tumors that are malignant in nature, including sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphatic cancers, etc. Examples of various types of cancer include, but are not limited to, lung cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, oral cancer, ovarian cancer, thyroid cancer, prostate cancer, uterine cancer, testicular cancer, neuroblastoma, squamous cell carcinoma of the head, neck, cervix and vagina, multiple myeloma, soft tissue and osteogenic sarcoma, colon cancer, liver cancer, renal cancer (e.g., RCC), pleural cancer, cervical cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, small intestine cancer, cancer of the central nervous system, skin cancer, choriocarcinoma; osteogenic sarcoma, fibrosarcoma, glioma, melanoma, etc.

[0028] When used in the context of "normal cells," the term "normal" is meant to refer to cells of a non-transformed phenotype or to indicate the morphology of non-transformed cells of the tissue type being examined (e.g., PBMCs). In some embodiments, as used herein, "normal samples" include non-tumor samples, such as saliva samples, skin samples, hair samples, etc. It should be noted that the methods of the present disclosure can be performed without the use of normal samples.

[0029] As used herein, the term "abnormality" generally refers to a state of a biological system that deviates from normal (e.g., wild type) to some degree. The abnormal state can occur at the physiological or molecular level. Representative examples include, for example, physiological conditions (diseases, pathologies) or genetic abnormalities (mutations, single nucleotide variations, copy number variations, gene fusions, indels, etc.). The pathology can be cancer or a precancerous pathology. The abnormal biological state can be associated with a degree of abnormality (e.g., a quantitative measure indicating the distance away from the normal state).

[0030] As used herein, "formalin-fixed, wax-embedded" or "formalin-fixed, paraffin-embedded" or "FFPE" tissue samples are broadly construed to refer to samples that have been fixed in formalin or an equivalent substance and embedded in wax, such as paraffin wax or an equivalent substance. FFPE tissues herein may be from any human, animal or plant source. An alternative to FFPE is a cryopreserved sample embedded in optimal cutting temperature compound (OCT).

[0031] A "slide" herein may be any type of surface capable of holding a sample or specimen, such as FFPE tissue, for analysis and may be made from any suitable material.

[0032] A "region of interest" or "ROI" herein refers to a portion of a sample on a substrate that a user may wish to analyze, e.g., evaluate changes in the sequence, structure, or expression levels of a gene. A sample on a substrate may consist of all ROIs, no ROIs, one ROI, or more than one ROI. A ROI is also referred to herein as an "S" sample or region. A "region of non-interest" or "RONI" sample on a substrate is referred to herein as an "X" sample or region. Macrodissection techniques, which involve the preparation of tissue slices of the region of interest as well as the surrounding tissue of the organ under study, are increasingly employed in many pathological studies, e.g., tumor classification, diagnosis of inflammatory diseases, and determination of degenerative diseases. In macrodissection, the patient tissue of interest (the "S" region) is taken from a histological sample, e.g., a sample on a substrate such as a glass slide, and the unwanted regions (the "X" region) are excluded, and only the "S" region is used as input material for downstream assays. In some cases, the definition of a complex "S" shape by the pathologist can result in a difficult peeling operation for the tissue technician. In some embodiments, the sample is a liquid biopsy sample, such as a blood sample. In such embodiments, the region of interest may be a liquid biopsy droplet (e.g., a blood droplet) that contains a cell of interest, and the region of non-interest may be a liquid biopsy droplet (e.g., a blood droplet) that does not contain a cell of interest.

[0033] As used herein, the term "particulate" material refers to material composed of particles, such as substantially spherical particles or particles of less than irregular shape. Typically, the particulate material has a diameter of about 10 nm to about 100 μm, preferably about 50 to about 400 nm, in particular about 100 to about 200 nm.

[0034] As used herein, the term "assay" is a test or examination for the amount, presence, or absence of a substance.

[0035] As used herein, the term "pressurized" air means air that has been compressed, for example at a pressure greater than atmospheric pressure. The "air" component in such compressed air is typically an inert gas selected from helium, argon, xenon, nitrogen, carbon dioxide, oxygen, or mixtures thereof. As is common in pressurized systems, the "air" component may be in liquid, semi-liquid, or gas form.

[0036] As used herein, "contacting" means that a composition containing a drug (e.g., a contacting medium) is introduced into a target, e.g., a sample containing a cellular target, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary tube, etc., and incubated at a temperature and for a time sufficient to allow interaction between the target and the drug.

[0037] In the context of in vivo diagnosis or treatment, "contacting" means introducing an active ingredient (e.g., a compound or drug) into a subject and allowing the active ingredient to come into contact with a target tissue (e.g., an epithelial tissue) of the subject in vivo.

[0038] As used herein, the term "subject" refers to any animal, including animals commonly used in clinical research, preferably a mammal such as a human, a veterinary or farm animal, a livestock animal, or a pet. In some embodiments, the subject is a human subject. In some embodiments, the subject is a human patient diagnosed with a disease, such as cancer. The subject may have, potentially have, or be suspected of having one or more characteristics or symptoms associated with the disease. The subject may be asymptomatic with respect to the disease, or may be undiagnosed with the disease. In some embodiments, the subject may have cancer. In some embodiments, the subject may exhibit a symptom(s) associated with cancer. In some embodiments, the subject may be free of symptoms associated with cancer, or the subject may not have been diagnosed with cancer.

[0039] As used herein, the term "noise" in its broadest sense refers to any disturbance (e.g., a signal not directly related to a true event) that is unwanted but may be processed or received as a true event. Noise is the sum of unwanted or disturbing energy introduced into a system from artificial and natural sources. Noise can distort a signal in such a way that it degrades or makes unreliable the information carried by the signal. This term is in contrast to a "signal," which is a function that conveys information about the behavior or attributes of a phenomenon, e.g., a probabilistic association between a marker (SNV, CNV, indel, SV) and a disease such as cancer.

[0040] As used herein, the term "estimate" in the context of marker levels is used broadly. As such, the term "estimate" may refer to an actual value (e.g., a variation of 1 per mbp DNA), a range of values, a statistical value (e.g., an average, a median, etc.), or other means of estimation (e.g., by using probability theory).

[0041] As used herein, the term "substantially" means sufficient to function for the intended purpose. Thus, the term "substantially" allows for minor, slight variations from absolute or perfect conditions, dimensions, measurements, results, etc., that would be expected by one of ordinary skill in the art, but that do not appreciably affect overall performance. When used in reference to a number or a parameter or characteristic that can be expressed as a number, "substantially" means within 10 percent.

[0042] As used herein, the term "component" refers to a component of a system. For example, in a cellular system, components can include polypeptides (e.g., small peptides and large proteins), nucleic acids (e.g., DNA or RNA), carbohydrates (e.g., simple sugars and macromolecules such as starch), lipids, and other components such as vitamins and cholesterol.

[0043] As used herein, the term "transfer" or "transferring" is used in its broadest sense to refer to any act or method of moving or copying a marking from one entity to another, or converting a marking from one form to another. In some embodiments, the term "transferring" refers to moving or copying a marking from one entity to another. In some embodiments, the term "transferring" refers to moving or copying a marking from one slide or an image thereof to another slide or an image thereof. For example, in some embodiments, "transferring" refers to moving or copying a marking from a stained slide or an image thereof to an unstained slide or an image thereof. In some embodiments, "transferring" refers to moving or copying a digital marking from an image of a stained slide to an image of an unstained slide. In some embodiments, "transferring" refers to converting a digital marking from an image of a stained slide to a marking on an unstained slide, such as a physical or manual marking on an unstained slide. In some embodiments, "transferring" refers to converting a marking from one form (e.g., a digital marking) to another form (e.g., coordinates). For example, in some embodiments, "transferring" refers to converting a digital marking on a slide or an image thereof into coordinates. As used herein, the term "covalent" interactions includes the sharing of electrons between bonded atoms. In contrast, "non-covalent" interactions can include, for example, ionic interactions, electrostatic interactions, hydrogen bonding interactions, physicochemical interactions, van der Waals forces, Lewis acid / Lewis base interactions, or combinations thereof.

[0044] As used herein, the term "analyte" generally refers to the target molecule(s) detected using the methods or systems disclosed herein. The analyte can be a DNA analyte, an RNA analyte, a nucleic acid analyte, a macromolecule, or a small molecule, as these terms are used in the art. In particular, the macromolecule can include, for example, a polynucleotide, a polypeptide, a carbohydrate, a lipid, or a combination of one or more of these. As a general rule, the molecular weight of a macromolecule is at least about 300 Daltons and can be several million Daltons. A small molecule is an organic compound with a molecular weight of up to about 300 Daltons. In a particular example, the analyte is a nucleic acid analyte.

[0045] As used herein, a "probe" is a substance, e.g., a molecule, that can recognize or be specifically recognized by a particular target. Potential probe / target or target / probe binding partner types include receptor / ligand; ligand / antiligand; nucleic acid (polynucleotide) interactions, including DNA / DNA, DNA / RNA, PNA (peptide nucleic acid) / nucleic acid; substrates, small molecules or effector molecules with enzymes, other catalysts, or other substances, and the like. Examples of probes contemplated by the present disclosure include, but are not limited to, peptides, enzymes (such as proteases or kinases), enzyme substrates, cofactors, drugs, lectins, sugars, nucleic acids (including oligonucleotides, DNA, RNA, PNA, or modified or substituted nucleic acids), oligosaccharides, proteins, enzymes, polyclonal and monoclonal antibodies, single-chain antibodies, or fragments thereof. Probe polymers can be linear or cyclic. Probes can distinguish between different targets, either by differences in activity, differences in binding, or identification from structural markers. Probes of the present disclosure are preferably nucleic acid molecules, particularly preferably DNA. In certain instances, a "probe" may function as a "target" and a "target" may function as a probe; for example, a complementary DNA (cDNA) may function as a probe that hybridizes to a portion of a target gene sequence, the mRNA product of which and the cDNA itself match, and therefore corresponds to the target sequence.

[0046] As used herein, the terms "analyzing" and "detecting" can refer to the qualitative or quantitative determination of a parameter of interest related to an analyte, such as the amount, level, concentration, or activity (both absolute and relative) of the analyte.

[0047] As used herein, the term "diagnosis" refers to a method that can determine whether a subject is likely to suffer from a given disease or condition. Those skilled in the art often make a diagnosis based on one or more diagnostic indicators (e.g., markers), the presence, absence, amount, or change in amount of the marker indicates the presence, severity, or absence of a disease or condition. Other diagnostic indicators can include patient history; physical symptoms, such as unexplained weight loss, fever, fatigue, pain, or skin abnormalities; phenotype; genotype; or environmental or genetic factors. Those skilled in the art will understand that the term "diagnosis" refers to an increase in the probability that a certain course or outcome will occur, i.e., an increase in the probability that a course or outcome is more likely to occur in a patient who exhibits a given characteristic, such as the presence or level of a diagnostic indicator, when compared to an individual who does not exhibit that characteristic. The diagnostic methods of the present disclosure can be used independently or in combination with other diagnostic methods to determine whether a course or outcome is more likely to occur in a patient who exhibits a given characteristic.

[0048] The term "nucleic acid" generally refers to DNA or RNA, whether it is the product of amplification, synthetic production, reverse transcription of RNA, or naturally occurring. Generally, nucleic acids are single-stranded or double-stranded molecules and are composed of naturally occurring nucleotides. A double-stranded nucleic acid molecule need not be or is not assumed to be completely double-stranded over its entire length, as it can have 3' or 5' overhangs. Furthermore, the term nucleic acid can be composed of non-naturally occurring nucleotides and / or modifications to naturally occurring nucleotides. Examples herein include, but are not limited to, phosphorylation of 5' or 3' nucleotides to allow ligation or blocking of exonuclease degradation / polymerase extension, respectively; amino-, thiol-, alkyne-, or biotinyl-modifications for covalent and quasi-covalent bonds; fluorophores and quenchers; phosphorothioate, methylphosphonate, phosphoramidite, and phosphorotiester linkages between nucleotides to block degradation; methylation; and modified bases.

[0049] As used herein, the term "polypeptide" is used interchangeably and means a peptide, protein, or polypeptide comprising a chain of amino acids of a given length, in which the amino acid residues are joined by covalent peptide bonds. The term polypeptide also refers to, but does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, protein processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0050] As used herein, the term "isolated" or "extracted" in the context of a molecule refers to a molecule that is substantially free of impurities. A molecule (e.g., DNA or RNA) is "isolated" or "extracted" when it is purified from other components in a sample. Purification refers to separating a target from one or more extraneous components that are also found in the sample. Components that are isolated, extracted or purified from a mixed specimen or sample are generally purified or enriched to at least 50%, at least 60%, at least 75%, at least 90%, or at least 98%, or even at least 99%, compared to a raw or unextracted sample.

[0051] The term "synthetic" refers to a molecule that is chemically synthesized using art-recognized techniques, for example, using phosphoramidite chemistry or synthetic chemistry.

[0052] In the context of nucleic acids, the term "hybrid" or "hybridize" is broadly meant to include not only double strands, but also molecules capable of forming such double strands. In this context, single stranded nucleic acids that base pair over multiple bases are said to "hybridize". Hybridization is generally determined under physiological or biological conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular: pH 7.4, 145 mM sodium ion).

[0053] As used herein, the term "analog" includes, but is not limited to, oligonucleotides that contain synthetically introduced residues or linkers therein, such as ribonucleic acid residues in a DNA sequence, branched linkers such as glycerol derivatives, or aminoalkyl linkers. "Adducts" include, for example, O6-alkyl-dG and O6-Me-dG. Similarly, in one embodiment, the term "conjugate" refers to a target recognition agent that is covalently or non-covalently attached to one or more polynucleotides. In another embodiment, the term "conjugate" refers to a linear, branched, or dendritic polynucleotide that is covalently or non-covalently attached to one or more fluorophores.

[0054] As used herein, "target" refers to a substance whose presence, activity and / or amount is desired to be determined and that has affinity for a given probe. Targets can be man-made or naturally occurring substances. They can also be used in their native state or as aggregates with other species. Targets can be attached, covalently or non-covalently bound to a binding member, either directly or via a specific binding substance. Examples of targets that can be used in the present disclosure include, but are not limited to, nucleic acids or polynucleotides (including mRNA, tRNA, rRNA, oligonucleotides, DNA, viral RNA or DNA, ESTs, cDNA, PCR amplification products derived from RNA or DNA, and mutations, variants or modifications thereof); proteins (including enzymes, such as those responsible for cleaving neurotransmitters, proteases, kinases, etc.); enzyme substrates; peptides; cofactors; lectins; sugars; polysaccharides; cells (which can include cell surface antigens); cell membranes; organelles, etc., as well as other such molecules or other such substances that can exist in the form of complexation, covalent binding, cross-linking, etc. Targets can also be referred to as anti-probes.

[0055] When a probe binds to a target sequence, its binding can be "specific" or "selective". In general, when a probe has one and only one binding partner (e.g., target), it has the property of "specificity". In fact, most probes are "selective" rather than "specific", since most probes bind to multiple targets, especially at high concentrations. Therefore, these terms are used interchangeably. The specificity and selectivity of binding can be determined using conventional methods. For example, if the target is a specific mRNA, the probe can be, for example, an oligonucleotide that specifically binds to the target but does not bind to interfering RNA or DNA under selected hybridization conditions. Those skilled in the art can use art-recognized methods to experimentally determine the characteristics of an oligonucleotide that hybridizes optimally to a target with minimal hybridization to non-specific interfering DNA or RNA (see, for example, above). In general, the length of the oligonucleotide probe used to distinguish target mRNA present in a background of a large excess of non-target RNA can range from about 8 to about 50 nucleotides in length, preferably about 18, 20, 22 or 25 nucleotides in length. The oligonucleotide probe can be shorter than 8 nucleotides when used in biochemical assays where the background of competing targets is not large. Using art-recognized procedures (e.g., the computer program BLAST), the sequences of the oligonucleotide probes can be selected to be unrelated to each other and different from potentially interfering sequences in known gene databases. Selection of hybridization conditions that allow specific hybridization of the oligonucleotide probe to the RNA target can be routinely determined using art-recognized procedures.

[0056] As used herein, the term "primer" refers to a short nucleic acid molecule, such as a DNA oligonucleotide, that contains 9 or more nucleotides, and in some instances is used to initiate the synthesis of a longer nucleic acid sequence. Longer primers can be about 10, 12, 15, 20, 25, 30, or 50 or more nucleotides in length. Primers can also be used for detection.

[0057] As used herein, "mechanical removal or ablation" of a particular sample from a substrate means either separating the sample from the substrate or vaporizing or otherwise decomposing the sample by mechanical means such that the sample is no longer present on the substrate.

[0058] As used herein, chemically "degrading" a macromolecule means denaturing or disrupting a macromolecule, such as RNA, DNA, and / or protein, or sufficiently chemically modifying the macromolecule so that it does not contaminate subsequent analysis of the RNA, DNA, and / or protein in the ROI tissue.

[0059] A "mark" made on a slide by a pathologist or laboratory user or other individual is referred to herein as a "manual mark." Such marks may be made by any available means, for example, by a physical or virtual pen or etching device. In contrast, marks or features made automatically by the systems herein may be referred to as "virtual marks" or "digital marks" or "digital features" to indicate that they are made not manually but through the use of one or more algorithms.

[0060] Terms such as "digital," "digitized," "automated," and "automatically" refer to actions performed by the systems herein, e.g., actions controlled by an algorithm and / or by interaction between a user and a computer user interface, as opposed to actions performed by a user without the assistance of an algorithm, computer, computer user interface, etc. In contrast, the term "manual" or "manually" refers to actions performed by a user / operator. In some embodiments, a "manual" action may include an operator directing an application, such as software, firmware, or hardware, to perform / implement a task. The terms "user" and "operator" are used interchangeably herein to refer to a human being. In some embodiments, a "user" or "operator" refers to a human being using a component of a system or performing a portion of a method described herein, including a histologist, pathologist, etc.

[0061] As used herein, the term "surface" refers to any material that provides sites that allow interaction between analytes or probes of interest. Preferably, the surface is the surface of a solid support (e.g., nitrocellulose), the walls of a well in a reaction tray, a multi-well plate, a test tube, polystyrene beads, magnetic beads, a membrane, and a microparticle (e.g., latex particle). Any suitable porous material with sufficient porosity to allow access by detector reagents and suitable surface affinity for immobilizing capture reagents (e.g., oligonucleotides) is contemplated by this term. For example, the porous structure of nitrocellulose has excellent absorption and adsorption properties for a wide variety of reagents, such as capture reagents. Nylon has similar properties and is suitable. Microporous structures are useful as are materials with gel structures in the hydrated state.Further examples of useful solid supports include natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters, especially those with nitric and carboxylic acids, mixed cellulose esters, and cellulose ethers; nitrogen-containing natural polymers, such as proteins and derivatives, including cross-linked or modified gelatin; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared in suitable porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and their partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers of the above polycondensates and terpolymers. Polymers such as polyesters, polyamides, and other polymers such as polyurethanes or polyepoxides; porous inorganic materials such as barium sulfate, calcium sulfate, calcium carbonate, alkali and alkaline earth metal silicates, alkaline earth metal and magnesium sulfates or carbonates, including aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolites, silica gel, or glass, which can be used as filters in conjunction with the above polymeric materials; as well as mixtures or copolymers of the above classes, such as graft copolymers obtained by initiating the polymerization of a synthetic polymer onto an existing natural polymer.

[0062] As used herein, the term "signature" refers to a set of markers that indicate a phenotype of interest, for example, a cancer signature that includes ≧3 mutations that indicate that the cell or tissue that harbors the mutations is a tumor cell. In some embodiments, the signature includes the presence, absence, and / or abundance of a combination of markers, for example, tumor markers. By combining various probe sets, a reliable method can be designed for detecting the phenotype of interest. Such a signature test performed as a single assay can provide great advantages for evaluating and understanding the interactions between various markers.

[0063] The term "amplification" generally refers to the production of multiple nucleic acid molecules from a target nucleic acid in which primers hybridize to specific sites on the target nucleic acid molecule to provide initiation sites for extension by a polymerase. Amplification can be performed by any method commonly known in the art, including, but not limited to, standard PCR, long PCR, hot start PCR, qPCR, RT-PCR, and real-time PCR.

[0064] As used herein, the term "antibody" refers to an intact immunoglobulin such as IgA, IgD, IgE, IgG or IgM, or a fragment of an antibody (particularly an antigen-binding fragment) such as a Fab, Fv or Fc, or a fusion antibody, a fusion antibody fragment or any other derivative of an antibody. The term "labeled antibody" refers to an antibody that is labeled with an enzyme, a fluorochrome, a chemiluminescent substance, biotin, avidin or a radioisotope.

[0065] The term "epitope" refers to an antigenic region of a compound such as a protein, carbohydrate, or lipid. An antigenic region generally consists of 5 to 8 amino acids. An epitope is specifically recognized by the antigen-binding site of a respective antibody.

[0066] The term "fixed tissue or cells" is used herein as known to those of skill in the art to refer to biological tissues or cells that are preserved from decay by chemical fixation methods. Such methods prevent autolysis or putrefaction within such biological tissues or cells. Fixation terminates biochemical reactions and increases the mechanical stability of the treated tissue.

[0067] The term "immunohistochemistry" or "IHC" refers to a technique for detecting the presence of an antigen in a histological sample by means of an antibody capable of specifically binding to that antigen. Detection of the antibody-antigen complex is usually performed by a color reaction with an enzyme-labeled antibody or by a fluorescently labeled antibody.

[0068] As used herein, the term "macrodissection" refers to the process of removing an area of ​​interest from a tissue section mounted on a solid support, such as a microscope slide, using a tool such as a scalpel, blade or spatula. As used herein, the term "microdissection" refers to the process of cutting and isolating one or more specific cells or areas of interest from a tissue sample. Microdissection can be performed, for example, by cutting the relevant area with a laser using laser capture microdissection (LCM).

[0069] As used herein, the term "membrane slide" refers to a solid support or microscope slide for use in laser capture microdissection (LCM). For microdissection, a glass slide covered by a membrane or a frame slide, consisting of a metal frame that can be covered with various membranes, can be used.

[0070] The term "polylysine" refers to a molecule that contains up to several hundred repeating units and is suitable for improving the affinity between a sample, such as a tissue section, and the membrane side to which the sample is attached. A polylysine according to the present invention is poly-L-lysine. A poly-L-lysine according to the present invention has a molecular weight of 70 to 300 kDa. Poly-L-lysine can be digested by proteases. Another polylysine according to the present invention is, for example, poly-D-lysine. A poly-D-lysine according to the present invention has a molecular weight of 70 to 300 kDa. Poly-D-lysine is resistant to digestion by proteases.

[0071] The term "qPCR" generally refers to a PCR technique known as real-time quantitative polymerase chain reaction, quantitative polymerase chain reaction, or kinetic polymerase chain reaction, which uses PCR to simultaneously amplify and quantify a target nucleic acid, with quantification being by intercalating a fluorescent dye or sequence-specific probe that contains a fluorescent reporter molecule that is detectable only when hybridized to the target nucleic acid.

[0072] The term "RNA" is used herein as known to those of skill in the art and refers to pre-mRNA, pre-mRNA transcripts, mRNA, transcript processing intermediates, mature mRNA used for translation and transcription from one or more genes, or nucleic acids derived therefrom. Transcript processing includes processes such as splicing, editing, modification, and degradation. mRNA containing samples include, for example, mRNA of one or more genes, mRNA transcripts, cDNA derived from mRNA using reverse transcription, RNA transcribed from amplified DNA, cRNA transcribed from cDNA, DNA amplified from genes, and the like.

[0073] A "container" that may contain a sample herein is broadly construed to mean any type, shape, or size of container, including a surface, well, tube, or vial. A container need not have any particular shape or size, made from any specific material, but simply function as a physical structure that allows for the analysis or manipulation of tissue located therein or thereon.

[0074] A "stained" slide / substrate, as used herein, refers to a substrate housing a sample that has been treated to aid in revealing differences between regions within the sample. In some embodiments, the sample is treated before being placed on the slide or otherwise disposed on it. In some embodiments, the sample is treated after being placed on the slide or otherwise disposed on it. References to a "stained" slide or a slide that has been "treated" are used herein to describe both of these examples, i.e., a slide housing tissue that has been treated before being placed on the slide, and a slide housing tissue that is treated after being placed on the slide. In some embodiments, a "stained" slide refers to a slide that has been treated to aid in revealing differences between regions of interest and regions of non-interest within the sample, such that a pathologist or other trained individual can mark the substrate to outline any ROIs on the substrate. In some embodiments, a "stained" slide is treated with a dye. Suitable dyes include, for example, hematoxylin, a basic dye that stains acidic structures resulting in a purple / blue hue, and eosin, which stains structures with a pink / red hue. Further exemplary stains include, but are not limited to, Giemsa stain, Periodic Acid Schiff reaction (PAS) stain, trichome stain, Congo red, Prussian blue, mucicarmine, Sudan black, Oil Red O, Nissl stain (also known as cresyl violet stain), and Papanicolaou stain. In some embodiments, the "stained" slide is treated with an ion. For example, in some embodiments, the "stained" slide is a silver stain slide. In some embodiments, the "stained" slide is an immunohistochemistry stain slide (e.g., a slide containing tissue that has undergone an immunohistochemistry procedure). For example, the "stained" slide can be contacted with an antibody to detect an antigen, if present in the sample. In some embodiments, the "stained" slide is treated with light to measure one or more properties of the sample on the slide. For example, the slide can be treated with light to measure one or more optical properties of the sample, including light scattering properties.An "unstained" slide / substrate is one that has not been treated in this manner, but which may or may not have been subjected to other types of treatment. Thus, an "unstained" slide / substrate may be one that has not been treated with dye or light.

[0075] As used herein, the term "separating" refers to any suitable means for isolating a particular region from a sample. In some embodiments, "separating" refers to isolating one or more regions of interest from a sample. In some embodiments, "separating" refers to isolating one or more regions of non-interest from a sample, leaving the regions of interest on a substrate. Suitable methods for "separating" include physical removal (e.g., cutting and removing), ablation (e.g., vaporization, ablation, or physical disintegration) of the regions, or chemical treatment of the regions. In some embodiments, "separating" refers to dissection performed on a tissue sample. Such dissection (e.g., macrodissection, microdissection) may be performed to remove one or more regions of interest or may be performed to remove one or more regions of non-interest. In some embodiments, "separating" refers to isolating one or more droplets present in a liquid sample (e.g., a blood sample). For example, "separating" may refer to isolating one or more droplets of a liquid sample that contain cells of interest. As another example, "separating" may refer to isolating one or more droplets of a liquid sample that do not contain the cells of interest, thereby leaving only the droplets containing the cells of interest on the substrate for further evaluation.

[0076] As used herein, the term "substrate" refers to a variety of slides, including, but not limited to, FFPE slides, tissue slides, standard slides, containers, stained slides, unstained slides, biological tissues, cell layers attached to glass or polymer containers, etc. In some embodiments, the substrate is a slide that contains a liquid biopsy sample (e.g., a blood sample).

[0077] A "computer processor" or "computing means" or "computer" is broadly construed herein to refer to any combination of hardware and / or software that performs the necessary functions. For example, the processor may be a programmable digital microprocessor, such as those available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). If the processor is programmable, the appropriate programming can be communicated from a user interface that is either built into the computer body or remote to the processor, or can be pre-stored in a computer program product (e.g., a portable or fixed computer-readable storage medium, whether magnetic, optical, or solid-state device based). For example, a magnetic medium or optical disk can carry the programming and be read by a suitable reader that communicates with each processor at its corresponding user interface. A "user interface" is broadly construed herein to mean a physical structure that allows a user to program the computer and thereby control certain operations of the system through the computer. Examples include the keyboard and monitor of a mainframe or laptop computer, other types of visual monitor and keyboard systems, such as pad-type or smartphone-type devices or other remote devices. The user interface may be either a physical part of the computer body, located elsewhere in the system, or located remotely from the computer, and may communicate with the computer processor via a wired or wireless connection.

[0078] Current processes for transferring markings from a stained slide to an unstained slide may rely on a fully manual physical alignment of the two slides. However, this physical alignment of the two slides can be difficult for a variety of reasons. For example, the tissue from the unstained slide may not be fully secured to the slide, so the tissue may slide, fold, stretch, or otherwise move on the slide during or before attempting physical alignment. Additionally, in some cases, the slides may have one or more labels (e.g., stickers) that can result in an uneven slide surface that can make manual alignment of the slides difficult. Visual and manual handling of the slides can be ergonomically challenging for the user (e.g., a histologist), leading to health issues such as carpal tunnel syndrome or eye strain. Additionally, the process of fully manual alignment of two slides (e.g., a stained slide and an unstained slide) is time consuming and is often limited by the number of slides that a user (e.g., a histologist) can successfully align within a given day. Furthermore, pen markings can become invisible over time and are difficult to correct if an error is noticed after the marking has been made. Therefore, a method of digitally marking an image of a slide, rather than physically marking the slide itself, is desirable.

[0079] Digital pathology annotation methods exist, but these methods require automatic alignment between two digital images. Such alignment is also called registration. Current image registration processes rely on algorithms that are prone to glitches and have not achieved anywhere near 100% success rates. Algorithms such as these often rely on pixel-by-pixel recognition and automatic alignment of two images based on image landmarks. However, image landmarks may move, shift, or look different in different slides based on tissue slicing, mounting, and staining, leading to poor registration. For example, in some cases, different slices from a tissue may stretch, fold, or otherwise deform during collection and / or mounting of the tissue on a slide. Such stretching or folding may result in poor image registration. As another example, some tissues contain a large amount of fat, which may be transparent in stained slides but not in unstained slides. Such differences in tissue opacity may also result in poor algorithm-based image registration. Examples of poor algorithm-based image registration processes are sometimes referred to as "fringe" cases. When an image registration algorithm is poor, it cannot successfully transfer the markings from the stained slide to the unstained slide or the image of the unstained slide. It is difficult to improve the success rate of the image registration algorithm.

[0080] The systems and methods described herein address the problems discussed above.

[0081] In some aspects, provided herein are systems, tools, and methods for transferring pathologist markings. In some embodiments, provided herein are systems and methods for physically marking slides. In some embodiments, provided herein are systems and methods for digitally marking slides. In some embodiments, the systems and methods described herein can be used for both physical and digital marking of slides. The systems and methods described herein are advantageous in that the methods are performed at least in part manually, without the need for error-prone image registration algorithms. Furthermore, the systems and methods described herein are advantageous in that the methods are performed at least in part digitally, using images of stained or unstained slides, thereby avoiding the need for purely manual transfer of markings from one physical slide to another.

[0082] In some aspects, the present disclosure of the present application is directed to systems, tools, and methods for transferring pathologist markings, e.g., suitable for histology and / or pathobiology. The methods and approaches disclosed herein can improve reliability and consistency in creating, converting, and / or transferring markings for use during automated downstream analysis or processing of biological samples. In general, sample analysis and dissection typically involves a series of slides (e.g., glass slides or microscope slides). In some embodiments, the series of slides includes parallel slices of a biological specimen (sample). In some embodiments, the slides include a liquid biological sample (e.g., a blood sample).

[0083] In some embodiments, one or more slides in the set may be stained, for example, to reveal individual cell nuclei and / or to aid in distinguishing between different types of cells, such as for oncology applications, cancer and non-cancer cells. The pathologist can inspect the substrate and mark regions of interest (ROIs) on the slide with a pen or other suitable marking device. In some embodiments, the markings are placed on a cover slide over the sample. In some embodiments, the markings are placed under the slide, such as for liquid biopsy samples that may not include a cover slide. These ROIs or associated pen markings can then map (rotate and align) the substrate from one or more adjacent slice(s) of the sample, which are ultimately analyzed to, for example, extract DNA for genomic sequencing, RNA for RNA expression analysis, or perform in situ analysis of cells, and so forth. In traditional sample dissection methods, the pathologist's pen marks are transferred by hand to the substrate, and a razor blade cuts out the ROI from the surrounding sample on the slide.

[0084] According to various embodiments described herein, a tool or system can be implemented for transferring markings, such as pathologist markings, either physically or digitally. In some embodiments, provided herein is a system for transferring markings, physically and / or digitally. In some embodiments, the system includes an imaging device for imaging the stained slides, and possibly the unstained slides. In some embodiments, the system also includes a display for displaying one or more images taken by the imaging device. Additionally, the system can include a mechanical stage, such as a scaffold, for positioning or orienting the stained slide relative to the unstained slide. In some embodiments, the mechanical stage is manually positioned. For example, the stage can be moved (e.g., raised, lowered, shifted, rotated) to position or orient the stained slide relative to the unstained slide. The stage can be moved manually directly by an operator. Alternatively, the movement of the stage can be controlled by an associated application, such as software, hardware, etc., but the operator can command the associated application to position the stage in an appropriate position determined by the operator. The term "manual" or "manually" when used with respect to positioning, movement, or manipulation of a platform / stage refers to each of these instances, i.e., direct positioning / movement / manipulation of the stage by an operator, and / or the operator directing an associated application to position / movement / manipulate the stage in a manner suitable to facilitate the desired alignment of the slide. This allows for physical marking of an unstained slide based on the markings on the stained slide. In some cases, an application, such as a custom-designed software program, can be used to digitally depict one or more features based on the physical markings and convert the physical markings into coordinates that can be used for further processing, such as dissection.

[0085] According to various embodiments, a tool or system for transferring pathologist markings can be designed. In some embodiments, provided herein is a system for transferring pathologist markings. In some embodiments, the system includes an imager for imaging stained and unstained slides, and a display for displaying one or more images of the stained and unstained slides. In some embodiments, the system is coupled to a first application for aligning the images to enable digital marking of the unstained slides, and a second application for digitally marking one or more features and converting the digital markings into coordinates. In some embodiments, the coordinates are to be used for further processing, for example, dissection, or isolation of one or more droplets from a liquid biopsy sample (e.g., a blood sample). In various embodiments, the first application can be used for semi-manual processing or semi-automation, while the second application can be used via algorithm-based processing. For example, in some embodiments, the first application is a software program operated by a user (e.g., an operator). An operator manually aligns the images using visual cues and utilizes a software application to align the images (e.g., shift, move, zoom, pan, rotate, mirror, etc.). The software program can track the amount of motion that occurs and store these motions as coordinates. Coordinates such as these can be used for further processing. For example, coordinates such as these can be applied to an image of an unstained slide or the unstained slide itself to mark the image of the unstained slide, to mark the unstained slide, and / or to assist in dissection of tissue from the unstained slide. Such a system / method is in contrast to methods that are algorithm-based, in which the operator does not need to perform manual alignment of the images.

[0086] For any of the embodiments described herein, a "stained" slide refers to a substrate that contains a sample that has been treated to help reveal differences between regions within the sample. In some embodiments, the "stained" slide is treated with a dye. Suitable dyes include, for example, hematoxylin, a basic dye that stains acidic structures resulting in a purple / blue hue, and eosin, which stains structures with a pink / red hue. Further exemplary stains include, but are not limited to, Giemsa stain, Periodic Acid Schiff reaction (PAS) stain, trichome stain, Congo red, Prussian blue, mucicarmine, Sudan black, Oil Red O, Nissl stain (also known as cresyl violet stain), and Papanicolaou stain. In some embodiments, the "stained" slide is treated with ions. For example, in some embodiments, the "stained" slide is a silver stain slide. In some embodiments, the "stained" slide is an immunohistochemistry stain slide (e.g., a slide containing tissue that has undergone an immunohistochemistry procedure).

[0087] In some embodiments, the "stained" slide is treated with light to measure one or more properties of the sample on the slide. For example, the slide can be treated with light and one or more optical properties of the sample can be measured, including light scattering properties. Exemplary light scattering properties that can be measured include, for example, wavelength-dependent light scattering (e.g., using elastic scattering spectroscopy, light scattering spectroscopy, diffuse reflectance modeling, spectroscopic optical coherence tomography (OCT), dark-field spectral scattering imaging, partial wave spectroscopy), angle-resolved light scattering, or Fourier domain methods. In contrast to the above, an "unstained" slide / substrate is a substrate that has not been treated in this manner, but may or may not have been subjected to other types of treatment. Thus, an "unstained" slide / substrate may be one that has not been treated with dye or light.

[0088] Further details are provided below with respect to Figures 1-6.

[0089] FIG. 1 is a schematic diagram of a system 100 for transferring markings, according to various embodiments. As shown in FIG. 1, the system 100 includes an intake stage 120, a manipulation stage 140, an output stage 160, and an optional auxiliary stage 180. As shown, the intake stage 120 represents a stained slide having a pathologist marking and / or a pre-marking on a sample (e.g., a biological specimen). In various embodiments, the stained slide can include a hematoxylin and eosin stained slide or an immunohistochemistry stained slide, among others. In various embodiments, an image of the stained slide having the markings can be captured via an imaging device (also referred to herein as an "electron microscope"), such as a microscope or optical microscope connected to a camera, such as a digital camera (CCD or CMOS camera) or a video camera, with the ability to zoom, pan, and / or focus in two- and three-dimensional space.

[0090] Upon input of the stained slide with pathologist markings or pre-markings to the manipulation stage 140, the manipulation stage 140 represents a manipulation process that includes at least marking, converting and / or transferring the markings onto the unstained slide. In various embodiments, the pre-markings can be used as a guideline when transferring the physical markings onto the unstained slide. In various embodiments, a software application can be used to recognize the physical markings and convert them into one or more features, e.g., digital features, that need to be transferred to the unstained slide. In various embodiments, the software application can be a metrology software program used to inspect the physical properties of the stained or unstained slides. In various embodiments, the physical markings can be one or more dots, straight or curved lines, or polylines that can be drawn by an operator on a display or projector or directly on the unstained slide. Such physical markings can be converted into one or more digital features. Thus, the digital features can be one or more dots, curved lines, or polylines.

[0091] During the manipulation stage 140, a software application can be used to align one or more images of stained or unstained slides. In various embodiments, alignment using the application can include positioning and orienting the images as well as performing one or more of the following operations: panning, rotating, zooming in or out (generally referred to herein as "zooming"), mirroring, or overlaying in any direction (e.g., two or three dimensions) of the images. In some embodiments, aligning one or more images of stained or unstained slides is performed by an operator using a software application. For example, an operator may manually align one or more images based on visual cues in each image. However, the alignment itself (e.g., moving, shifting, rotating the images) is performed by the operator using a software application, which induces the desired movement selected by the operator in the images of the slides. This is in contrast to other methods of aligning images of stained and unstained slides that are fully automated (e.g., performed using an algorithm rather than manually performed by an operator with the assistance of a software application).

[0092] In various embodiments, the second software application can be used to digitally mark one or more features and convert the digital marking of one or more features to coordinates. In various embodiments, a low-resolution image of the stained slide can be used to recognize the physical markings and convert the physical markings to features. Low resolution refers to an image taken at a magnification of 20X (i.e., 20 times) or less. For example, a low-resolution image is an image taken at a magnification of 2X, 3X, 5X, 7X, 10X, 15X, or 20X or less. In various embodiments, one or more boundary edges or corners of the low-resolution image can be used in the recognition of the physical markings and conversion of the physical markings to one or more features.

[0093] 1, the output of the unstained slide onto which the markings have been transferred represents stage 160. Once the markings have been transferred onto the unstained slide, the sample can be further processed during one or more downstream processes (e.g., dissection), represented by stage 180.

[0094] FIG. 2 illustrates an exemplary embodiment 240 of a system for transferring a marking, according to various embodiments. In the embodiment 240 illustrated in FIG. 2, a stained slide 210 having a marking 220 is shown aligned with an unstained slide 250, as indicated by the sign "+", resulting in an overlay of the two slides 210 and 250. In various embodiments, an image of the stained slide 210 can be used instead of the stained slide 210. In various embodiments, an image of the unstained slide 250 can be used instead of the unstained slide 250. As illustrated on the right side of FIG. 2, this overlay shows the unstained slide 250 rotated and shifted upwards relative to the stained slide 210 to align the marking 220. In some embodiments, such alignment (e.g., rotation and shifting to align the marking 220) is performed manually by an operator based on visual cues in the image, rather than using a fully automated method (e.g., rather than using an algorithm).

[0095] FIG. 3 illustrates another exemplary embodiment 340 of a system for transferring a marking, according to various embodiments. In the embodiment 340 illustrated in FIG. 3, an image of a stained slide 310 is shown having a digital marking 320 disposed thereon. The digital marking 320 is then copied from the image of the stained slide 310 and pasted onto the image of an unstained slide 350. Once pasted onto the image of the unstained slide 350, the digital marking 320 can be manipulated (e.g., positioned, oriented, panned, rotated, zoomed, mirrored, etc.). After manipulation, the digital marking 320 is considered a marking 360 transferred onto the image of the unstained slide 350. FIG. 3 also illustrates a case where a fully automated alignment (e.g., an algorithm-based alignment) cannot properly align the two images. In this case, the digital marking 320, with the image of the stained slide 310 disposed thereon, would be improperly transferred to the image of the unstained slide 350 if relying solely on the algorithm-based alignment. This is illustrated in the top right image of FIG. 3. However, the digital transfer of the markings 360 on the image of the unstained slide 350 is much more accurate when it is performed by an operator manually aligning the images based on visual cues. This is shown in the bottom image of FIG. 3. A further example of the inability of fully automatic alignment is illustrated in FIG. 7. As shown in the figure, a first slice (shown in the light rectangle on the left) is expected to overlap with a successive slice (dark rectangle) with a moderate degree of slice-to-slice variation. Such expected variation is shown in the graph on the top right. However, if a large variation occurs between the first and second slice, for example a slice with a large rotation is combined with a slice with a large translation, image registration becomes impossible using algorithm-based methods.In contrast, the methods described herein are advantageous in that such large variations between slices can be taken into account by an operator based on visual inspection of successive slides and appropriately handled to facilitate transfer or marking from one slide or image thereof to a second slide or image thereof.

[0096] 4 is a flow chart of an exemplary method S100 for transferring markings, according to various embodiments. Method S100 includes projecting an image of a stained slide with pathologist markings on a display in step S102, placing an unstained slide on a scaffold above the display in step S104, aligning the unstained slide with the projected image of the stained slide by moving the scaffold in step S106, and physically transferring the pathologist markings onto the unstained slide in step 108. Step S106 is performed manually by an operator based on visual cues in the stained slide and the unstained slide.

[0097] In various embodiments, the stained slide further comprises one or more pre-markings. In various embodiments, the one or more pre-markings are used as a guideline during the physical transfer of the pathologist markings onto the unstained slide. In various embodiments, the physical transfer of the pathologist markings onto the unstained slide comprises depicting one or more features or markings on the unstained slide. In various embodiments, the one or more features comprise one or more dots, straight or curved lines, or polylines. In various embodiments, the projected image of the stained slide is panned, rotated, zoomed, or overlaid to aid in alignment with the unstained slide. In various embodiments, the projected image of the stained slide is flipped to aid in alignment with the unstained slide. In some embodiments, such movements (panning, rotating, zooming, overlaying, flipping, etc.) are performed manually by an operator rather than using a fully automated algorithm.

[0098] In various embodiments, the method S100 can optionally include recognizing the pathologist markings via an application in step S110, optionally converting the pathologist markings into one or more features in step S112, and optionally transferring the one or more features onto an unstained slide in step S114.

[0099] In various embodiments, the low-resolution image of the stained slide is used in recognizing the pathologist markings and converting the pathologist markings into one or more features. In various embodiments, one or more boundary edges or corners of the low-resolution image are used in recognizing the pathologist markings and converting the pathologist markings into one or more features. In various embodiments, projecting the image of the stained slide onto the display is performed from behind, above, or to the side of the stained slide. In various embodiments, the transferred pathologist markings are used to peel off a portion of the biological sample. In various embodiments, the stained slide comprises a hematoxylin and eosin stained slide or an immunohistochemistry stained slide.

[0100] 5 is a flow chart of an exemplary method S200 for transferring markings according to various embodiments. The method S200 includes acquiring an image of a stained slide under an imager in step S202, projecting the image of the stained slide on a display in step S204, marking the image of the stained slide in step S206, transferring the markings of the stained slide to an unstained slide in step S208, and capturing the marked unstained slide with the markings transferred in step S210.

[0101] In various embodiments, the stained slide includes one or more pre-markings. In various embodiments, marking the image of the stained slide includes using the one or more pre-markings as a reference guideline. In various embodiments, marking the image of the stained slide includes depicting one or more features on the display. In various embodiments, marking the image of the stained slide includes depicting one or more dots, straight or curved lines, or polylines on the display, recognizing the depicted one or more dots or lines or depicted polylines via an application, and converting the recognized one or more dots or lines or recognized polylines into one or more digital features.

[0102] In various embodiments, the image of the stained slide is a low-resolution image of the stained slide, and the low-resolution image is used in the recognition of the one or more dots or lines or polylines and in the conversion of the one or more dots or lines or polylines into one or more digital features. In various embodiments, one or more boundary edges or corners of the low-resolution image are used in the recognition of the one or more dots or lines or polylines and in the conversion of the one or more dots or lines or polylines into one or more features.

[0103] In some embodiments, transferring the marking of the stained slide to the unstained slide includes overlaying the unstained slide on the stained slide, reorienting the unstained slide to align and overlay one or more digital features on the unstained slide, and marking the unstained slide. In some embodiments, the unstained slide is overlaid on the stained slide and manually (e.g., by an operator) reoriented to align and overlay one or more features. In various embodiments, transferring the marking of the stained slide to the unstained slide includes overlaying the unstained slide on the stained slide with the marking, reorienting the unstained slide to align and overlay one or more digital features of the marking of the stained slide on the unstained slide, and marking the unstained slide with a marking of a physical tissue feature of the stained slide. In some embodiments, the unstained slide is overlaid on the stained slide and manually (e.g., by an operator) reoriented to align and overlay one or more features.

[0104] In various embodiments, an image of the unstained slide is used in place of the unstained slide during transfer of the stained slide markings to the unstained slide. In some embodiments, the image of the unstained slide is manually aligned with the stained slide or the image of the stained slide. In various embodiments, transferring the stained slide markings to the unstained slide includes copying the stained slide markings and pasting the copied stained slide markings onto the unstained slide.

[0105] In various embodiments, the image of the unstained slide is slightly transparent, allowing for simultaneous observation of the image of the stained slide and the image of the unstained slide during the transfer of the stained slide markings to the unstained slide. In various embodiments, the reorientation of the image of the unstained slide further comprises simultaneously zooming in or out the image of the stained slide and the image of the unstained slide by the same amount. In various embodiments, the reorientation of the image of the unstained slide further comprises performing one or more of the following operations: panning, rotating, zooming, or mirroring the image of the unstained slide. In various embodiments, the reorientation of the image of the unstained slide further comprises overlaying the image of the unstained slide on the image of the stained slide. Any of these movements (e.g., zooming in or out, panning, rotating, zooming, or mirroring) can be performed by an operator, including an operator using the assistance of a first application (e.g., software). In some embodiments, these movements are determined by an operator based on visual cues. This is in contrast to performing the alignment using a fully automated method, such as an algorithm, which may rely on pixel-by-pixel analysis to determine the appropriate movement to attempt to align the slide / image of the slide.

[0106] In various embodiments, method S200 can optionally include panning the stained slide image and the unstained slide image, storing translation coordinates of the pan, and associating the translation coordinates of the pan with the unstained slide (not shown). In various embodiments, method S200 can optionally include rotating the stained slide image and the unstained slide image, storing translation coordinates of the rotation, and associating the translation coordinates of the rotation with the unstained slide (not shown).

[0107] In various embodiments, method S200 can optionally include marking a first area ("S") and a second area ("X") on the marking unstained slide and saving an image of the unstained slide with the marking image coordinates and the markings of the first and second areas overlaid (not shown). In various embodiments, method S200 can optionally include associating the saved image of the unstained slide with the marking image coordinates and the markings of the first and second areas overlaid with the unstained slide (not shown).

[0108] In various embodiments, the transfer of the markings can be repeated as necessary for additional unstained slides. In various embodiments, the systems and methods disclosed herein can be used to mark multiple tissue samples on a slide. In some embodiments, the systems and methods disclosed herein can be used to mark multiple droplets of a liquid biopsy sample on a slide. In various embodiments, an operator or physician can remotely mark stained slides and / or transfer digitized stained images to a laboratory to process unstained slides. In various embodiments, the laboratory can send back digitized stained slides to present to the physician, or vice versa. In various embodiments, the disclosed systems and methods allow for digital stitching of images of slides with broken microscope glass. In various embodiments, the disclosed systems and methods can process tissues of various sizes. In various embodiments, there are no constraints on the size of tissue samples. In various embodiments, the disclosed systems and methods can allow for overlaying slides of various stain types and processing different areas differently. In various embodiments, the disclosed systems and methods can keep track of the stain markings and the transformation matrix for the unstained image.

[0109] 6 is a block diagram illustrating a computer system 600 that may implement embodiments of the present teachings. In various embodiments of the present teachings, the computer system 600 may include a bus 602 or other communication mechanism for communicating information and a processor 604 coupled with the bus 602 for processing information. In various embodiments, the computer system 600 may also include a memory, which may be a random access memory (RAM) 606 or other dynamic storage device coupled to the bus 602 for determining instructions to be executed by the processor 604. The memory may also be used to store temporary variables or other intermediate information during execution of instructions to be executed by the processor 604. In various embodiments, the computer system 600 may further include a read only memory (ROM) 608 or other static storage device coupled to the bus 602 for storing static information and instructions for the processor 604. A storage device 610, such as a magnetic disk or optical disk, may be provided and coupled to the bus 602 for storing information and instructions.

[0110] In various embodiments, computer system 600 may be coupled via bus 602 to a display 612, such as, for example, a cathode ray tube (CRT), liquid crystal display (LCD), or light emitting diode (LED) display, for displaying information to a computer user. An input device 614, including alphanumeric and other keys, may be coupled to bus 602 for communicating information and command selections to processor 604. Another type of user input device is a cursor control 616, such as a mouse, trackball, or cursor direction keys, for communicating directional information and command selections to processor 604 and for controlling movement of a cursor on display 612. This input device 614 generally has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allow the device to specify a position in a plane. However, it should be understood that input devices 614 that allow three-dimensional (x, y, and z) cursor movement are also contemplated herein.

[0111] In accordance with a particular implementation of the present teachings, results may be provided by computer system 600 in response to processor 604 executing one or more sequences of one or more instructions contained in memory 606. Such instructions may be read into memory 606 from another computer-readable medium or computer-readable storage medium, such as storage device 610. Execution of the sequences of instructions contained in memory 606 causes processor 604 to perform the processes described herein. Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementation of the present teachings is not limited to any specific combination of hardware circuitry and software.

[0112] As used herein, the terms "computer-readable medium" (e.g., data store, data storage, etc.) or "computer-readable storage medium" refer to any medium that participates in providing instructions to the processor 604 for execution. Such media may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Examples of non-volatile media may include, but are not limited to, optical disks, solid-state disks, magnetic disks, such as the storage device 610. Examples of volatile media may include, but are not limited to, dynamic memory, such as the memory 606. Examples of transmission media may include, but are not limited to, coaxial cables, copper wire, and fiber optics, including the wires that comprise the bus 602.

[0113] Common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with a pattern of holes, RAM, PROM, and EPROM, FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0114] In addition to computer-readable media, instructions or data may be provided as signals on a transmission medium included in a communication device or system that may provide a sequence of one or more instructions to the processor 604 of the computer system 600 for execution. For example, a communication device may include a transceiver having signals indicative of instructions and data. The instructions and data are configured to cause one or more processors to implement the functions outlined in this disclosure herein. Representative examples of data communication transmission connections may include, but are not limited to, a telephone modem connection, a wide area network (WAN), a local area network (LAN), an infrared data connection, an NFC connection, and the like.

[0115] It will be understood that the methodologies, flowcharts, figures, and accompanying disclosure described herein can be implemented using computer system 600 as a stand-alone device or on a distributed network of shared computer processing resources, such as a cloud computing network.

[0116] The methodologies described herein may be implemented by various means depending on the application. For example, the methods may be implemented, at least in part, in hardware, firmware, software, or any combination thereof. In the case of a hardware implementation, the processing unit may be implemented within one or more application specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field programmable gate arrays (FPGAs), processors, controllers, microcontrollers, microprocessors, electronic devices, other electronic units designed to perform the functions described herein, or combinations thereof.

[0117] In various embodiments, the methods of the present teachings may be implemented, at least in part, as firmware and / or software programs and applications written in conventional programming languages ​​such as C, C++, Python, etc. When implemented as firmware and / or software, the embodiments described herein may be implemented on a non-transitory computer-readable medium having stored thereon a program for causing a computer to perform the above-described methods. It should be understood that the various engines described herein may be provided on a computer system, such as computer system 600 of FIG. 6, such that processor 604 performs the analysis and decisions provided by these engines according to instructions provided by any one or combination of memory components 606 / 608 / 610 and user input provided via input device 614.

[0118] According to various embodiments, a non-transitory computer readable medium is provided having stored thereon a program for causing a computer to execute a method for transferring markings. According to various embodiments, the method includes acquiring an image of a stained slide under an imager, projecting the image of the stained slide onto a display, marking the image of the stained slide with digitally marking one or more features and converting the digital marking of the one or more features into coordinates, and capturing a marked unstained slide to which the markings have been transferred.

[0119] Description of the embodiments Embodiment 1. A system for transferring pathologist markings, comprising: an imaging device configured to image the stained and unstained slides; a display for displaying one or more images of the stained slide and the unstained slide; a first application for use in aligning the one or more images of the stained slide or the unstained slide; a second application for use in transferring the digital marking of one or more features; The system comprising:

[0120] Embodiment 2. The system of embodiment 1, wherein the second application is used to transfer the digital markings of the one or more features from an image of the stained slide to an image of the unstained slide.

[0121] Embodiment 3. The system of embodiment 1, wherein the second application is used to convert the digital markings of the one or more features into coordinates for use in further processing.

[0122] Embodiment 4. The system of any one of the preceding embodiments, wherein aligning the one or more images is performed manually based on visual cues.

[0123] Embodiment 5. The system of embodiment 1 or embodiment 2, wherein the imaging device is an electron microscope.

[0124] Embodiment 6. The system of any one of the preceding embodiments, wherein the stained slide comprises a hematoxylin and eosin stained slide, a slide containing a sample assessed by one or more light scattering properties, or an immunohistochemistry stained slide.

[0125] Embodiment 7. The system of any one of embodiments 3 to 6, wherein the further processing comprises one or more downstream processes comprising at least dissection or separation of a portion of the biological sample.

[0126] Embodiment 8. A system described in any one of the preceding embodiments, wherein the first application is a metrology software program used to inspect the physical properties of the stained or unstained slide.

[0127] Embodiment 9. A system as described in any one of the preceding embodiments, wherein the second application is a marking software program used in digitally marking the one or more features.

[0128] Embodiment 10. A system described in any one of the preceding embodiments, wherein the alignment of the one or more images includes positioning and / or orienting the one or more images of the stained slide or the unstained slide, and the positioning and / or orienting of the one or more images is performed manually by an operator.

[0129] Embodiment 11. The system of embodiment 10, wherein the orienting of the one or more images of the stained slide or the unstained slide includes one or more of panning, rotating, zooming, or mirroring the one or more images of the stained slide or the unstained slide.

[0130] Embodiment 12. The system of embodiment 10, wherein the one or more images of the stained or unstained slides are overlaid on top of each other to obtain alignment of the one or more images of the stained or unstained slides.

[0131] Embodiment 13. A system described in any one of the preceding embodiments, wherein the one or more features include one or more dots, straight or curved lines, or polylines.

[0132] Embodiment 14. The system of embodiment 13, wherein the one or more features are manually depicted by an operator.

[0133] Embodiment 15. A system as described in embodiment 13 or embodiment 14, wherein the second application is used to recognize the one or more dots or lines, or the polylines, and convert the one or more dots or lines, or the polylines into the digital marking of the one or more features.

[0134] Embodiment 16. The system of embodiment 15, wherein the one or more images of the stained slide include a low-resolution image of the stained slide.

[0135] Embodiment 17. The system of embodiment 16, wherein one or more boundary edges or corners of the low resolution image are used in the recognition of the one or more dots or lines, or the polylines, and in the conversion of the one or more dots or lines, or the polylines into the digital markings of the one or more features.

[0136] Embodiment 18. A method for digitally transferring a marking, comprising: acquiring an image of the stained slide under an imager; displaying the image of the stained slide on a display; transferring the markings of the stained slide to an unstained slide; capturing the unstained slide with the marking transferred thereto; and The method comprising:

[0137] Embodiment 19. The method of embodiment 18, further comprising marking the image of the stained slide prior to transferring the marking of the stained slide to an unstained slide.

[0138] Embodiment 20. The method of embodiment 18 or embodiment 19, wherein the stained slide comprises one or more pre-markings.

[0139] Embodiment 21. The method of embodiment 20, wherein marking the image of the stained slide comprises using the one or more pre-markings as a reference guideline.

[0140] Embodiment 22. The method of any one of embodiments 18 to 21, wherein marking the image of the stained slide comprises depicting one or more features on the display.

[0141] Embodiment 23. Marking the image of the stained slide comprises: drawing one or more dots, straight or curved lines, or polylines on said display; recognizing, by an application, one or more of the drawn dots or lines, or the drawn polyline; converting the recognized dot or lines, or the recognized polyline, into one or more digital features; The method according to any one of embodiments 18 to 23, comprising:

[0142] Embodiment 24. The method of embodiment 23, wherein the image of the stained slide is a low-resolution image of the stained slide, and the low-resolution image is used during the recognition of the one or more dots or lines, or the polylines, and during the conversion of the one or more dots or lines, or the polylines, into the one or more digital features.

[0143] Embodiment 25. The method of embodiment 24, wherein one or more boundary edges or corners of the low resolution image are used in the recognition of the one or more dots or lines, or the polylines, and in the conversion of the one or more dots or lines, or the polylines into the one or more digital features.

[0144] Embodiment 26. The transfer of the markings of the stained slide to the unstained slide comprises: overlaying the unstained slide over the image of the stained slide with markings; reorienting the unstained slide to align and overlay the one or more digital features of the markings on the image of the stained slide on the unstained slide; marking the unstained slide with a marking of a physical texture characteristic of the stained slide; 26. The method according to any one of embodiments 23 to 25, comprising:

[0145] Embodiment 27. The method of embodiment 26, wherein overlaying the unstained slide onto the image of the stained slide and / or reorienting the unstained slide is performed manually by an operator.

[0146] Embodiment 28. The method of embodiment 26 or embodiment 27, wherein an image of the unstained slide is used in place of the unstained slide during the transfer of the markings of the stained slide to the unstained slide.

[0147] Embodiment 29. The transfer of the markings of the stained slide to the unstained slide comprises: copying the markings of the stained slide; pasting the copied markings of the stained slide onto the unstained slide; The method according to any one of embodiments 18 to 25, comprising:

[0148] Embodiment 30. The method of embodiment 29, wherein an image of the unstained slide is used in place of the unstained slide during the transfer of the markings of the stained slide to the unstained slide.

[0149] Embodiment 31. The method of embodiment 30, wherein the image of the unstained slide is slightly transparent, thereby allowing simultaneous observation of the image of the stained slide and the image of the unstained slide during the transfer of the marking of the stained slide to the unstained slide.

[0150] Embodiment 32. The method of embodiment 30 or 31, wherein the redirecting of the image of the unstained slide further comprises simultaneously zooming in or out the image of the stained slide and the image of the unstained slide by the same amount.

[0151] Embodiment 33. The method of embodiment 30 or 31, wherein the reorienting of the image of the unstained slide further comprises performing one or more of the following operations: panning, rotating, zooming, or mirroring the image of the unstained slide.

[0152] Embodiment 34. The method of embodiment 30, wherein the redirecting of the image of the unstained slide further comprises performing an overlay of the image of the unstained slide onto the image of the stained slide.

[0153] Embodiment 35. Panning the image of the stained slide and the image of the unstained slide; storing the pan transformation coordinates; relating the transformed coordinates of the pan to the unstained slide; 31. The method of embodiment 30, further comprising:

[0154] Embodiment 36. Rotating the image of the stained slide and the image of the unstained slide; storing transformed coordinates of the rotation; relating the transformed coordinates of the rotation to the unstained slide; 31. The method of embodiment 30, further comprising:

[0155] Embodiment 37. Marking a first area ("S") and a second area ("X") on the marked unstained slide; saving an image of the unstained slide with marking image coordinates and markings of the first and second regions overlapping; The method of any one of embodiments 18 to 36, further comprising:

[0156] Embodiment 38. The method described in embodiment 37, further comprising associating the marking image coordinates of the first region and the second region and the image of the unstained slide in which the markings are superimposed and stored, with the unstained slide.

[0157] Embodiment 39. A system for transferring pathologist markings, comprising: an imaging device configured to image stained or unstained slides; a display for displaying one or more images of the stained or unstained slide; and a mechanical stage / staff to position or orient the stained slide relative to the unstained slide to allow for physical marking of the unstained slide; an application used to digitally represent one or more features based on said physical markings and convert said physical markings into coordinates that are used for further processing; The system comprising:

[0158] Embodiment 40. The system of embodiment 39, wherein the mechanical stage / scaffold is manually manipulated to position or orient the stained slide relative to the unstained slide.

[0159] Embodiment 41. A system described in embodiment 39 or embodiment 40, wherein the imaging device is an electron microscope.

[0160] Embodiment 42. A system described in any one of embodiments 39 to 41, wherein the application is a metrology software program used to inspect the physical properties of the stained or unstained slides.

[0161] Embodiment 43. The system of any one of embodiments 39 to 42, wherein the stained slide comprises a hematoxylin and eosin stained slide, a slide containing a sample evaluated by one or more light scattering properties, or an immunohistochemistry stained slide.

[0162] Embodiment 44. The system of any one of embodiments 39 to 43, wherein the further processing comprises one or more downstream processes comprising at least dissection or separation of a portion of the biological sample.

[0163] Embodiment 45. A system described in any one of embodiments 39 to 44, wherein the one or more features include one or more dots, straight or curved lines, or polylines.

[0164] Embodiment 46. A system described in any one of embodiments 39 to 45, wherein the stained slide further comprises one or more pre-markings.

[0165] Embodiment 47. The system of embodiment 46, wherein the one or more pre-markings are used as guidelines during the transfer of the physical marking onto the unstained slide.

[0166] Embodiment 48. A system described in any one of embodiments 39 to 47, wherein the application is used to recognize the physical marking and convert the physical marking into the one or more features.

[0167] Embodiment 49. The system of embodiment 48, wherein the one or more images of the stained slide include a low-resolution image, which is used in recognizing the physical marking and converting the physical marking into the one or more features.

[0168] Embodiment 50. The system of embodiment 49, wherein one or more boundary edges or corners of the low resolution image are used in the recognition of the physical marking and in the conversion of the physical marking into the one or more features.

[0169] Embodiment 51. A method for transferring a marking, comprising: projecting an image of the stained slide with pathologist markings on a display; placing an unstained slide on a scaffold above said display; aligning the unstained slide with the projected image of the stained slide by positioning the scaffold; physically transferring said pathologist markings onto said unstained slide; The method comprising:

[0170] Embodiment 52. The method of embodiment 51, wherein the scaffold is manually positioned by an operator.

[0171] Embodiment 53. The method of embodiment 51 or 52, wherein the stained slide further comprises one or more pre-markings.

[0172] Embodiment 54. The method of embodiment 53, wherein the one or more pre-markings are used as guidelines during the physical transfer of the pathologist markings onto the unstained slide.

[0173] Embodiment 55. The method of any one of embodiments 51 to 54, wherein the physical transfer of the pathologist marking onto the unstained slide comprises depicting one or more features or markings on the unstained slide.

[0174] Embodiment 56. The method of embodiment 55, wherein the one or more features include one or more dots, straight or curved lines, or polylines.

[0175] Embodiment 57. A method according to any one of embodiments 51 to 56, wherein the projected image of the stained slide is panned, rotated, zoomed and / or overlaid to assist in the alignment with the unstained slide.

[0176] Embodiment 58. The method of any one of embodiments 51 to 57, wherein the projected image of the stained slide is flipped to assist in the alignment with the unstained slide.

[0177] Embodiment 59. The method described in embodiment 57 or 58, wherein the projected images of the stained slide are manually panned, rotated, zoomed, overlaid, and / or flipped by an operator.

[0178]

[0046] Embodiment 60. Recognizing the pathologist marking by an application; converting the pathologist markings into one or more features; transferring the one or more features onto the unstained slide; and The method of any one of embodiments 51 to 60, further comprising:

[0179] Embodiment 61. The method of embodiment 60, wherein a low resolution image of the stained slide is used in the recognition of the pathologist marking and in converting the pathologist marking into the one or more features.

[0180] Embodiment 62. The method of embodiment 23, wherein one or more boundary edges or corners of the low resolution image are used in the recognition of the pathologist marking and in the conversion of the pathologist marking into the one or more features.

[0181] Embodiment 63. A method according to any one of embodiments 51 to 62, wherein projecting the image of the stained slide onto the display is performed from behind, above, or to the side of the stained slide.

[0182] Embodiment 64. The method of any one of embodiments 51 to 63, wherein the transferred pathologist marking is used to exfoliate a portion of the biological sample.

[0183] Embodiment 65. The method of any one of embodiments 51 to 64, wherein the stained slide comprises a hematoxylin and eosin stained slide, a slide containing a sample evaluated by one or more light scattering properties, or an immunohistochemistry stained slide.

[0184] Although the present specification contains many specific implementation details, these should not be construed as limitations on the scope of any disclosure or the scope that may be claimed, but rather as descriptions of features specific to particular implementations of particular embodiments. Certain features described in the present specification in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation can also be implemented separately in multiple implementations or in any suitable subcombination. Furthermore, although features may be described above as functioning in a particular combination and even initially claimed as such, one or more features from the claimed combination can, in some cases, be excluded from the combination, and the claimed combination may be directed to a subcombination or a variation of the subcombination.

[0185] Similarly, although operations are shown in a particular order in the figures, this should not be understood as requiring that such operations be performed in the particular order or sequential order shown, or that all of the operations shown be performed, to achieve desirable results. In certain situations, multitasking and parallel processing may be advantageous. Furthermore, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the program components and systems described may generally be integrated into a single software product or packaged into multiple software products.

[0186] References to "or" may be construed as inclusive, such that any term described using "or" may refer to either one, more than one, or all of the described term. Labels such as "first," "second," "third," etc. are not necessarily meant to denote an order, but are generally used merely to distinguish among like or similar items or elements.

[0187] Various modifications to the implementations described in this disclosure may be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations without departing from the spirit or scope of the present disclosure. Thus, the claims are not intended to be limited to the implementations shown herein, but are to be accorded the widest scope consistent with the present disclosure, the principles and novel features disclosed herein.

Claims

1. A system for transferring a pathologist's marking, comprising: an imaging device configured to image a stained slide and an unstained slide; a display for displaying one or more images of the stained slide and the unstained slide; a first application used when aligning the one or more images of the stained slide or the unstained slide; a second application used when transferring digital markings of one or more features; wherein aligning the one or more images is performed manually based on visual cues, the system.

2. The system according to claim 1, wherein the second application is used when transferring the digital markings of the one or more features from an image of the stained slide to an image of the unstained slide.

3. The system according to claim 1, wherein the second application is used when converting the digital markings of the one or more features into coordinates for use in further processing.

4. The system according to claim 3, wherein the further processing includes one or more downstream processes having at least dissection or separation of a portion of a biological sample.

5. The system according to any one of claims 1 to 4, wherein the first application is a measurement software program used for inspecting physical properties of the stained slide or the unstained slide.

6. The system according to any one of claims 1 to 4, wherein the second application is a marking software program used when digitally marking the one or more features.

7. The system according to any one of claims 1 to 4, wherein the alignment of the one or more images includes positioning and / or orienting the one or more images of the stained slide or the unstained slide, and the positioning and / or the orienting of the one or more images are performed manually by an operator.

8. The system according to claim 7, wherein the orienting of the one or more images of the stained slide or the unstained slide includes one or more operations of panning, rotating, zooming, or mirroring the one or more images of the stained slide or the unstained slide.

9. The system according to claim 7, wherein the one or more images of the stained slide or the unstained slide are overlaid on top of each other to obtain alignment of the one or more images of the stained slide or the unstained slide.

10. The system according to any one of claims 1 to 4, wherein the one or more features include one or more dots, straight or curved lines, or polylines.

11. The system according to claim 10, wherein the one or more features are manually depicted by an operator.

12. The system according to claim 10, wherein the second application is used when recognizing the one or more dots or lines, or the polylines and converting the one or more dots or lines, or the polylines into the digital markings of the one or more features.

13. The system according to claim 12, wherein the one or more images of the stained slide include a low-resolution image of the stained slide.

14. The system according to claim 13, wherein one or more boundary edges or corners of the low-resolution image are used during the recognition of the one or more dots or lines, or the polylines and during the conversion of the one or more dots or lines, or the polylines into the digital markings of the one or more features.

15. A method for digitally transferring markings, comprising: acquiring an image of a stained slide under an imager; displaying the image of the stained slide on a display; marking the image of the stained slide; transferring the marking of the stained slide to an unstained slide; including transferring the marking of the stained slide to the unstained slide includes one or more steps manually performed by an operator.

16. The method according to claim 15, wherein marking the image of the stained slide includes depicting one or more features on the display.

17. Marking the image of the stained slide includes depicting one or more dots, straight or curved lines, or polylines on the display; recognizing the one or more depicted dots or lines, or the depicted polylines by an application; converting the one or more recognized dots or lines, or the recognized polyline, into one or more digital features; The method according to claim 15 or 16, comprising:

18. The image of the stained slide is a low-resolution image of the stained slide, and the low-resolution image is used in the recognition of the one or more dots or lines, or the polyline, and in the conversion of the one or more dots or lines, or the polyline, into the one or more digital features. The method according to claim 17.

19. The image of the stained slide is a low-resolution image of the stained slide, and one or more boundary edges or corners of the low-resolution image are used in the recognition of the one or more dots or lines, or the polyline, and in the conversion of the one or more dots or lines, or the polyline, into the one or more digital features. The method according to claim 17.

20. The transfer of the marking of the stained slide to the unstained slide overlaying the unstained slide over the image of the stained slide with the marking; aligning the one or more digital features on the unstained slide and reorienting the unstained slide to overlay; marking the unstained slide; comprising: The overlaying of the unstained slide over the image of the stained slide and / or the reorienting of the unstained slide are performed manually by an operator. The method according to claim 17.

21. The reorienting includes placing the unstained slide on a scaffold on the display and positioning the scaffold to align the unstained slide with the projected image of the stained slide. The scaffold is manually operated by a user. The method according to claim 20.

22. A system for transferring a pathologist's marking, comprising: an imaging device configured to image a stained slide or an unstained slide; a display for displaying one or more images of the stained slide or the unstained slide; A mechanical stage / staging for positioning or orienting the stained slide relative to the unstained slide to enable physical marking of the unstained slide, An application used to digitally depict one or more features based on the physical marking and convert the features into coordinates for use in further processing, The mechanical stage / staging is manually operated to position or orient the stained slide relative to the unstained slide, the system.

23. The system according to claim 22, wherein the application is a measurement software program used for inspecting the physical properties of the stained slide or the unstained slide.

24. The system according to claim 22 or 23, wherein the further processing includes one or more downstream processes having at least dissection or separation of a portion of the biological sample.

25. A method for transferring a marking, comprising: Projecting an image of a stained slide with a pathologist's marking onto a display; Placing an unstained slide on a staging on the display; Aligning the unstained slide with the projected image of the stained slide by positioning the staging; Physically transferring the pathologist's marking onto the unstained slide; Including, The method, wherein the staging is manually positioned by an operator.

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