Contactless fluid mixture on slide

Abstract

To efficiently mix a reagent for dying a biological tissue on a microscope slide and the tissue by using a small amount of reagent.SOLUTION: A microscope slide holder (5) includes: a slide supporting member (20); and at least one acoustic source (30) for introducing an elastic wave to a microscope slide (10) leading to the slide supporting member (20) so that at least one fluid on the microscope slide (10) is mixed contactlessly. The acoustic source operates in the range of approximately 1 Hz to 1 kHz.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0001] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 511,390, filed May 26, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002]

[0002] Many organizations do not retain enough color after processing to make the components of the tissue visible under bright - field microscopy. Thus, it is common practice to add color and contrast to the components of the tissue by staining the tissue with various reagents. Previously, the step of staining tissue samples for histological or cytological analysis was performed manually, and this process is inherently inconsistent. Inconsistent staining makes it difficult for pathologists or other medical personnel to read slides and to compare different samples. Thus, numerous devices and methods have been described that help automate the staining process and reduce staining non - consistency.

[0003]

[0003] Devices for automated staining, particularly for high-volume staining using conventional reagents such as hematoxylin and eosin (H&E), are mainly of the "dip and dunk" type in which a slide rack is automatically lowered into and removed from a series of reagent tanks. For example, U.S. Patent No. 4,911,098 to Tabata describes an automated staining apparatus where a microscope slide holding a tissue specimen is successively immersed in a number of chemical solution containers. The slide is vertically mounted within a slide holder basket and a clamp that engages and disengages with respect to this basket is used to move the slide from solution to solution. The clamp can include a mechanism for tilting the basket, which helps remove excess solution before the basket is immersed in the next solution. Further automated staining devices of the "dip and dunk" type are described in U.S. Patent No. 5,573,727 to Keefe, U.S. Patent No. 6,080,363 to Takahasi et al., U.S. Patent No. 6,436,348 to Ljungmann et al., and U.S. Patent Application Publication No. 2001 / 0019703 named inventors Thiem et al.

[0004]

[0004] Another type of automated staining apparatus delivers fresh reagent directly to individual slides. For example, U.S. Patent No. 6,387,326 to Edwards et al. describes an apparatus for staining slides where slides are released one at a time from a slide storage device and are individually processed at various staining stations as they move along a conveyor belt transport device. Further devices for automatically staining individual slides are described in U.S. Patent No. 6,180,061 to Bogen et al., PCT Publication WO03 / 045560 named inventors Tseung et al., and U.S. Patent Application Publication No. U.S.2004 / 0052685 named inventors Richards et al.

[0005]

[0005] Efficient mixing of fluids is an important step in many industrial, chemical, and pharmaceutical processes, as well as in biotechnological applications. Mixing in small amounts is often a difficult task. In some embodiments, molecular diffusion is the main mixing mechanism, which slows down the entire process. The integration of active mixers is often difficult, increases the cost of any such device, and results in cross-contamination between samples.

Prior Art Documents

Patent Documents

[0006]

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Non - Patent Document

[0007]

Non - Patent Document 1

Summary of the Invention

[0008]

[0006] Disclosed herein are systems and methods for non-contact dispensing, replenishing, and / or mixing of one or more fluids on a substrate. Applicants have found that by using low-frequency acoustic or vibrational energy (e.g., at a frequency that does not damage cells, a frequency below 2000 Hz, etc.) to replenish, distribute, and / or mix one or more fluids present on the surface of a substrate, high mixing efficiency, reduced process time, and prevention of contamination can be achieved while maintaining low manufacturing and maintenance costs. Applicants have also surprisingly found that the introduction of such low-frequency acoustic energy promotes mixing in small volumes due to the absorption of acoustic energy by waves generated within one or more fluids. Applicants have also unexpectedly found that the aforementioned benefits can be achieved without damaging the cells and / or tissues present on the substrate (e.g., by lysis or cell membrane disruption) and without unnecessarily heating the sample. Applicants have also found that mixing fluids according to the methods described herein (i) reduces the risk of staining artifacts and (ii) enables a uniform antibody reagent concentration across cells and / or tissues during staining. It is also contemplated that the use of the devices and methods disclosed herein may enable the use of lower concentrations of detection probes (e.g., antibodies) in any staining procedure.

[0009]

[0007] One aspect of the present disclosure is a substrate holder or carrier having at least one support member and one or more acoustic sources (e.g., one or more transducers) for non-contact mixing, dispensing, or replenishing a fluid present on the surface of a substrate (e.g., a microscope slide containing a specimen), wherein the acoustic source communicates with the support member or the substrate. "Communicates with the support member or the substrate" means, for example, that the acoustic source is at least partially in contact with the support member or the substrate, or can be moved so as to be placed in at least partial contact with the support member or the substrate. In some embodiments, the acoustic source is embedded within the support member, or the acoustic source is in contact with another element that contacts the support member or the substrate. In some embodiments, the acoustic source is a mechanical transducer or a piezoelectric transducer that operates at a low frequency (e.g., a frequency ranging from about 1 Hz to about 1 kHz). In some embodiments, it is a microscope slide. Other examples of the substrate include surface enhanced laser desorption / ionization ("SELDI") and matrix assisted laser desorption / ionization ("MALDI") chips, and silicon wafers.

[0010]

[0008] Another aspect of the present disclosure is a slide holder for non-contact mixing, dispensing, or replenishing one or more fluids (including fluids already present on the surface of the slide) on a slide containing a specimen, comprising a slide support member and at least one transducer in communication with the slide support member, wherein the at least one transducer operates at a frequency ranging from about 1 Hz to about 1 kHz. In some embodiments, the at least one transducer communicates with the lower surface of the slide support member. In some embodiments, the at least one transducer is positioned along the slide support member in a region that substantially corresponds to the center of the specimen end of the slide.

[0011]

[0009] In some embodiments, at least two transducers communicate with a slide support. In some embodiments, each of the at least two transducers is configured such that a first elastic wave (or a first series of elastic waves) supplied by a first transducer among the at least two transducers is not canceled by a second elastic wave (or a second series of elastic waves) supplied by a second transducer among the at least two transducers. In some embodiments, the at least two transducers are out of phase with each other.

[0012]

[0010] In some embodiments, at least one transducer operates at a frequency ranging from about 1 Hz to about 500 Hz. In some embodiments, the frequency ranges from about 50 Hz to about 500 Hz. In some embodiments, the frequency ranges from about 100 Hz to about 200 Hz. In some embodiments, the power supplied to at least one transducer ranges from about 40 mVpp to about 350 mVpp.

[0013]

[0011] In some embodiments, the slide support has a support surface configured to support at least a portion of the back surface of the slide, and the back surface is on the opposite side of the surface containing the specimen. In some embodiments, the slide support further comprises a heating element. In some embodiments, the slide support further comprises a controller in electrical communication with at least one transducer. In some embodiments, the controller includes an amplifier and a signal generator. In some embodiments, the slide holder is part of a staining device or a cover glass coating device, as further described herein. In other embodiments, the slide support is further configured to include heating and / or cooling elements such as resistive heating elements or Peltier heating and cooling elements.

[0014] Another aspect of the present disclosure is a slide support and at least one acoustic source for introducing low-frequency vibrations into the slide such that one or more fluids present on the surface of the slide are mixed without contact. In some embodiments, the low-frequency vibrations are elastic waves. In some embodiments, the acoustic source for introducing the low-frequency vibrations is selected from the group consisting of a mechanical transducer, a piezoelectric transducer, and a surface acoustic wave device.

[0015] Another aspect of the present disclosure is a slide tray comprising a plurality of the above slide holders. In some embodiments, each slide holder is positioned at spaced positions substantially horizontally and in the same plane. In other embodiments, each slide holder is positioned along an arc. Another aspect is a movable slide support including the above slide holder.

[0016] Another aspect of the present disclosure is a staining device comprising at least one of the slide holders (or slide trays) described herein and at least one dispenser (or other device) capable of introducing one or more fluids onto a surface containing a specimen on the slide. In some embodiments, the staining device further comprises a feedback control device (e.g., a camera) for monitoring the mixing of one or more fluids introduced onto the surface of the slide containing the specimen. In some embodiments, dispensing of the reagent occurs without contact with any mixing device or gas stream in the reservoir or sample. In some embodiments, the staining device further comprises an active mixing device that contacts a fluid reservoir on the surface of the slide. Fluid dispensers and other components for the automated processing of biological specimens are disclosed in WO2015 / 086484, WO2010 / 080287, and U.S. Patent No. 7,615,371, the disclosures of which are incorporated herein by reference in their entirety.

[0017] Another aspect of the present disclosure is a method of non - contact mixing, replenishing, or distributing a fluid present on the surface of a slide containing a specimen, wherein the mixing, replenishing, or distributing of the fluid occurs without causing damage to any specimen present on the surface of the slide. In some embodiments, the mixing, replenishing, or distributing of the fluid is achieved by an acoustic source operating at a low frequency. In some embodiments, the method includes the steps of dispensing a first fluid and a second fluid onto the slide, and non - contact mixing the first fluid and the second fluid by elastic waves.

[0018] Another aspect of the present disclosure is a method of processing a slide containing a specimen, the method including (i) contacting a sample on the slide containing the specimen with a first reagent, and (ii) uniformly distributing the first reagent over the specimen - containing slide by introducing low - frequency elastic waves into the slide containing the specimen. In some embodiments, the low - frequency elastic waves are generated by at least one transducer in communication with the slide containing the specimen. In some embodiments, the first reagent is dispensed into an existing fluid present on the slide containing the specimen, and the elastic waves generated by the at least one transducer uniformly mix the first reagent within the existing fluid. In some embodiments, the existing fluid is a buffer solution.

[0019]

[0017] In some embodiments, at least one transducer is configured to operate at a frequency ranging from about 1 Hz to about 1 kHz. In some embodiments, the frequency ranges from about 50 Hz to about 500 Hz. In some embodiments, the frequency ranges from about 100 Hz to about 200 Hz. In some embodiments, the elastic wave is introduced into the sample during a time interval ranging from about 1 second to about 120 seconds. In some embodiments, the time interval ranges from about 1 second to about 60 seconds. In some embodiments, the time interval ranges from about 1 second to about 30 seconds. In some embodiments, the time interval ranges from about 2 seconds to about 15 seconds. In some embodiments, the bottom surface of the slide containing the specimen is at least partially in contact with the substrate, and at least one transducer is coupled to the substrate.

[0020]

[0018] In some embodiments, the first reagent is a detection probe specific to a first target in the sample. In some embodiments, the method further comprises contacting the sample with a first detection reagent to facilitate detection of the first detection probe. In some embodiments, the method further comprises contacting the sample with a second detection probe specific to a second target. In some embodiments, the second detection probe is introduced simultaneously with the first detection probe, and at least one transducer mixes the first detection probe and the second detection probe with the existing fluid. In some embodiments, the first detection probe and the second detection probe are antibodies.

[0021] Another aspect of the present disclosure is a method of replenishing a reagent, comprising: (i) contacting a sample on a slide containing a specimen with a first reagent; (ii) ensuring a time for the first reagent to react with the sample or to be absorbed by the sample; and (iii) introducing an elastic wave onto the slide containing the specimen to uniformly distribute the first reagent on the slide containing the specimen, thereby replenishing the reagent in a region where the reagent is at least partially depleted. In some embodiments, the method optionally includes introducing an additional aliquot of the first reagent prior to uniformly distributing the first reagent by introduction of the elastic wave. In some embodiments, the process may be repeated with additional reagent.

[0022] Another aspect of the present disclosure is a method of staining a sample, comprising: (a) dispensing a reagent into a fluid pool present on a slide containing a sample; and (b) dispensing the reagent non - contact into the fluid pool using a low - frequency elastic wave, wherein the step of dispensing the reagent occurs without damaging cells or tissue within the sample. In some embodiments, the reagent is dispensed substantially uniformly into the fluid pool within about 30 seconds after initiation of dispensing of the reagent. In some embodiments, non - contact dispensing of the reagent is achieved using an acoustic source selected from the group consisting of a mechanical transducer and a piezoelectric transducer. In some embodiments, the acoustic source operates at a frequency ranging from about 100 Hz to about 200 Hz. In some embodiments, the reagent is a specific binding site. In some embodiments, the specific binding site comprises an antibody. In some embodiments, the method further comprises dispensing a second reagent into the fluid pool.

Brief Description of the Drawings

[0023]

Figure 1A

[0021] Side view of a microscope slide holder in communication with a transducer.

Figure 1B

[0022] Top view of a microscope slide holder in communication with a transducer.

Figure 1C

[0023] Side view of a microscope slide holder in communication with a plurality of transducers.

Figure 1D

[0024] Top view of a microscope slide holder in communication with a plurality of transducers.

Figure 1E

[0025] Side view of a microscope slide holder in communication with a transducer.

Figure 1F

[0026] Top view of a microscope slide holder in communication with a transducer, where the support member is larger than the microscope slide in at least one dimension.

Figure 1G

[0027] Top view of a microscope slide holder in communication with a transducer, where the support member is smaller than the microscope slide in at least one dimension.

Figure 1H

[0028] Side view of a microscope slide holder in communication with a transducer.

Figure 1I

[0029] Side view of a microscope slide holder in communication with a transducer.

Figure 1J

[0030] Top view of a microscope slide holder in communication with a transducer.

Figure 2A

[0031] Side view of a microscope slide holder in communication with a transducer.

Figure 2B

Figure 2C

[0032] Top view of a microscope slide holder in communication with a transducer.

Figure 3

[0033] Top view of a slide tray comprising a plurality of microscope slide holders, each slide holder being in communication with at least one transducer.

Figure 4A

[0034] Side view of a microscope slide holder in communication with a transducer, where the slide holder includes a cantilever portion.

Figure 4B

[0035] Top view of a microscope slide holder in communication with a transducer, where the slide holder includes a cantilever portion.

Figure 4C

[0036] Side view of a microscope slide holder in communication with a transducer, where the slide holder includes a cantilever portion and a heating plate.

Figure 5A

[0037] Side view of a microscope slide holder in communication with a transducer, where the slide holder includes a heating plate.

Figure 5B

[0038] Side view of a microscope slide holder in communication with a plurality of transducers, where the slide holder includes a heating plate.

Figure 6

[0039] Side view of a system comprising a microscope slide holder in communication with a transducer, the system also including elements for feedback control. The slide processing station (SPS) heating plate is a fixture on which the slide is placed. The amount of fluid on the slide is mixed by the displacement of the transducer. The signal generator supplies the frequency and voltage for driving the transducer. The UV light, camera, and enclosure are used to visualize and quantify the fluid mixing by enhancing the fluorescence signal and removing interference from ambient light.

Figure 7A

[0040] Diagram illustrating over time the distribution of dye placed at the corner of a slide when an elastic wave is introduced.

Figure 7B

Figure 7C

Figure 7D

Figure 8A

[0041] It is a figure that illustrates over time the distribution of the dye placed at the corner of the slide when an elastic wave is introduced.

Figure 8B

[0042] (a) It illustrates that the standard deviation of the green values of the pixels within the region of interest (ROI) is calculated for each frame, and (b) it provides a plot of the standard deviation of the green values within the ROI during the mixing process.

Figure 9

[0043] It is a figure that illustrates the result of sweeping the input voltage from a first voltage to a higher second voltage, and illustrates that the area of the standing wave pattern increases as the input voltage increases.

Figure 10A

[0044] It is a figure that illustrates over time the distribution of the dye when an elastic wave is introduced.

Figure 10B

[0045] It is a figure that illustrates over time the distribution of the dye when an elastic wave is introduced.

Figure 11

[0046] It is a figure that illustrates the relationship between the total transducer period time, the on-time, and the off-time.

DETAILED DESCRIPTION OF THE INVENTION

[0024]

[0047] Definitions

[0048] It should also be understood that in any method claimed in this specification that includes more than one step or act, unless the contrary is specifically indicated otherwise, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0025]

[0049] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively such that "comprising A or B" means including A, B, or both A and B.

[0026]

[0050] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., at least one inclusion, but including two or more of several elements or a list of elements, and optionally, additional unenumerated items as well. Conversely, only terms explicitly indicated, such as "only one of", "exactly one of", or "consisting of" when used in a claim, refer to the inclusion of exactly one element of several elements or a list of elements. Generally, the term "or" as used in this specification, when followed by an exclusive term such as "either", "one of", "only one of", or "exactly one of", should be interpreted to indicate only an exclusive alternative (i.e., "one or the other but not both"). "Consisting essentially of" shall have its ordinary meaning as used in the field of patent law when used in a claim.

[0027]

[0051] As used in this specification and the claims, the expression "at least one" related to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically enumerated in the list of elements, and is not to be construed as excluding any combination of elements in the list of elements. This definition also allows for the possibility that elements, optionally, whether or not related to the specifically identified elements in the list of elements referred to by the expression "at least one", may exist. Thus, as a non-limiting example, "at least one of A and B" (which is equivalent to "at least one of A or B", which is equivalent to "at least one of A and / or B") may, for example, in one embodiment, optionally include two or more A and no B (and 、 may refer to at least one (optionally including elements other than B), and in another embodiment, may refer to at least one including two or more Bs and no A (and optionally including elements other than A), and in yet another embodiment, may refer to at least one (optionally including two or more As and further optionally including two or more Bs and optionally including other elements).

[0028]

[0052] As used herein, the terms "comprising," "including," "having," and the like are used synonymously and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used synonymously and have the same meaning. Specifically, each of these terms is defined in accordance with the general U.S. patent statutory definition of "comprising," and thus is construed as an open term meaning "at least the following" and is also construed not to exclude additional features, limitations, aspects, etc. Thus, for example, a "device having components a, b, and c" means a device including at least components a, b, and c. Similarly, a "method involving steps a, b, and c" means a method including at least steps a, b, and c. Further, although steps and processes may be schematically shown herein in a particular order, one of ordinary skill in the art will recognize that the order of steps and processes can vary.

[0029]

[0053] As used herein, the term "elastic wave" encompasses sonic energy, acoustic energy, acoustic pulses, sound energy, sound waves, sonic pulses, pulses, waves, or vibrational energy, or any other grammatical form of these terms, and any other type of energy having characteristics similar to acoustic energy. Each of these terms may be used synonymously herein.

[0030]

[0054] As used herein, the term "antibody" refers to, by way of example and without limitation, immunoglobulins or immunoglobulin-like molecules, including IgA, IgD, IgE, IgG, and IgM, and combinations thereof, as well as similar molecules generated during an immune response in any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice), and antibody fragments (such as F(ab’)2 fragments, Fab’ fragments, Fab’-SH fragments, and Fab fragments, etc., recombinant antibody fragments (sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)’2 fragments, single-chain Fv proteins ("scFv"), disulfide-stabilized Fv proteins ("dsFv"), bispecific antibodies, and trispecific antibodies (as known in the art), and camelid antibodies) that specifically bind to a molecule of interest (or a group of molecules closely resembling the molecule of interest) until substantially excluding binding to other molecules. An antibody further refers to a polypeptide ligand that includes at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody can be composed of a heavy chain and a light chain, each of the heavy and light chains having a variable region called the variable heavy (VH) region and the variable light (VL) region. The VH region and the VL region together are responsible for binding the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins, as well as variants and portions thereof well known in the art.

[0031]

[0055] As used herein, the term "biological sample" or "tissue sample" refers to any sample containing biomolecules (such as proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (humans; domestic animals such as cats, dogs, horses, cows, and pigs; and laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears of cells obtained by microdissection, Pap smears or blood smears or samples of cells), or cell fractions, cell fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (such as obtained by surgical biopsy or needle biopsy), nipple aspirate fluid, earwax, breast milk, vaginal fluid, saliva, rinse fluid (such as oral rinse fluid), or any substance containing biomolecules derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion of a tumor obtained from a subject. Examples include cell fractions, cell fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (such as obtained by surgical biopsy or needle biopsy), nipple aspirate fluid, earwax, breast milk, vaginal fluid, saliva, rinse fluid (such as oral rinse fluid), or any substance containing biomolecules derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion of a tumor obtained from a subject.

[0032]

[0056] As used herein, the term "fluid" refers to any fluid including water, solvents, solutions (e.g., buffer solutions), etc. The term "fluid" also refers to any mixture, colloid, suspension, etc. The term "fluid" also encompasses reagents, stains, and other specimen processing agents (e.g., adhesives, fixatives, etc.) that can be applied to microscope slides and / or specimens. The fluid may be aqueous or non-aqueous. Further examples include solutions or suspensions of antibodies, solutions or suspensions of nucleic acid probes, and solutions or suspensions of dyes or staining molecules (e.g., H&E staining solution, Pap staining solution, etc.). Still further examples of fluids include solvents and / or solutions for deparaffinizing paraffin-embedded biological specimens, aqueous washing solutions, and hydrocarbons (e.g., alkanes, isoalkanes, and aromatic compounds such as xylene). Still further examples of fluids include solvents (and mixtures thereof) used for dehydrating or rehydrating biological specimens.

[0033]

[0057] As used herein, the term "plurality" refers to two or more, for example, three or more, four or more, five or more, etc.

[0058] As used herein, the term "reagent" refers to any liquid or liquid composition used in a specimen processing operation involving adding a liquid or liquid composition to a slide. Examples of reagents and processing liquids include solutions, emulsions, suspensions, and solvents (either pure substances or mixtures thereof). These examples and others may be aqueous or non-aqueous. Further examples include solutions or suspensions of specific binding entities, antibodies, solutions or suspensions of nucleic acid probes, and solutions or suspensions of dyes or staining molecules (e.g., H&E staining solution, Pap staining solution, etc.). Still further examples include solvents and / or solutions for deparaffinizing paraffin-embedded biological specimens, aqueous washing solutions, and hydrocarbons (e.g., alkanes, isoalkanes, and aromatic compounds such as xylene).

[0034]

[0059] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen within a tissue sample. A primary antibody is generally the first antibody used in immunohistochemical methods. Epitope-labeled antibodies, unmodified antibodies, or antibody conjugates, each as described herein, are examples of primary antibodies. Thus, a primary antibody serves as a "detection probe" for detecting a target within a tissue sample.

[0035]

[0060] As used herein, the term "secondary antibody" refers to an antibody that specifically binds to a detection probe or a portion thereof (e.g., a hapten or a primary antibody) herein, and thereby forms a bridge between the detection probe and a subsequent reagent (e.g., a label, an enzyme, etc.) if such a subsequent reagent is present. A secondary antibody may be used to indirectly detect a detection probe, e.g., a primary antibody. Examples of secondary antibodies include anti-tag antibodies, anti-species antibodies, and anti-label antibodies, each as described herein.

[0036]

[0061] As used herein, the term "slide" refers to any suitable substrate of any suitable dimension (e.g., a substrate made in whole or in part of glass, quartz, plastic, silicon, etc.) on which a biological specimen is placed for analysis, and more particularly refers to a "microscope slide" such as a standard 7.62 cm (3 inch) × 2.54 cm (1 inch) microscope slide or a standard 75 mm × 25 mm microscope slide. Examples of biological specimens that can be placed on a slide include, but are not limited to, cytological smears, thin tissue sections (such as from a biopsy), and arrays of biological specimens, e.g., tissue arrays, cell arrays, DNA arrays, RNA arrays, protein arrays, or any combination thereof. Thus, in one embodiment, a tissue section, a DNA sample, an RNA sample, and / or a protein is placed at a specific location on a slide. In some embodiments, the term "slide" may refer to SELDI and MALDI chips, as well as silicon wafers.

[0037]

[0037]

[0062] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by binding to each other until binding to other molecules is substantially excluded (e.g., the specific binding pair may have a binding constant that is at least 103 M-1, 104 M-1, or 105 M-1 greater than the binding constant of either of the two members of the binding pair with other molecules in a biological sample). Specific examples of specific binding sites include specific binding proteins (e.g., antibodies, lectins, avidin such as streptavidin, and protein A). A specific binding site may also include a molecule (or a portion thereof) that is specifically bound by such a specific binding protein. Specific binding entities include the primary antibodies or nucleic acid probes described above.

[0038]

[0063] As used herein, the terms "stain", "staining" or the like generally refer to any treatment of a biological sample that detects and / or differentiates the presence, location, and / or amount (such as concentration) of a particular molecule (such as a lipid, protein, or nucleic acid) or a particular structure within a biological sample (such as a normal or malignant cell, cytosol, nucleus, Golgi apparatus, or cytoskeleton). For example, staining can provide contrast between a particular molecule or a particular cellular structure and the surrounding portion of the biological sample, and the intensity of the staining can provide a measure of the amount of a particular molecule within the sample. Staining can be used to assist in the observation of molecules, cellular structures, and organisms using not only bright-field microscopes, but also other observation instruments such as phase-contrast microscopes, electron microscopes, and fluorescence microscopes. The staining performed by System 2 can be used to visualize the cell contour. Other staining performed by System 2 may depend on a particular cellular component (such as a molecule or structure) being stained without or with relatively less staining of other cellular components. Examples of types of staining methods performed by System 2 include, but are not limited to, histochemical methods, immunohistochemical methods, and other methods based on intermolecular reactions (including non-covalent interactions) such as hybridization reactions between nucleic acid molecules. Specific staining methods include, but are not limited to, primary staining methods (such as H&E staining, Pap staining, etc.), enzyme-linked immunohistological methods, and in situ RNA and DNA hybridization methods such as fluorescence in situ hybridization (FISH).

[0039]

[0064] As used herein, the term "substantially" means a qualitative situation representing the full or almost full scale or degree of the characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena, when they exist, rarely proceed to completion and / or to perfection, or achieve or avoid absolute results. In some embodiments, "substantially" means within about 20%. In some embodiments, "substantially" means within about 15%. In some embodiments, "substantially" means within about 10%. In some embodiments, "substantially" means within about 5%.

[0040]

[0065] As used herein, "target" means any molecule whose presence, location, and / or concentration is determined or determinable. Examples of targets include nucleic acid sequences and proteins such as those disclosed herein.

[0041]

[0066] As used herein, the term "transducer" refers to any device capable of converting a first type of energy, such as electrical energy, into acoustic energy, sonic energy, or vibrational energy. Typically, the first type of energy is electrical energy, electromagnetic energy, or electrostatic energy. The transducer may be of any type, including single-element type, multi-element type, array type, mechanically focused type, acoustically lens-type, mechanically unfocused type, mechanically collimated type, mechanically defocused type, mechanically scanned type, electronically scanned type, etc.

[0042]

[0067] As used herein, the abbreviation "Vpp" means peak-to-peak voltage.

[0068] Devices and Systems

[0069] While not wishing to be bound by any particular theory, non-contact on-slide distribution, replenishment, and / or mixing of fluids on a microscope slide is thought to be able to improve staining quality, reduce total assay time, save costs by requiring lower concentrations of reagents (e.g., antibodies), and eliminate the risk of cross-contamination. In view of this, one aspect of the present disclosure is a microscope slide holder comprising a slide support member and at least one acoustic source for introducing elastic waves into a microscope slide in communication with the slide support member such that one or more fluids present on the surface of the microscope slide are mixed non-contact. In some embodiments, the microscope slide contains a biological sample containing cells and / or tissue, and the mixing using elastic waves occurs without damaging the cells and / or tissue within the sample. In some embodiments, the mixing occurs without substantially increasing the temperature of any fluid present on the surface of the microscope slide and / or without increasing the temperature of the sample (e.g., without increasing the temperature of the fluid or sample by more than 10 degrees).

[0043]

[0070] In some embodiments, the elastic waves are generated by an acoustic source. In some embodiments, the acoustic source is a transducer. In some embodiments, the transducer is a mechanical transducer. In other embodiments, the transducer is a piezoelectric transducer. In some embodiments, the transducer consists of a piezoelectric wafer that generates mechanical vibrations. In some embodiments, the transducer is a unidirectional transducer that operates within a single axis, i.e., generates surface acoustic waves in only one direction, e.g., along the y-axis. In other embodiments, the transducer operates in multiple directions, e.g., along the x-axis and the y-axis.

[0044]

[0071] In some embodiments, the surface transducer is used to distribute or mix a fluid volume on the slide. Without wishing to be bound by any particular theory, it is believed that the low-frequency elastic waves generated by the surface transducer are radiated through a material assembly (such as those described herein) into the fluid volume on the slide to distribute and / or mix the solution. In some embodiments, an electromagnet driven by a signal generator moves the surface transducer up and down (e.g., from 0.1 mm to about 3 mm). More specifically, the surface transducer converts the input frequency and voltage into a vertical diaphragm displacement by the electromagnet. The oscillation of the transducer diaphragm propagates through the slide fixture to distribute and / or mix the fluid on the slide.

[0045]

[0072] In some embodiments, the acoustic source is a surface transducer having a diameter of about 5 mm to about 60 mm. In other embodiments, the acoustic source is a surface transducer having a diameter of about 10 mm to about 50 mm. In still other embodiments, the acoustic source is a surface transducer having a diameter of about 20 mm to about 50 mm.

[0046]

[0073] In some embodiments, the acoustic source operates at frequencies ranging from about 0.5 Hz to about 2000 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 0.5 Hz to about 1000 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 1 Hz to about 1000 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 1 Hz to about 750 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 1 Hz to about 500 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 1 Hz to about 250 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 1 Hz to about 200 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 1 Hz to about 150 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 10 Hz to about 200 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 50 Hz to about 200 Hz. In some embodiments, the acoustic source operates at frequencies ranging from about 100 Hz to about 200 Hz. In some embodiments, the acoustic source operates at a frequency of about 150 Hz.

[0047]

[0074] In some embodiments, the frequencies used in certain embodiments of the present disclosure are affected by the energy absorption characteristics of the sample or support member for a particular frequency. To the extent that a particular frequency is better absorbed or preferentially absorbed by the sample, it may be preferable for the effect of the elastic wave on mixing to be better affected. In some embodiments, the elastic wave can be delivered in the form of short pulses or as a continuous field over a defined length of time. In some embodiments, the pulses can be bundled or regularly spaced.

[0048]

[0075] In some embodiments, the acoustic source operates at a first frequency during a first time interval and then at a second frequency during a second time interval. In other embodiments, the acoustic source may initially operate at a first frequency, which may increase or decrease over time (e.g., increase or decrease over time at a predetermined interval). For example, the first frequency may be 100 Hz, the second frequency may be 200 Hz, and the frequency may be increased from 100 Hz to 200 Hz at 10 Hz intervals every 0.5 seconds until the 200 Hz frequency is reached. In other embodiments, the acoustic source employs frequency modulation, and thus the frequency deviates by an amount that is a value of + / - 20% of a predetermined frequency value. For example, an acoustic source operating at 100 Hz may deviate its frequency by + / - 10 Hz. In other embodiments, the acoustic source can operate at multiple frequencies simultaneously, each of which may be modulated.

[0049]

[0076] In embodiments where multiple acoustic sources are utilized, each acoustic source may operate at the same frequency (or range of frequencies) or at different frequencies (or different ranges of frequencies). In embodiments where multiple acoustic sources are utilized, each acoustic source may operate during different time intervals. For example, a first acoustic source may operate over a first time interval, followed by the operation of a second acoustic source over a second time interval (both time intervals may be at the same frequency or different frequencies).

[0050]

[0077] In embodiments where multiple acoustic sources are utilized, each acoustic source may operate in phase or out of phase with each other. Those skilled in the art will understand that by operating multiple acoustic sources, each acoustic source can be adjusted to operate in a phase - shifted manner, such that the elastic waves generated by the acoustic sources do not cancel each other out, thereby enabling efficient mixing of one or more fluids present on the surface of the microscope slide. Those skilled in the art will also understand that the frequency of each of the transducers can be modulated throughout their operation to be understood.

[0051]

[0078] In some embodiments, the input amplitude ranges from about 0.1 to about 1,000 mVpp. In other embodiments, the input amplitude ranges from about 40 to about 350 mVpp. In some embodiments, the input amplitude can be modulated over time. For example, the acoustic source can initially operate at a first amplitude, which can increase or rise over time until it reaches a final amplitude. In some embodiments, the amplitude is increased by a predetermined amount over a predetermined amount of time. In some embodiments, the input amplitude is selected such that the fluid present on the slide is retained, i.e., not displaced from the slide.

[0052]

[0079] FIG. 1A depicts a slide holder 5 having a slide support member 20 configured to support at least a portion of the back surface of the microscope slide 10. In some embodiments, the slide support member 20, such as that depicted in FIG. 1A, is substantially the same size as the microscope slide 10. Of course, those skilled in the art will understand that the slide support member 20 may be larger or smaller than the microscope slide 10 in any dimension. For example, as also depicted in FIG. 1F, the slide support member 20 is larger than the microscope slide 10, while in FIG. 1G, the slide support member 20 (dashed line) is depicted as being smaller than the microscope slide 10. In some embodiments, the support member has a thickness ranging from about 0.1 mm to about 20 mm.

[0053]

[0080] In some embodiments, the support member 20 is constructed of a material that enables elastic waves from the acoustic source 30 to be radiated, conducted, and / or transferred through the support surface 20 and to the specimen or fluid located on the slide 10. For example, the support member can be constructed of a metal (e.g., copper, aluminum, brass, chromium, silver, gold, platinum, and titanium, or an alloy containing any of the metals specified above).

[0054]

[0081] In some embodiments, the support member 20 comprises a heating element or other means for heating the microscope slide 10. In some embodiments, the support member 20 is a heating plate such as that depicted in FIG. 5A. In some embodiments, the support member 20 comprises a cooling element or other means for removing heat from the microscope slide 10. In other embodiments, a heat regulating plate comprising an element for heating or cooling the microscope slide is disposed between the support member 20 and the microscope slide 10.

[0055]

[0082] In some embodiments, the slide holder 5 comprises one or more retaining members 22, such as a spring, clip, or tab, for releasably securing the microscope slide 10 to the slide support member 20. In some embodiments, the retaining member 22 restricts axial, lateral, and vertical movement of the slide 10 when the slide is mounted on the support member 20.

[0056]

[0083] In some embodiments, the acoustic source 30 (or its upper surface 31) is in communication with the slide support member 20. In some embodiments, the acoustic source 30 is in contact with the bottom surface 27 of the slide support member 20 (see FIGS. 1A - 1G). In some embodiments, the acoustic source 30 is in contact only while the acoustic source is operating. In some embodiments, the acoustic source moves up and down to contact the slide support when operating. In other embodiments, the transducer is at least partially embedded within the slide support member 20 and, for example, is integrated within the slide support member 20 such that the slide support and the transducer form a single element.

[0057]

[0084] In other embodiments, the acoustic source (or the upper portion of the acoustic source) is the slide support member 2 Although it is fitted within 0, it still communicates with the bottom surface 26 of the support member 20 (see Fig. 1H). In yet other embodiments, the transducer communicates directly with the slide. For example, the sound source (or the upper part of the sound source) may be fitted within the slide support member 20 and communicate directly with the bottom surface 28 of the microscope slide 10 (see Fig. 1I). Fig. 1K illustrates a support member 20 having a cavity 29 for inserting a properly sized sound source.

[0058]

[0085] In some embodiments, the support member 20 communicates with a single transducer 30, as illustrated in Figs. 1A, 1E, and 1F. In other embodiments, the support member may communicate with a plurality of transducers. For example, Fig. 1C illustrates a slide support member 20 that communicates with two transducers 30A and 30B. Of course, those skilled in the art will understand that any number of transducers may communicate with the slide support member 20. Regardless of the number of transducers incorporated, those skilled in the art will understand that one or more transducers are configured such that the generated elastic waves, such as surface acoustic waves, are permitted to radiate to the microscope slide 10 and affect fluid mixing.

[0059]

[0086] It is to be understood by those skilled in the art that the transducer 30 can be placed at any position relative to the microscope slide 10. In some embodiments, the microscope slide 10 includes a label end 40 and a specimen-containing end 50, and the transducer is placed within the boundary of the specimen-containing end 50. In some embodiments, the transducer 30 is placed at a position approximating the center of the specimen-containing end 50 (see FIG. 1B). In other embodiments, the transducer is placed at a position along the slide 10 where most of the fluid will be dispensed. In some embodiments, the transducer is placed at least 1 cm from the end of the microscope slide. In some embodiments, the transducer is placed at least 1.5 cm from the end of the microscope slide. In other embodiments, the transducer is placed approximately 1 cm from the label end of the slide.

[0060]

[0087] In embodiments having multiple transducers, the transducers can be placed at any position relative to the slide. In some embodiments, as also illustrated in FIG. 1C, the transducers 30A and 30B can be placed at both ends of the support member 20. In other embodiments, as also illustrated in FIG. 1D, the upper portions 31A and 31B of the two transducers are positioned at the diagonal corners of the specimen-containing end 50 of the slide 10. In embodiments where multiple transducers are provided, the transducers can be spaced from each other by 0.1 cm to 10 cm.

[0061]

[0088] FIGS. 1A and 1C illustrate a transducer 30 having an upper surface 31 in a plane substantially parallel to the plane of the support member 20 or the slide 10, but it is to be understood by those skilled in the art that the upper surface 31 can be offset at any angle relative to the support member 20 or the slide 10.

[0062]

[0089] In some embodiments, one or more acoustic sources in communication with the microscope slide are surface transducers, and the total surface area of the transducers is at least about 5% of the surface area of the microscope slide. In other embodiments, one or more acoustic sources in communication with the microscope slide are surface transducers, and the total surface area of the transducers is at least about 10% of the surface area of the microscope slide. In still other embodiments, one or more acoustic sources in communication with the microscope slide are surface transducers, and the total surface area of the transducers is at least about 15% of the surface area of the microscope slide. In further embodiments, one or more acoustic sources in communication with the microscope slide are surface transducers, and the total surface area of the transducers is at least about 20% of the surface area of the microscope slide.

[0063]

[0090] In some embodiments, the acoustic source is movable relative to the microscope slide (e.g., by a stepper motor). For example, the acoustic source can be moved from a first position to a second position (along either the x-axis, the y-axis, or both) relative to the microscope slide 10 such that elastic waves are introduced along various portions of the microscope slide. Those skilled in the art will understand that movement of the acoustic source during operation can facilitate uniform mixing of the fluid on the surface of the microscope slide. Those skilled in the art will also understand that the acoustic source can be repositioned depending on the location where the specimen and / or fluid is provided on the surface of the microscope slide. In other embodiments, the acoustic source can be moved along the z-axis relative to the support member, such as to accommodate different support members, to enhance contact with the acoustically conductive substrate, and the like.

[0064]

[0091] In other embodiments, the microscope slide is movable relative to the acoustic source. For example, the microscope slide can be moved from a first position to a second position on a slide tray. For example, the first position can be a dispensing station such that the slide receives one or more fluids, and the second position can be a fluid distribution or mixing station having an acoustic source transducer for distributing, replenishing, and / or mixing the one or more dispensed fluids (as further described herein).

[0065]

[0092] In other embodiments, the slide holder 5 can comprise a plurality of support members 21. FIGS. 2A and 2B depict a microscope slide 10 supported by support members 21A and 21B located at each end of the slide 10. Alternatively, as shown in FIG. 2C, the microscope slide 10 can be supported by rails (not depicted) that extend substantially along the longitudinal edges of the slide. In these embodiments, the transducer 30, or its upper surface 31, may communicate directly with the bottom surface 28 of the microscope slide 10.

[0066]

[0093] Alternatively, the transducer 30, or its upper surface 31, may communicate indirectly with the microscope slide 10. For example, a conductive material 23 may be placed between the microscope slide 10 and the transducer 30 so as to enhance or distribute the elastic waves supplied to the microscope slide (such that the transducer would communicate with the bottom surface 27 of the conductive material 23). In some embodiments, the conductive material 23 is a substrate (e.g., a disposable substrate) having an outer shell and containing water, liquid, gel, hydrogel within the boundaries of the shell. In other embodiments, the conductive material 23 may be a liquid or gel placed between the slide 10 and the acoustic source 30. In still other embodiments, the conductive material is an acoustically conductive solid.

[0067]

[0094] In other embodiments, also referring to FIGS. 4A and 4B, the slide holder includes a support member 20 positioned between the microscope slide 10 and the cantilever 70. In these embodiments, the cantilever comprises at least a distal portion 71 in communication with the acoustic source 30. In some embodiments, neither the support member 20 nor the microscope slide 10 is positioned over the distal portion 71. In some embodiments, a single acoustic source 30 is placed within the boundaries of the distal portion 71. In other embodiments, multiple acoustic sources 30 are placed within the boundaries of the distal portion 71, and each of the multiple acoustic sources 30 can be spaced apart from or arranged relative to each other in any manner. In still other embodiments, one or more acoustic sources 30 are placed within the boundaries of the distal portion 71, and at least one additional acoustic source 30 is placed in communication with the cantilever 70 at a location under the support member 20.

[0068]

[0095] In some embodiments, the distal portion 70 extends from the support member 20 by at least 2.54 cm (1 inch). In other embodiments, the distal portion 70 extends from the support member 20 by at least 5.08 cm (2 inches). In still other embodiments, the distal portion 70 extends from the support member 20 by at least 7.62 cm (3 inches). In other embodiments, the distal portion 70 extends from the support member 20 by at least 10.16 cm (4 inches).

[0069]

[0096] In some embodiments, the cantilever 70 is constructed from a material selected from the group consisting of plastics (e.g., polyethylene terephthalate (PET), high density polyethylene (HDPE), polypropylene (PP), and low density polyethylene (LDPE)), and metals (such as aluminum, copper, stainless steel, etc.).

[0070]

[0097] In other embodiments, also referring to FIG. 4C, the slide holder includes a support member 20 positioned between the microscope slide 10 and the cantilever portion 70, and a fluid (e.g., LCS (Liquid Cover Slip), Ventana Medical Systems, Inc., Tucson, AZ, USA) is positioned between the support member 20 (identified as a heating plate) and the slide 10. In some embodiments, the surface of the microscope slide may include a plurality of individual liquid droplets dispensed separately on the surface of the slide, and the acoustic source 30 enables the distribution and / or mixing of those separate fluids.

[0071]

[0098] In some embodiments, the acoustic source may be coupled to an arm (or other similar member), and this arm may be positioned such that the acoustic source coupled to the arm contacts the support member or the slide, thereby enabling movement to introduce low-frequency elastic waves into the slide or the specimen on the slide.

[0072]

[0099] Slide tray

[0100] Referring to FIG. 3, the present disclosure also holds a plurality of microscope slides 10 Contemplated is a slide tray 100 configured such that each individual microscope slide 10 within the tray 100 is held in a separate horizontal position (but within the same plane) relative to adjacent slides, and each individual microscope slide 10 is in communication with an acoustic source 30 to enable non-contact mixing of one or more fluids present on the microscope slide. In some embodiments, each position within the slide tray may comprise a slide holder having an acoustic source, such as illustrated in any of FIGS. 1A-1G, FIGS. 2A-2C, FIGS. 4A-4C, FIGS. 5A-5B, or FIG. 6. In fact, each position within the slide tray may comprise a slide support member, one or more retaining members, and at least one acoustic source, as described above. A suitable slide tray may have any suitable shape, and the microscope slides held within a given slide tray may be arranged in any suitable manner to hold any suitable number of slides, such as 5 or more slides, or 10 or more slides, or 20 or more slides, or 30 or more slides. Some examples of slide trays of different shapes and holding capabilities are disclosed in U.S. Patent No. 7,468,161, which is hereby incorporated by reference in its entirety.

[0073]

[0100] In some embodiments, the slide tray is a generally rectangular tray configured to hold two rows of slides held side-by-side on opposite sides of a central longitudinal axis of the slide tray such that the longitudinal length of the slides is disposed outwardly from the long central axis of the tray. Each acoustic source 30 within the slide tray 100 may be communicatively coupled to a controller 60. In some embodiments, a material may be placed between each adjacent microscope slide or adjacent support member within the slide tray to reduce the amount of acoustic energy transmitted between adjacent slide holders, i.e., to prevent or mitigate the transmission of elastic waves from a first transducer at a first position to a microscope slide at a second position adjacent the first position.

[0074]

[0101] Automatic slide processing system

[0102] Another aspect of the present disclosure is a slide processing apparatus comprising at least one fluid dispenser configured to dispense fluid onto a slide, and a slide holder or slide tray comprising at least one acoustic source for non-contact mixing. Of course, those skilled in the art will understand that slide trays such as the above, which incorporate acoustic sources, can be utilized in the staining systems and specimen processing apparatuses described in U.S. Patent Nos. 8,663,991, 8,048,373, and 7,468,161, as well as U.S. Patent Publication No. 2016 / 0282239, and the disclosures of each of these patents are hereby incorporated by reference in their entirety.

[0075]

[0103] In some embodiments, the specimen processing apparatus incorporating the slide holder or slide tray described herein is the assignee of a number of U.S. patents that disclose systems and methods for performing automated analysis, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, as well as U.S. Published Patent Applications Nos. 20030211630 and 20040052685. It is an automated apparatus such as the BENCHMARK XT instrument, SYMPHONY instrument, BENCHMARK ULTRA instrument, etc., sold by Ventana Medical Systems, Inc., and the disclosures of each of these patents are hereby incorporated by reference in their entirety. Alternatively, the specimen can be processed manually.

[0076]

[0104] Examples of commercially available H&E stainers include the VENTANA SYMPHONY (individual slide stainer) and VENTANA HE600 (individual slide stainer) series H&E stainers from Roche, the Dako CoverStainer (batch stainer), the Leica ST4020 Small Linear from Leica Biosystems Nussloch GmbH Examples of the stainer include a Stainer (batch stainer), a Leica ST5020 Multistainer (batch stainer), and a Leica ST5010 Autostainer XL series (batch stainer) H&E stainer.

[0077]

[0105] The specimen processing apparatus can apply a fixative to the specimen. The fixative can include a cross-linking agent (aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde, and non-aldehyde cross-linking agents), an oxidizing agent (metal ions and metal complexes such as osmium tetroxide and chromic acid), a protein denaturant (such as acetic acid, methanol, and ethanol), a fixative of unknown mechanism (such as mercuric chloride, acetone, and picric acid), a combination reagent (such as Carnoy fixative, methacarn, Bouin fixative, B5 fixative, Rossman solution, and Gendre solution), microwaves, and miscellaneous fixatives (such as excluded volume fixation and vapor fixation). An acoustic source in communication with the microscope slide can be used to uniformly distribute any of these fixatives within the slide or another fluid, as detailed herein.

[0078]

[0106] If the specimen is a paraffin-embedded sample, the sample can be deparaffinized by the specimen processing apparatus using an appropriate deparaffinizing fluid. After the waste remover removes the deparaffinizing fluid, any number of substances can be successively applied to the specimen. This substance can be for pretreatment (such as protein cross-linking, nucleic acid exposure, etc.), denaturation, hybridization, washing (such as Stringency Wash), detection (such as linking a visual or marker molecule to a probe), amplification (such as amplification of proteins, genes, etc.), counterstaining, coverslipping, or the like. Again, any of these substances applied can be mixed or distributed by the use of an acoustic source.

[0079]

[0107] The specimen processing device can apply a wide range of substances to the specimen, which can then be uniformly distributed and / or mixed using an acoustic source in communication with the slide holder. The substances include, but are not limited to, stains, probes, reagents, rinse solutions, and / or conditioners. The substances can be a fluid (e.g., gas, liquid, or gas / liquid mixture), or the like. The fluid can be a solvent (e.g., polar solvent, non-polar solvent, etc.), a solution (e.g., aqueous solution, or other type of solution), or the like. The reagents can include, but are not limited to, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen recovery fluids (e.g., aqueous or non-aqueous based antigen recovery solutions, antigen recovery buffers, etc.), or the like. The probe can be an isolated nucleic acid or an isolated synthetic oligonucleotide attached to a detectable label. The label can include a radioisotope, an enzyme substrate, a cofactor, a ligand, a chemiluminescent or fluorescent agent, a hapten, and an enzyme.

[0080]

[0108] An automated IHC / ISH slide stainer typically includes at least a reservoir for various reagents used in the staining protocol, a reagent dispensing unit in fluid communication with the reservoir for dispensing the reagents onto the slide, a waste removal system for removing used reagents and other waste from the slide, and a control system for coordinating the operation of the reagent dispensing unit and the waste removal system. In addition to performing the staining steps, many automated slide stainers can also perform (or are compatible with a separate system for performing) auxiliary steps of staining, including slide baking (to adhere the sample to the slide), dewaxing (also referred to as deparaffinization), antigen retrieval, counterstaining, dehydration and clearing, and coverslipping. Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578-1582 (2014), which is hereby incorporated by reference in its entirety, describes some specific examples of automated IHC / ISH slide stainers, including intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND, and Lab Vision Autostainer (Thermo Scientific), and their various features. In addition, Ventana Medical Systems, Inc. is the assignee of several U.S. patents disclosing systems and methods for performing automated analysis, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Published Patent Applications Nos. 20030211630 and 20040052685, each of which is hereby incorporated by reference in its entirety.

[0081]

[0109] Commercially available staining units typically operate on one of the following principles: (1) Open individual slide staining where the slide is positioned horizontally and the reagent is dispensed as a pool on the surface of the slide containing the tissue sample (such as those implemented in the DAKO AUTOSTAINER Link 48 (Agilent Technologies) and intelliPATH (Biocare Medical) stainers), (2) Liquid overlay techniques where the reagent is either covered by or dispensed through an inert fluid layer deposited on top of the sample (such as those implemented in the VENTANA BenchMark and DISCOVERY stainers), (3) The slide surface is placed in proximity to another surface (which may be another slide or a cover plate) to create a narrow gap, and this gap causes capillary forces to rise and keep the liquid reagent in contact with the sample such as the staining principle used by capillary gap staining (DAKO TECHMATE, Leica BOND, and DAKO OMNIS stainers). Several repetitions of capillary gap staining do not mix the fluid in the gap (such as in DAKO TECHMATE and Leica BOND). In a variant of capillary gap staining called dynamic gap staining, capillary forces are used to apply the sample to the slide, and then the parallel surfaces are translated relative to each other to agitate the reagent during incubation for effective reagent mixing (such as the staining principle implemented in the DAKO OMNIS slide stainer (Agilent)). In translational gap staining, a translatable head is positioned over the slide. The lower surface of the head is spaced from the slide by a first gap small enough to allow a liquid meniscus to arise from the liquid on the slide during translation of the slide. A mixing extension having a lateral dimension smaller than the width of the slide extends from the lower surface of the translatable head, defining a second gap smaller than the first gap between the mixing extension and the slide. During translation of the head, the lateral dimension of the mixing extension is sufficient to create a lateral movement of the liquid on the slide in a direction generally extending from the second gap to the first gap. See WO2011-139978 A1. In recent years, it has been proposed to deposit reagents on slides using inkjet technology. See WO2016-170008 A1. This list of staining techniques is not intended to be comprehensive, and either a slide holder or a slide tray incorporating acoustic mixing can be used in conjunction with such a system to achieve distribution and mixing of the staining reagent.

[0082]

[0110] In some embodiments, an apparatus for automatically processing biological specimens, the apparatus comprising at least one slide tray (such as those described herein) for holding a plurality of slides in a substantially horizontal position, wherein a biological specimen is located on the slide and each of the plurality of slides is in communication with at least one acoustic source to enable introduction of an elastic wave into the slide and / or the sample; at least one slide tray; one or more workstations for receiving the slide tray and performing one or more slide processing operations on the plurality of slides held within the slide tray; a transport device for moving the slide tray into and out of the one or more workstations; a fluidics module in fluid communication with the one or more workstations for supplying reagents to the one or more workstations; an aerodynamics module in fluid communication with the one or more workstations and the fluidics module, the aerodynamics module supplying vacuum and / or pressurized gas to the one or more workstations and the fluidics module; and a control module in electrical communication with the transport device, the one or more workstations, the fluidics module, and the aerodynamics module, the control module coordinating the functions of the components of the apparatus (including the acoustic source) during processing of the biological specimen.

[0083]

[0111] In some embodiments, an apparatus for automatically processing a biological specimen further comprises a control system for independently controlling each acoustic source so as to independently vary the operating parameters of the acoustic source, and the controller includes at least a signal generator. In some embodiments, an apparatus for automatically processing a biological specimen further comprises one or more sensors or other feedback mechanisms to enable monitoring of the mixing and / or distribution of fluids dispensed on the surface of the slide. In some embodiments, the control system comprises a microprocessor and one or more microcontrollers, and the one or more microcontrollers receive instructions from the microprocessor and separately control one or more of the workstations, fluidics modules, one or more acoustic sources, and / or transport devices. In some embodiments, the slide tray comprises a plurality of slide holders, such as a slide holder as described herein or illustrated in FIGS. 1A-1G.

[0084]

[0112] In some embodiments, at least one of the workstations comprises a movable nozzle assembly, the nozzle assembly including one or more nozzles through which reagents are delivered to the slide. The nozzle may be a dispensing nozzle or a lifting nozzle.

[0085]

[0113] In some embodiments, the workstation can perform slide processing operations on one or more individual slides within the slide tray, e.g., at least two or four slides within the slide tray, or the workstation can simultaneously perform slide processing operations (including mixing operations by an acoustic source) on all of the slides within the slide tray. In some embodiments, one or more workstations dispense a reagent onto a slide within the slide tray such that a significant amount of the reagent contacting the first slide does not contact the second slide, thereby minimizing cross - contamination between the slides. Such a workstation can include one or more directional nozzles for dispensing the reagent onto the slide. For example, the one or more directional nozzles can include a pair of directional nozzles that dispense the reagent in opposite directions across the surface of the slide. In more specific embodiments, the one or more directional nozzles can further include a directional nozzle that dispenses the reagent towards the bottom surface of the slide. In other embodiments, one or more workstations can simultaneously dispense a reagent (e.g., the same reagent) onto at least two slides held within the slide tray within a given workstation, or one or more workstations can simultaneously dispense a reagent (e.g., the same reagent) onto all of the slides held within the slide tray within a given workstation. Following the dispensing of the fluid or reagent, an acoustic source can be activated to distribute and / or mix the fluid on the surface of the slide.

[0086]

[0114] In some embodiments, an automated method for processing a plurality of slides containing biological tissue samples, the method comprising performing slide processing operations on the plurality of slides at one or more workstations while the plurality of slides are held in a substantially horizontal position in a spatially co-planar manner within a slide tray, each of the plurality of slides being in communication with at least one acoustic source to enable introduction of elastic waves into the slide and / or the sample, the set of slide processing operations comprising at least flowing one or more staining agents from at least one reagent container, through a fluidics module, and out of at least one dispensing nozzle positioned over the slide tray to stain the sample on the slide in a substantially horizontal position in a spatially co-planar manner, and performing slide processing operations including solvent exchange; transporting the slide tray holding the plurality of slides to an automated coverslipper workstation after performing the set of slide processing operations including at least staining and solvent exchange; coverslipping the plurality of slides held within the slide tray using separate respective coverslips while the plurality of slides are held in a substantially horizontal position in a spatially co-planar manner within the slide tray such that the coverslips on the slides are separated from each other using the automated coverslipper workstation; and removing the slide tray holding the coverslipped slides from the automated coverslipper workstation.In some embodiments, the process comprises: (i) baking the sample under a radiative heating device; (ii) deparaffinizing the sample; (iii) staining the sample by delivering one or more staining agents through one or more fluidic components and out of one or more nozzles positioned generally over the slide tray, wherein the one or more fluidic components fluidically couple at least one reagent container holding the one or more staining agents to the one or more nozzles; (iv) solvent-exchanging the sample; and (v) coverslipping the sample using a separate coverslip, wherein the foregoing steps are automatically performed by an apparatus comprising two or more workstations, and the slide tray holding the slides is moved between these two or more workstations during processing. -shion are moved.

[0087]

[0115] In some embodiments, after the specimen has been processed, the user can transport the slide containing the specimen to an imaging device. In some embodiments, the imaging device is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the imaging device is a digital pathology device as disclosed in International Patent No. PCT / US2010 / 002772 entitled IMAGING SYSTEM AND TECHNIQUES (Publication No. WO / 2011 / 049608) or U.S. Patent Application Publication No. 2014 / 0178169, filed Feb. 3, 2014, entitled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International Patent Application No. PCT / US2010 / 002772 and U.S. Patent Application Publication No. 2014 / 0178169 are hereby incorporated by reference in their entirety. In other embodiments, the imaging device comprises a digital camera coupled to a microscope.

[0088]

[0116] Control system

[0117] Referring to FIG. 1A, transducer 30 can be controlled by controller 60. In some embodiments, as also depicted in FIG. 1E, the transducer can be communicatively coupled to a switch or timer 61, an amplifier 62, and / or a signal generator 63. In some embodiments, the control system comprises a printed device circuit board assembly (PCBA) including an amplifier and a signal generator. In some embodiments, the control system comprises a power subsystem that provides an electrical input for use by the signal generator. In some embodiments, the signal generator generates an oscillating electrical signal of sufficient amplitude and frequency to drive an acoustic source (e.g., a transducer). In some embodiments, the acoustic source (e.g., a transducer) converts the electrical signal supplied by the signal generator into acoustic vibrations. In some embodiments, the control system is contained within a casing. In some embodiments, the control system can be communicatively coupled to a computer 64. In some embodiments, the control system for the transducer is a controller used to control an automatic staining device.

[0089]

[0118] In some embodiments, the control system can further comprise one or more sensors for monitoring elastic waves provided to a microscope slide. In other embodiments, the control system can comprise one or more feedback mechanisms for monitoring the mixing of one or more fluids present on the surface of the slide.

[0090]

[0119] Optical or video detection and analysis can be used to optimize mixing. As an example, optical or video detection can be used to detect color changes as a coloring reagent mixes into a transparent fluid. Other optical measurements such as spectral excitation, absorption, light scattering, fluorescence, luminescence, radiation, polarized microscopy, Raman scattering, and spectral analysis can also be used to monitor the mixing of fluids in contact with a sample on the surface of a microscope slide. Data can be acquired and analyzed by a computer or control system that controls the mixing process. For example, based on the received data, if sufficient mixing is achieved, the transducer can be turned off. As another example, based on the received data, if mixing is insufficient, the controller can increase the time the transducer is turned on, or the controller can change the frequency of the transducer, or the power on an additional transducer.

[0091]

[0120] Method

[0121] Another aspect of the present disclosure provides a method of distributing and / or mixing a fluid on the surface of a microscope slide by introducing a low-frequency elastic wave (e.g., a frequency that does not damage cells, a frequency below 2000 Hz, etc.) into the microscope slide. In some embodiments, the introduction of the elastic wave facilitates the formation of a substantially uniform distribution of the fluid over a biological sample provided on the surface of the microscope slide. For example, the fluid can be dispensed into a predetermined region on the surface of the microscope slide, and upon activation of the acoustic source and introduction of the elastic wave, this fluid can be distributed beyond the initial region of dispensing. In some embodiments, the distribution of the fluid on the surface of the microscope slide by the introduction of the elastic wave can be used to facilitate replenishment of the fluid (e.g., a reagent) onto a biological sample loaded on the surface of the slide. For example, the biological sample can absorb (or can absorb unevenly) the reagent deposited on the surface, and ultimately, the amount of reagent in contact with the biological sample can be substantially depleted (or can be depleted from a particular region or portion of the sample). Activation of the acoustic source can facilitate the distribution of another aliquot of the reagent onto the biological sample, thus replenishing the reagent in contact with the biological sample. Activation of the acoustic source can also facilitate redistribution of the reagent from other regions of the slide to the biological sample faster than by diffusion means, thus replenishing the reagent in contact with the biological sample.

[0092]

[0122] In other embodiments, the first fluid may already be present on the surface of the microscope slide (e.g., a fluid pool), and following the introduction of a second fluid, e.g., a reagent, the second fluid may be substantially uniformly distributed within the first fluid following the introduction of an elastic wave. In some embodiments, being substantially uniformly distributed means that the reagent concentrations at two separate locations on the slide differ by no more than 15% in magnitude. In some embodiments, being substantially uniformly distributed means that the reagent concentrations at two separate locations on the slide differ by no more than 10% in magnitude. In other embodiments, being substantially uniformly distributed means that the reagent concentrations at two separate locations on the slide differ by no more than 5% in magnitude. In still other embodiments, being substantially uniformly distributed means that the reagent concentrations at two separate locations on the slide differ by no more than 2% in magnitude. Of course, one of ordinary skill in the art will understand that any number of fluids may be deposited on the surface of the microscope slide, and each of these fluids may be mixed, i.e., substantially uniformly distributed, within each other following the introduction of an elastic wave.

[0093]

[0123] In some embodiments, the methods disclosed herein are suitable for distributing and / or mixing any volume of fluid on the surface of a slide. In some embodiments, the volume of fluid that can be distributed and / or mixed according to the methods disclosed herein ranges from about 50 μL to about 2000 μL. In some embodiments, the volume of fluid that can be distributed and / or mixed according to the methods disclosed herein ranges from about 50 μL to about 1000 μL. In other embodiments, the volume of fluid that can be distributed and / or mixed according to the methods disclosed herein ranges from about 50 μL to about 750 μL. In yet other embodiments, the volume of fluid that can be distributed and / or mixed according to the methods disclosed herein ranges from about 50 μL to about 500 μL. In yet other embodiments, the volume of fluid that can be distributed and / or mixed according to the methods disclosed herein ranges from about 100 μL to about 500 μL. One of ordinary skill in the art can select an appropriate acoustic source, including all operating parameters (e.g., duration of operation, frequency, amplitude, amplitude modulation, frequency modulation, position of the acoustic source relative to the sample, etc.), such that the total volume present on the slide is distributed and / or mixed substantially uniformly as desired.

[0094]

[0124] Generally, the method includes (i) introducing a fluid onto the surface of a slide and (ii) introducing a low-frequency elastic wave onto the slide. In some embodiments, the method includes, but is not limited to, (a) a detection step for feedback control of the acoustic source, (b) a fluid removal step, and / or (c) a further fluid dispensing step. and may include additional steps.

[0095]

[0125] In some embodiments, a method of processing a slide containing a specimen includes (i) contacting a sample on the slide containing the specimen with a first reagent, and (ii) introducing an elastic wave into the slide containing the specimen to uniformly distribute the first reagent over the specimen. In some embodiments, the uniform distribution enables the fluid to penetrate into regions of the slide that are free of fluid. As described herein, the elastic wave can be generated by a transducer in communication with the sample. In some embodiments, the reagent is introduced into or proximate to a first fluid pool (e.g., a pool of liquid containing a buffer) on the surface of the slide. In some embodiments, the reagent is a detection probe. In some embodiments, the detection probe is a specific binding site for a particular target (e.g., an antibody, a nucleic acid) in a biological sample. In some embodiments, the detection probe utilized is a primary antibody, specifically, a primary antibody that enables the detection of a protein target (or an epitope of a protein target) in the sample. In some embodiments, the primary antibody is conjugated to a detectable label such as a fluorophore, a hapten, or an enzyme. In other embodiments, the detection probe is a nucleic acid probe that enables the detection of a nucleic acid sequence target in the sample. In other embodiments, the specific binding site is a nucleic acid probe, and this nucleic acid probe is conjugated to a detectable label or reporter such as a chromogen, a fluorophore, a hapten, or an enzyme.

[0096]

[0126] In some embodiments, following introduction of the reagent, low-frequency elastic waves are introduced into the sample. In some embodiments, the elastic waves are introduced over a time interval ranging from about 0.5 seconds to about 6 hours. In some embodiments, the elastic waves are introduced over a time interval ranging from about 0.5 seconds to about 240 seconds. In other embodiments, the elastic waves are introduced over a time interval ranging from about 1 second to about 180 seconds. In other embodiments, the elastic waves are introduced over a time interval ranging from about 1 second to about 120 seconds. In other embodiments, the elastic waves are introduced over a time interval ranging from about 1 second to about 60 seconds. In other embodiments, the elastic waves are introduced over a time interval ranging from about 1 second to about 30 seconds. In other embodiments, the elastic waves are introduced over a time interval ranging from about 1 second to about 15 seconds. In other embodiments, the elastic waves are introduced over a time interval ranging from about 5 seconds to about 10 seconds.

[0097]

[0127] In some embodiments, the method further includes detecting whether the fluid and / or reagent is appropriately distributed or mixed (e.g., by using a feedback mechanism as described herein). If the detection step determines that the fluid and / or reagent is not appropriately mixed, the operating parameters of the acoustic source can be adjusted (e.g., frequency, amplitude, duration of operation, pulsed versus continuous introduction of elastic waves, or any combination thereof).

[0098]

[0128] In some embodiments, the sample is pulsed with elastic waves. In some embodiments, the pulsed vibration of the sample by elastic waves may be at regular intervals. For example, the sample can be pulsed with elastic waves over a particular predetermined amount of time (e.g., about 0.5 second intervals), followed by a predetermined amount of time (e.g., about 1 second intervals) during which no elastic waves are introduced. In other embodiments, the pulsed vibration of the sample by elastic waves may be at irregular intervals. In other embodiments, the determination as to whether to pulse the sample with elastic waves can be made by using a detector that provides feedback regarding the degree of mixing, or a detector that can detect whether a slide or a portion of the sample needs replenishment.

[0099]

[0129] In some embodiments, the sample can be pulsed with elastic waves throughout the entire time interval during which the reagent is in contact with the sample (e.g., during an incubation period). For example, if an antibody is introduced into the sample and the protocol calls for the antibody to remain in contact with the sample over a time interval of about 360 seconds (e.g., an incubation period), elastic waves can be introduced into the sample at set intervals over a predetermined amount of time during the incubation period. For example, pulses of elastic waves can be introduced at 5 - second intervals at times 0, +30 seconds, +60 seconds, +90 seconds, +120 seconds, +150 seconds, +180 seconds, +210 seconds, +240 seconds, +270 seconds, +300 seconds, and +330 seconds after the introduction of the antibody. Of course, rather than pulsing the sample with elastic waves at predetermined intervals or over a predetermined amount of time, a feedback control device (such as those described herein) may be utilized to determine whether the introduction of pulses of elastic waves is necessary, including the length of time of the pulses.

[0100]

[0130] In other embodiments, the slide or sample can be pulsed with elastic waves each time fluid is dispensed onto the slide or sample and for any purpose (i.e., fluid replenishment, fluid dispensing, and / or fluid mixing). For example, if the protocol calls for adding a specific aliquot of fluid every two minutes, the elastic waves can be applied to the slide and / or sample for at least a predetermined amount of time each time an aliquot is added. Of course, additional pulses can be applied during the dispensing cycle as needed and as described above.

[0101]

[0131] In addition, when a particular system or device requires moving a slide between different stations or processing areas of the system or device (e.g., a sample staining area, a sample incubation area), elastic waves can be pulsed into the sample before and / or after the movement of the slide to ensure that the fluid is properly distributed and / or mixed before, during, and after such movement. Following the mixing of the first reagent into the first fluid pool, the mixed first reagent / fluid pool can be removed from the surface of the slide. Subsequently, the first detection reagent can be introduced and then distributed on the surface of the slide or mixed with a second fluid pool present on the slide. In some embodiments, the first detection reagent is specific to the label of the detection probe. For example, if the label is an enzyme, the substrate of the enzyme (a detectable moiety, e.g., a chromogenic moiety) can be introduced such that a colored precipitate can be detected. In still other embodiments, an anti-label antibody (secondary antibody) is introduced to elicit detection, and this anti-label antibody is specific to the label of the detection probe. For example, if the label is a hapten, an anti-hapten antibody specific to the hapten label is introduced, and this anti-hapten antibody contains a detectable moiety. In some embodiments, the detectable moiety of the anti-hapten antibody is an enzyme, and the substrate of the enzyme is further introduced to detect the detection probe and the target. The detectable moiety can then be detected according to processes known to those skilled in the art. The introduction of the detection probe and / or the detection reagent can be repeated "n" times to correspond to any desired number of targets within the sample.

[0102]

[0132] The methods disclosed herein are also suitable for multiplex assays. For example, a first detection probe specific for a first target and a second detection probe specific for a second target can be introduced simultaneously or sequentially. When both the first and second detection probes are introduced into the sample, an elastic wave can be introduced into the sample such that the first and second detection probes are mixed and uniformly distributed. Without wishing to be bound by any particular theory, it is believed that the distribution of the first and second detection probes can facilitate a uniform detection probe concentration during staining and / or reduction of staining artifacts. One of ordinary skill in the art will understand that any number of detection probes can be introduced into the sample on the surface of the slide simultaneously or sequentially, and that "n" detection probes can be mixed by the introduction of an elastic wave. As described herein, the elastic wave can be generated by an acoustic source in communication with the sample or as otherwise described herein. In some embodiments, the elastic wave is introduced over a time interval ranging from about 0. 5 seconds to about 6 hours. In some embodiments, the elastic wave is introduced over a time interval ranging from about 0.5 seconds to 240 seconds. Following the introduction of the detection probe, one or more detection reagents can also be introduced here, either simultaneously and / or sequentially, and can also be mixed here by introduction of an elastic wave.

[0103]

[0133] In some embodiments, a method of replenishing a fluid or reagent includes: (i) contacting a sample on a slide containing a specimen with a first reagent; (ii) ensuring a time for the reagent to react with the sample or be absorbed by the sample; and (iii) introducing a low-frequency elastic wave into the slide containing the specimen to uniformly distribute the first reagent over the specimen containing slide, thereby replenishing the reagent in regions where the reagent is at least partially depleted. In some embodiments, the method optionally includes introducing an additional aliquot of the first reagent prior to uniformly distributing the first reagent by introduction of the elastic wave. As described herein, the elastic wave can be generated by a transducer in communication with the sample. In some embodiments, the reagent is introduced into a first fluid pool (e.g., a liquid pool containing a buffer) on the surface of the slide. In some embodiments, the reagent is a detection probe. In some embodiments, the detection probe is a binding site specific for a particular target in a biological sample. In some embodiments, the detection probe utilized is a primary antibody, specifically a primary antibody that enables detection of a protein target (or an epitope of a protein target) in the sample. In some embodiments, the primary antibody is conjugated to a detectable label such as a fluorophore, hapten, or enzyme. In other embodiments, the detection probe is a nucleic acid probe that enables detection of a nucleic acid sequence target in the sample. In other embodiments, the specific binding site is a nucleic acid probe, and this nucleic acid probe is conjugated to a detectable label or reporter such as a chromogen, fluorophore, hapten, or enzyme.

[0104]

[0134] In some embodiments, a method of processing a slide containing a specimen includes distributing a first fluid over the specimen by (i) dispensing the first fluid onto a first portion of a microscope slide and (ii) introducing an elastic wave into the slide containing the specimen. In some embodiments, the first fluid is distributed from the first portion of the microscope slide to at least a second portion of the microscope slide. In some embodiments, the first portion of the slide is a portion that does not contain a sample, and the second portion of the slide contains a biological sample. In some embodiments, the fluid includes a detection probe. In some embodiments, the detection probe is a binding site specific to a particular target within the biological sample. In some embodiments, the method further includes introducing an additional aliquot of the fluid onto the slide (in any region) and then distributing the fluid by introducing an elastic wave.

[0105]

[0135] Examples

[0136] General experimental protocol

[0137] To mix the fluid on the slide, a 45 mm diameter transducer (4 ohm, 5W, Adafruit product ID = 1784) was used. To power the transducer, an amplifier IC board (Adafruit product ID = 1552, TPA2012) was required. The input to the amplifier was a signal generator sine wave, and the amplifier output to the transducer was a PWM wave. The amplifier IC, TPA2012D2, is a stereo class D audio amplifier with 2 x 2.1 outputs as shown in FIG. 3. The board had pins to adjust the gain of the amplifier, and all experiments were performed with a 24 dB gain. Two shutdown pins (SDR and SDL) were used to turn the transducer on / off via the PRISM board and Atlas software. This provided the advantage of specifying the length of time to mix and understanding how the transducer on:off ratio and period time length affected mixing. The signal generator used was an Agilent 33512B. Channel 1 of the signal generator was specifically... It was used to output a sine wave having a fixed frequency and amplitude. The frequency was the resonance of the assembly.

[0106]

[0138] The camera used was Edmunds Optics EO-0413C LE. The software for viewing images is uEye cockpit, which can be downloaded from the Edmunds optics website. The fps to be recorded can be specified within the software property options.

[0107]

[0139] The transducer is screwed to the slide processing station (SPS) hot plate, and the bottom of the transducer is fixed to the experimental bench. The wet slide is placed on the SPS heater plate. The label end of the slide is taped to the slide alignment plate and the SPS hot plate.

[0108]

[0140] The setting drawing is shown in Fig. 6.

Example

[0109]

[0141] Mixing of fluorescein into the buffer

[0142] Overview

[0143] A total of 500 μL of fluid volume, fluorescein dye, and salt buffer were pipetted onto the slide for mixing. The signal generator was configured to generate a sine wave of approximately 150 Hz and approximately 100 mVpp to operate the transducer vertically up and down, thereby enabling mixing of the fluid on the slide.

[0110]

[0144] To capture and visualize fluid mixing, UV light, fluorescein dye, a camera, and an enclosure were required (see Fig. 6), although none of these components were necessarily required for mixing. Fluorescein was placed in volumes of 10 μL in five regions on the slide, four corners and the center.

[0111]

[0145] General procedure

[0146] 1. To improve the connection between the slide and the SPS heating plate, pipette 15 μL of liquid coverslip (LCS) onto the SPS heating plate.

[0112]

[0147] 2. Wet the slide - Immerse the slide in the reagent and gently flick the slide.

[0148] 3. Place the slide on the heating plate with the label side up. Tape the label end of the slide to the processing mark on the slide alignment plate.

[0113]

[0149] 4. Add 450 μL of salt buffer and add 10 μL of fluorescein to each of the five spots at the four corners and the center to visualize the mixing.

[0150] 5. Drive the transducer at 150 Hz and 100 mVpp and turn on the signal generator to mix the fluid on the slide.

[0114]

[0151] 6. Turn off the transducer, remove the slide, and wipe the SPS heating plate with alcohol to clean it.

[0152] Results

[0153] The experimental setup parameters are shown in Table 1.

[0115]

Table 1

[0116]

[0154] The results of the mixing are illustrated in FIG. 7. The individual frames of FIG. 7 illustrate fluorescein dye spreading across the fluid volume on the slide. Approximately 1 second prior to reaching mixing, the dyes at the four corners of the free end of the slide and in the center of the slide were mixed. The dyes at the four corners closest to the taped end of the slide initiated mixing, but such mixing at those locations took longer, which is thought to be due to their ends being more mechanically rigid as they are pushed down by the tape. Qualitatively, it was estimated that it took approximately 7 seconds for the dye to spread uniformly across the fluid over the slide. The Applicants have shown that the non-contact mixing means disclosed herein advantageously enables mixing of fluids without contacting the sample with any physical device or gas stream. This is thought to eliminate concerns of potential cross-contamination between slides when this technology is incorporated into a staining platform. Here, since the frequency for mixing is relatively low, about 150 Hz, there is no expectation of tissue damage. While not wishing to be bound by any particular theory, cell damage occurs in the ultrasonic range (above about 18,000 Hz) and in commercially available sonication devices, cell damage is thought to be caused by shear forces generated by bubble collapse. In the system provided herein, no cavitation or bubble collapse was observed.

Example

[0117]

[0155] Quantification of Mixing

[0156] The motivation for this experiment was to quantify over time the degree of mixing in the stained area. Due to ripples, waves, and shadowing caused by the vibration waves across the fluid, it was necessary to turn off the transducer to capture frames where the pixel values were free of interference.

[0118]

[0157] The experimental setup was the same as that presented in Example 1 within this specification. To define mixing To quantify, data from individual frames of the video data (see Figure 8A) was extracted and the standard deviation of the pixels within the region of interest was determined (see Figure 8B). More specifically, Figure 8A provides (a) an image of the fluid volume on the slide before mixing. The white box designates the region of interest (ROI). The ROI is 398 x 181 pixels, which is the same as 70% of the slide staining area or approximately 50 x 20 mm. The ROI designates the area of the frame that will be analyzed to quantify mixing. (b) and (c) 3.3 seconds of mixing and (c) 13.3 seconds of mixing. These frames were captured when the transducer was posed to capture the fluid without mixing artifacts. More specifically, Figure 8B illustrates that (a) the standard deviation of the green values of the pixels within the region of interest (ROI) is calculated for each frame and (b) provides a plot of the green value standard deviation within the ROI during the mixing process. In short, when fluorescein was mixed across the entire predefined region of interest, the individual green values of each pixel began to trend towards the green neighboring pixels of the same value. Ideally, a uniformly mixed solution was assumed to have a standard deviation of 0.

Example

[0119]

[0158] Input voltage sweep

[0159] The motivation for this experiment was to visualize and observe wave propagation within the fluid on the slide at different input voltages from 45 to 90 mVpp. The transducer was turned on and the voltage was continuously swept at five different input values of 45 mVpp, 50 mVpp, 55 mVpp, 60 mVpp, and 90 mVpp. As the voltage increased, it was observed that the area of the standing wave across the slide increased (see Figure 9). At 45 mVpp, the standing wave was limited to one corner of the slide, but at 90 mVpp, the standing wave was present across the entire slide. At 50 mVpp, the standing wave was seen at the end of the slide where it propagated most freely vertically. This suggested that the slide could function as a cantilever (see Figure 4 within this specification for embodiments employing a cantilever design).

Example

[0120]

[0160] Fluid volume on the slide

[0161] In this set of embodiments, the fluid volumes on the slides tested were 300 μL and 100 μL to observe coating and mixing. The transducer electronic settings and procedures were as described in Example 1 within this specification. The inputs to the transducer were 150 Hz (resonance) and 100 mVpp. The dye used to observe mixing and coating was fluorescein. The dye was 10% of the total fluid volume on the slide.

[0121]

[0162] A fluid volume of 300 μL on the slide demonstrated coating and mixing as shown in FIG. 10A. More specifically, FIG. 10A illustrates (a) 30 μL of fluorescein dye and 270 μL of Apollo buffer volume on the slide before mixing, (b) 6 seconds of transducer on. The fluid front is driven towards the labeled end of the slide, spreading the fluid across the slide. (c) 20 seconds of mixing (d) 2 minutes 30 seconds of mixing - the fluid covers almost the entire slide and the dye is mixed by the solution. A fluid volume of 100 μL on the slide demonstrated mixing as shown in 10B, but did not demonstrate coating. More specifically, FIG. 10B illustrates (a) 10 μL of fluorescein dye and 90 μL of Apollo buffer solution before mixing. (b) 20 seconds of mixing. The fluid pools at one end of the slide due to the SPS heater and transducer not being flat. (c) 1 minute 20 seconds of mixing. The fluid is mixed, but the fluid does not spread across the slide. (d) Visible light image of the mixed fluid. A summary of the results from the 500 μL, 300 μL, and 100 μL mixing experiments is shown in Table 1 below.

[0122]

Table 2

Example

[0123]

[0163] Transducer position

[0164] The purpose of this experimental set was to sweep the transducer position along the SPS heating plate in order to qualitatively determine the location of the transducer that demonstrated the fastest mixing. Four locations were tested, such as those illustrated in Figure 5B. The set-up and procedure for these experiments were as described in Example 1. To visualize the mixing, fluorescein was used as the dye, and a total of 50 μL of fluorescein was distributed in 450 μL of saline buffer volume, 10 μL at a time, at the four corners and the center of the slide.

[0124]

[0165] The results are summarized in Table 2 below. Each setting was evaluated over time to qualitatively determine whether the fluid on the slide was homogeneously mixed. Mixing the fluid using the transducer at either end, the free end, or the labeled end of the SPS heating plate required a higher input voltage and time to completely mix the solution on the slide. The transducer appeared to mix the fluid on the slide the fastest (in about 7 seconds) when positioned in the center of the slide.

[0125]

Table 3

Example

[0126]

[0166] Transducer period vs on:off ratio

[0167] In the previous mixing experiments using the transducer, it was observed that by turning the transducer on and off, respectively, the fluid on the slide appeared to be spread (pushed outwards) and contracted (relaxed). These experiments examined whether this spreading and contraction could enhance mixing.

[0127]

[0168] Mixing using the pause in the transducer introduced three parameters - 1) total transducer period - the length of time of a single on and off cycle, 2) transducer on time, and 3) transducer off time.

[0128]

[0169] The set of experiments was designed to explore multiple transducer period times and extreme transducer on:off ratios. The settings for these experiments are as described with reference to Example 1. The on:off time intervals were set using software (see Figure 11).

[0129]

[0170] Table 3 summarizes the results of the study, where the approximate time to mixing is the time for the fluid to be uniformly mixed on the slide. This was determined qualitatively. Since uniform mixing on the slide was achieved in 7 seconds in this setting, 5 seconds was selected as the maximum period time.

[0130]

Table 4

[0131]

[0171] The results showed that longer transducer on times promoted mixing. Since this was a qualitative observation, and also because the transducer may have generated ripples in the fluid while on, it was difficult to know for certain when the solution was homogeneously mixed. However, a transducer period of about 5 seconds at an on ratio of about 90% was predicted to mix the fluid at about the same rate as when the transducer was on 100% of the time. Therefore, turning the transducer off does not appear to affect accelerating the mixing time.

[0132]

[0172] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments may be modified if further embodiments need to be provided using concepts from various patents, applications, and publications.

[0133]

[0173] Although the present disclosure has been described with reference to several exemplary embodiments, it is understood that numerous other modifications and embodiments may be devised by those skilled in the art that fall within the spirit and scope of the principles of the present disclosure. More specifically, suitable variations and modifications are possible within the scope of the foregoing disclosure, drawings, and appended claims without departing from the spirit of the present disclosure in the arrangement of component parts and / or the combination and arrangement of objects. In addition to variations and modifications in component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

1. 1. A slide holder for non-contact mixing of fluids on a microscope slide containing a specimen, comprising a slide support and at least one transducer in communication with the slide support, the at least one transducer operating at a frequency ranging from about 1 Hz to about 1 kHz.

2. The slide holder of claim 1 , wherein the at least one transducer is in communication with a lower surface of the slide support.

3. 3. The slide holder of claim 1 or 2, wherein the at least one transducer is positioned along the slide support in an area substantially corresponding to the center of a specimen edge of a microscope slide containing the specimen.

4. 4. A slide holder according to claim 1, wherein at least two transducers are in communication with the slide support.

5. 5. The slide holder of claim 4, wherein each of the at least two transducers is configured such that a first acoustic wave provided by a first transducer of the at least two transducers is not canceled by a second acoustic wave provided by a second transducer of the at least two transducers.

6. The slide holder of claim 5 , wherein the at least two transducers are out of phase with each other.

7. 7. The slide holder of claim 1, wherein the frequency of the at least one transducer ranges from about 1 Hz to about 500 Hz.

8. 8. The slide holder of claim 1, wherein the frequency of the at least one transducer ranges from about 50 Hz to about 500 Hz.

9. 9. The slide holder of claim 1, wherein the frequency of the at least one transducer ranges from about 100 Hz to about 200 Hz.

10. 10. The slide holder of claim 1, wherein the power supplied to the at least one transducer ranges from about 40 mVpp to about 350 mVpp.

11. 11. A slide holder according to claim 1, wherein the slide support has a support surface configured to support at least a portion of a back side of a microscope slide containing the specimen, the back side being opposite the surface containing the specimen.

12. The slide holder of claim 11 , wherein the slide support further comprises a heating element.

13. 13. The slide holder of claim 1, further comprising a controller in electrical communication with the at least one transducer.

14. The slide holder of claim 13 , wherein the controller includes an amplifier and a signal generator.

15. A slide holder comprising a slide support and at least one acoustic source for introducing low frequency vibrations to a microscope slide such that one or more fluids present on a surface of the microscope slide are mixed in a contactless manner.

16. 16. The slide holder of claim 15, wherein the low frequency vibration is an elastic wave.

17. 18. A slide holder according to any one of claims 15 to 17, wherein the acoustic source for introducing low frequency vibrations is selected from the group consisting of a mechanical transducer, a piezoelectric transducer, and a surface acoustic wave device.

18. 18. A tray comprising a plurality of slide holders according to any one of claims 1 to 17, wherein each slide holder of the plurality of slide holders is positioned in substantially horizontal and coplanar spaced apart positions.

19. 1. A method of processing a slide containing a specimen, comprising: (i) contacting a sample on the slide containing the specimen with a first reagent; and (ii) uniformly distributing the first reagent on the slide containing the specimen by introducing low frequency acoustic waves into the slide containing the specimen, the low frequency acoustic waves having a frequency ranging from about 1 Hz to about 1 Khz.

20. 20. The method of claim 19, wherein the low frequency acoustic waves are generated by at least one transducer in communication with a slide containing the specimen.

21. 21. The method of claim 19 or 20, wherein the first reagent is dispensed into an existing fluid present on a slide containing the specimen, and the elastic waves generated by the at least one transducer homogenously mix the first reagent within the existing fluid.

22. 22. The method of claim 21, wherein the pre-existing fluid is a buffer solution.

23. 21. The method of claim 20, wherein the at least one transducer is configured to operate at a frequency spanning the range of about 1 Hz to about 500 Hz.

24. 24. The method of claim 23, wherein the frequency ranges from about 50 Hz to about 500 Hz.

25. 24. The method of claim 23, wherein the frequency ranges from about 100 Hz to about 200 Hz.

26. 26. The method of any one of claims 19 to 25, wherein elastic waves are introduced into the sample for a time interval ranging from about 1 second to about 120 seconds.

27. 27. The method of claim 26, wherein the time interval ranges from about 1 second to about 60 seconds.

28. 27. The method of claim 26, wherein the time interval ranges from about 1 second to about 30 seconds.

29. 27. The method of claim 26, wherein the time interval ranges from about 2 seconds to about 15 seconds.

30. 21. The method of claim 20, wherein a bottom surface of the slide containing the specimen is at least partially in contact with a substrate, and the at least one transducer is coupled to the substrate. 。

31. 22. The method of claim 21, wherein the first reagent is a detection probe specific for a first target in the sample.

32. 32. The method of any one of claims 19 to 31, further comprising contacting the sample with a first detection reagent to facilitate detection of the first detection probe.

33. 33. The method of any one of claims 19 to 32, further comprising contacting the sample with a second detection probe specific for a second target.

34. 34. The method of claim 33, wherein the second detection probe is introduced simultaneously with the first detection probe, and the at least one transducer mixes the first detection probe and the second detection probe.

35. 34. The method of claim 33, wherein the first detection probe and the second detection probe are antibodies.

36. 1. A method for staining a sample, comprising: (a) dispensing a reagent into a fluid reservoir present on a microscope slide containing the sample; and (b) non-contact dispensing of the reagent into the fluid reservoir by introducing low frequency acoustic waves into the slide, wherein dispensing the reagent occurs without damaging cells or tissue within the sample.

37. 37. The method of claim 36, wherein the reagent is substantially uniformly dispensed within the fluid reservoir within about 30 seconds after introducing the low frequency acoustic waves.

38. 38. The method of claim 36 or 37, wherein the acoustic source for inducing the low frequency acoustic waves is selected from the group consisting of a mechanical transducer, a piezoelectric transducer, and a surface acoustic wave device.

39. 40. The method of claim 38, wherein the acoustic source operates at a frequency spanning the range of about 100 Hz to about 200 Hz.

40. 40. The method of any one of claims 36 to 39, wherein the reagent is a specific binding site.

41. 41. The method of claim 40, wherein the specific binding site comprises an antibody.

42. 41. The method of claim 40, further comprising dispensing a second reagent into the fluid reservoir.

43. A staining device comprising one or more slide holders according to claim 1 and at least one dispenser capable of introducing one or more fluids to a specimen-containing surface of a microscope slide containing the specimen.

44. 44. The staining apparatus of claim 43, further comprising a feedback control device for monitoring mixing of the one or more fluids introduced to the surface of the microscope slide containing the specimen.

45. 45. The dye solution of claim 43 or 44, wherein dispensing the one or more fluids occurs without contacting the reservoir or sample with any mixing device or gas flow. Color device.

46. 45. The staining apparatus of claim 43 or 44, further comprising an active mixing device in contact with a fluid reservoir on the surface of the microscope slide containing the specimen.

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