Non-contact on-slide fluid mixing

Abstract

To provide efficient mixing of fluids on slides in automated staining devices.SOLUTION: A microscope slide holder (5) comprises a slide support member (20) and at least one acoustic source (30) for introducing acoustic waves to a microscope slide (10) in communication with the slide support member (20) such that one or more fluids present on the surface of the microscope slide (10) are contactlessly mixed.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 incorporated herein by reference in its entirety. [Background technology]

[0002]

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

[0003]

[0003] Devices for automated staining, particularly for high-volume staining with conventional reagents such as hematoxylin and eosin (H&E), are primarily of the "dip-and-dunk" type, in which a rack of slides is automatically lowered into and removed from a series of reagent baths. For example, U.S. Patent No. 4,911,098 to Tabata describes an automated staining apparatus in which microscope slides holding tissue specimens are immersed successively into multiple containers of chemical solution. The slides are mounted vertically in a slide holder basket, and clamps that engage and disengage from the basket are used to move the slides from solution to solution. The clamps may include a mechanism for tilting the basket, which helps remove excess solution before the basket is immersed in the next solution. Additional automated staining devices of the "dip and dunk" type are described in U.S. Pat. No. 5,573,727 to Keefe, U.S. Pat. No. 6,080,363 to Takahasi et al., U.S. Pat. No. 6,436,348 to Ljungmann et al., and U.S. Patent Application Publication No. 2001 / 0019703, which names Thiem et al. as inventors.

[0004] Another type of automated stainer delivers fresh reagents directly to individual slides. For example, U.S. Patent No. 6,387,326 to Edwards et al. describes an apparatus for staining slides in which slides are released one at a time from a slide storage device and individually processed at various staining stations as the slides move along a conveyor belt transport device. Additional devices for automatically staining individual slides are described in U.S. Patent No. 6,180,061 to Bogen et al., PCT Publication WO 03 / 045560 naming Tseung et al. as inventors, and U.S. Patent Application Publication No. US 2004 / 0052685 naming Richards et al. as inventors.

[0005] Efficient mixing of fluids is a critical step in many industrial, chemical, and pharmaceutical processes, as well as biotechnological applications. Mixing in small volumes is often a difficult task. In some embodiments, molecular diffusion is the predominant mixing mechanism, which slows down the overall process. Integration of active mixers is often difficult, increasing the cost of any such device and resulting in cross-contamination between samples. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 4,911,098 [Patent Document 2] U.S. Patent No. 5,573,727 [Patent Document 3] U.S. Patent No. 6,080,363 [Patent Document 4] U.S. Patent No. 6,436,348 [Patent Document 5] US Patent Application Publication No. 2001 / 0019703 [Patent Document 6] U.S. Patent No. 6,387,326 [Patent Document 7] U.S. Patent No. 6,180,061 [Patent Document 8] PCT Publication No. WO03 / 045560 [Patent Document 9] US Patent Application Publication No. US2004 / 0052685 [Patent Document 10] WO2015 / 086484 [Patent Document 11] WO2010 / 080287 [Patent Document 12] U.S. Patent No. 7,615,371 [Patent Document 13] U.S. Patent No. 7,468,161 [Patent Document 14] U.S. Patent No. 8,663,991 [Patent Document 15] U.S. Patent No. 8,048,373 [Patent Document 16] U.S. Patent No. 7,468,161 [Patent Document 17] U.S. Patent Publication No. 2016 / 0282239 [Patent Document 18] U.S. Patent No. 5,650,327 [Patent Document 19] U.S. Patent No. 5,654,200 [Patent Document 20] U.S. Patent No. 6,296,809 [Patent Document 21] U.S. Patent No. 6,352,861 [Patent Document 22] U.S. Patent No. 6,827,901 [Patent Document 23] U.S. Patent No. 6,943,029 [Patent Document 24] U.S. Published Patent Application No. 20030211630 [Patent Document 25] U.S. Published Patent Application No. 20040052685 [Patent Document 26] WO2011-139978 A1 [Patent Document 27] WO2016-170008 A1 [Patent Document 28] International Patent Number: PCT / US2010 / 002772 (Patent Publication Number: WO / 2011 / 049608) [Patent Document 29] US Patent Application Publication No. 2014 / 0178169 [Non-patent literature]

[0007] [Non-Patent Document 1] Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578-1582 (2014) 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 discovered that using low frequency acoustic or vibrational energy (e.g., at frequencies that do not damage cells, such as frequencies below 2000 Hz) to replenishing, distributing, and / or mixing one or more fluids present on the surface of a substrate allows for high mixing efficiency, reduced process time, and prevention of contamination while maintaining low manufacturing and maintenance costs. Applicants have also surprisingly discovered 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 discovered that the aforementioned benefits can be realized without causing damage (e.g., by lysis or cell membrane disruption) to cells and / or tissues present on the substrate, and without unnecessarily heating the sample. Applicants have also discovered that mixing fluids according to the methods described herein (i) reduces the risk of staining artifacts and (ii) allows for uniform antibody reagent concentrations across cells and / or tissues during staining. It is also believed that use of the devices and methods disclosed herein may allow for the use of lower concentrations of detection probes (e.g., antibodies) in any staining procedure.

[0009]

[0007] One aspect of the 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 fluids present on a surface of a substrate (e.g., a microscope slide containing a specimen), where the acoustic source is in communication with the support member or substrate. "In communication with the support member or substrate" means, for example, that the acoustic source is at least partially in contact with the support member or substrate, or can be moved to be placed in at least partially in contact with the support member or 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 substrate. In some embodiments, the acoustic source is a mechanical or piezoelectric transducer operating at low frequencies (e.g., frequencies ranging from about 1 Hz to about 1 kHz). In some embodiments, it is a microscope slide. Other examples of substrates include surface-enhanced laser desorption / ionization ("SELDI") and matrix-assisted laser desorption / ionization ("MALDI") chips, and silicon wafers.

[0010] Another aspect of the 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 specimen-containing slide comprising a slide support member and at least one transducer in communication with the slide support member, the at least one transducer operating at a frequency ranging from about 1 Hz to about 1 kHz. In some embodiments, the at least one transducer is in communication with a lower surface of the slide support member. In some embodiments, the at least one transducer is positioned along the slide support member in an area substantially corresponding to a center of the specimen end of the slide.

[0011] In some embodiments, the at least two transducers are in communication with the slide support. In some embodiments, each of the at least two transducers is configured such that a first acoustic wave (or a first series of acoustic waves) provided by a first transducer of the at least two transducers is not canceled by a second acoustic wave (or a second series of acoustic waves) provided by a second transducer of the at least two transducers. In some embodiments, the at least two transducers are out of phase with each other.

[0012] In some embodiments, the 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 the at least one transducer ranges from about 40 mVpp to about 350 mVpp.

[0013] In some embodiments, the slide support has a support surface configured to support at least a portion of the backside of the slide, the backside being opposite 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 the at least one transducer. In some embodiments, the controller comprises an amplifier and a signal generator. In some embodiments, the slide holder is part of a staining apparatus or a coverslipping apparatus, as further described herein. In other embodiments, the slide support is further configured to include a heating and / or cooling element, such as a resistive heating element or a Peltier heating and cooling element.

[0014] Another aspect of the disclosure is a slide support and at least one acoustic source for introducing low frequency vibrations to the slide such that one or more fluids present on the surface of the slide are mixed in a non-contact manner. 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 slide holders described above. In some embodiments, each slide holder is positioned at substantially horizontal and coplanar spaced apart locations. In other embodiments, each slide holder is positioned along an arc. Another aspect is a movable slide support including the slide holders described above.

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

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

[0018] Another aspect of the disclosure is 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. In some embodiments, the low frequency acoustic 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 acoustic 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] In some embodiments, the 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 acoustic waves are introduced into the sample for 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 in at least partial contact with a substrate, and the at least one transducer is coupled to the substrate.

[0020] 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 disclosure is a method of replenishing a reagent, comprising: (i) contacting a sample on a slide containing the specimen with a first reagent; (ii) allowing time for the first reagent to react with or be absorbed by the sample; and (iii) uniformly distributing the first reagent on the slide containing the specimen by introducing elastic waves onto the slide containing the specimen, thereby replenishing regions where the reagent has been 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 introducing elastic waves. In some embodiments, the process may be repeated with additional reagent.

[0022] Another aspect of the disclosure is a method of staining a sample, comprising: (a) dispensing a reagent into a fluid reservoir present on a slide containing the sample; and (b) non-contact dispensing the reagent into the fluid reservoir using low frequency acoustic waves, where dispensing the reagent occurs without damaging cells or tissues within the sample. In some embodiments, the reagent is dispensed substantially uniformly into the fluid reservoir within about 30 seconds after initiating dispensing of the reagent. In some embodiments, the non-contact dispensing of the reagent is accomplished 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 moiety. In some embodiments, the specific binding moiety comprises an antibody. In some embodiments, the method further comprises dispensing a second reagent into the fluid reservoir. [Brief description of the drawings]

[0023] [Figure 1A] FIG. 1 is a side view of a microscope slide holder in communication with a transducer. [Figure 1B] FIG. 1 is a top view of a microscope slide holder in communication with a transducer. [Figure 1C] FIG. 1 is a side view of a microscope slide holder in communication with multiple transducers. [Figure 1D]

[0024] FIG. 1 is a top view of a microscope slide holder in communication with multiple transducers. [Figure 1E]

[0025] FIG. 1 is a side view of a microscope slide holder in communication with a transducer. [Figure 1F]

[0026] FIG. 1 is a top view of a microscope slide holder in communication with a transducer, in which the support member is larger in at least one dimension compared to the microscope slide. [Figure 1G]

[0027] FIG. 1 is a top view of a microscope slide holder in communication with a transducer, the support member being smaller in at least one dimension compared to the microscope slide. [Figure 1H]

[0028] FIG. 1 is a side view of a microscope slide holder in communication with a transducer. [Figure 1I]

[0029] FIG. 1 is a side view of a microscope slide holder in communication with a transducer. [Figure 1J]

[0030] FIG. 1 is a top view of a microscope slide holder in communication with a transducer. [Figure 2A]

[0031] FIG. 1 is a side view of a microscope slide holder in communication with a transducer. [Figure 2B] FIG. 1 is a side view of a microscope slide holder in communication with a transducer. [Figure 2C]

[0032] FIG. 1 is a top view of a microscope slide holder in communication with a transducer. [Diagram 3]

[0033] FIG. 1 is a top view of a slide tray comprising multiple microscope slide holders, each slide holder in communication with at least one transducer. [Figure 4A]

[0034] FIG. 1 is a side view of a microscope slide holder in communication with a transducer, where the slide holder includes a cantilever portion. [Figure 4B]

[0035] FIG. 1 is a top view of a microscope slide holder in communication with a transducer, where the slide holder includes a cantilever portion. [Figure 4C]

[0036] FIG. 1 is a side view of a microscope slide holder in communication with a transducer, the slide holder including a cantilever and also including a heating plate. [Figure 5A]

[0037] FIG. 1 is a side view of a microscope slide holder in communication with a transducer, the slide holder also including a heating plate. [Figure 5B]

[0038] FIG. 1 is a side view of a microscope slide holder in communication with multiple transducers, where the slide holder also includes a heating plate. [Figure 6]

[0039] FIG. 1 is a side view of a system with a microscope slide holder in communication with a transducer, the system also including elements for feedback control. The slide processing station (SPS) heated plate is the fixture on which the slide rests. The fluid volume on the slide is mixed by the displacement of the transducer. A signal generator provides the frequency and voltage to drive the transducer. A UV light, camera, and enclosure are used to visualize and quantify the fluid mixing by enhancing the fluorescent signal and removing interference from ambient light. [Figure 7A]

[0040] FIG. 1 illustrates the distribution of a dye placed at the corner of a slide over time as an elastic wave is introduced. [Figure 7B] FIG. 1 illustrates the distribution of a dye placed at the corner of a slide over time as an elastic wave is introduced. [Figure 7C] FIG. 1 illustrates the distribution of a dye placed at the corner of a slide over time as an elastic wave is introduced. [Figure 7D] FIG. 1 illustrates the distribution of a dye placed at the corner of a slide over time as an elastic wave is introduced. [Figure 8A]

[0041] FIG. 1 illustrates the distribution of a dye placed at the corner of a slide over time as an elastic wave is introduced. [Figure 8B]

[0042] 13A illustrates that the standard deviation of green values ​​of pixels within a region of interest (ROI) is calculated for each frame; and FIG. 13B provides a plot of the green value standard deviation within the ROI during the blending process. [Figure 9]

[0043] FIG. 13 illustrates the results of sweeping the input voltage from a first voltage to a second, higher voltage, illustrating that the area of ​​the standing wave pattern increases as the input voltage increases. [Figure 10A]

[0044] FIG. 1 illustrates the distribution of dye over time when an elastic wave is introduced. [Figure 10B]

[0045] FIG. 1 illustrates the distribution of dye over time when an elastic wave is introduced. [Figure 11]

[0046] FIG. 13 is a diagram illustrating the relationship between total transducer period time, on time, and off time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024]

[0047] definition

[0048] It is also to be understood that, unless otherwise expressly indicated to the contrary, in any method claimed herein that includes more than one step or act, 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 indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0026]

[0050] As used herein and in 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" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including two or more of a number of elements or a list of elements, and, optionally, additional unlisted items. Only terms expressly indicated to the contrary, such as "only one of," "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a number of elements or a list of elements. In general, the term "or," as used herein, shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when followed by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0027]

[0051] As used herein and in the claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements may, optionally, be present other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, in other words, "at least one of A or B"; or, in other words, "at least one of A and / or B") may refer, for example, in one embodiment, to at least one that optionally includes more than one As and no Bs (and, optionally, includes elements other than B); in another embodiment, to at least one that includes more than one Bs and no As (and, optionally, includes elements other than A); and in yet another embodiment, to at least one that optionally includes more than one As, and further, optionally includes more than one Bs (and, optionally, includes other elements).

[0028]

[0052] As used herein, the terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Specifically, each of these terms is defined consistent with the general U.S. Patent Law definition of "comprising," and thus is interpreted as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means a device that includes at least components a, b, and c. Similarly, "a method with steps a, b, and c" means a method that includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined herein in a particular order, one of ordinary skill in the art will recognize that the order of steps and processes may 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 forms of these terms, as well as any other type of energy having similar properties to acoustic energy. Each of these terms may be used interchangeably herein.

[0030]

[0054] As used herein, the term "antibody" refers to any immunoglobulin or immunoglobulin-like molecule, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules generated during an immune response in any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice), as well as recombinant antibody fragments (sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv fragments, bispecific F ... Antibody refers to antibodies that are specific for a particular antigen, such as a fusion protein ("scFv"), a disulfide stabilized Fv protein ("dsFv"), a diabody, and a triabody (as known in the art), as well as camelid antibodies. Antibody further refers to a polypeptide ligand comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody may be composed of a heavy and a light chain, each of which has a variable region called the variable heavy (VH) region and the variable light (VL) region. The VH and VL regions 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 that are 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 (such as 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 tissues), cell samples (such as cytological smears, such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, cell fragments, or cell organelles (such as those obtained by lysing cells and centrifuging or otherwise separating their components). Other examples of biological samples include blood, serum, urine, semen, fecal material, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, earwax, breast milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample prepared from a tumor or a portion of a tumor obtained from a subject (such as a homogenized or liquefied sample).

[0032]

[0056] As used herein, the term "fluid" refers to any fluid, including water, solvents, solutions (e.g., buffer solutions), and the like. The term "fluid" also refers to any mixture, colloid, suspension, and the like. The term "fluid" also encompasses reagents, stains, and other specimen treatments (e.g., adhesives, fixatives, and the like) that may be applied to a microscope slide and / or specimen. Fluids 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 solutions, Pap staining solutions, and the like). Still further examples of fluids include solvents and / or solutions for deparaffinizing paraffin-embedded biological specimens, aqueous wash solutions, and hydrocarbons (e.g., aromatic compounds such as alkanes, isoalkanes, and xylenes). Still further examples of fluids include solvents (and mixtures thereof) used to dehydrate or rehydrate 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 that involves adding a liquid or liquid composition to a slide. Examples of reagents and processing liquids include solutions, emulsions, suspensions, and solvents (either pure or mixtures thereof). These and other examples 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 solutions, Pap staining solutions, etc.). Still further examples include solvents and / or solutions for deparaffinizing paraffin-embedded biological specimens, aqueous wash solutions, and hydrocarbons (e.g., aromatic compounds such as alkanes, isoalkanes, and xylenes).

[0034]

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

[0035]

[0060] As used herein, the term "secondary antibody" refers herein to an antibody that specifically binds to a detection probe or a portion thereof (e.g., a hapten or a primary antibody), thereby forming a bridge between the detection probe and a subsequent reagent, if any (e.g., a label, an enzyme, etc.). 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 of which is described herein.

[0036]

[0061] As used herein, the term "slide" refers to any substrate of any suitable dimensions (e.g., a substrate made in whole or in part of glass, quartz, plastic, silicon, etc.) upon which a biological specimen is placed for analysis, and more particularly refers to a substrate having a size of about 1 / 4" (3 cm) or less than 1 / 2" (7.62 cm). The term "slide" refers to a "microscope slide," such as a 1 inch by 2.54 cm (1 inch) microscope slide or a standard 75 mm by 25 mm microscope slide. Examples of biological specimens that may 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, such as tissue arrays, cell arrays, DNA arrays, RNA arrays, protein arrays, or any combination thereof. Thus, in one embodiment, tissue sections, DNA samples, RNA samples, and / or proteins are placed in specific locations on a slide. In some embodiments, the term slide may refer to SELDI and MALDI chips, as well as silicon wafers.

[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 that are characterized by binding to each other to the substantial exclusion of binding to other molecules (e.g., a specific binding pair may have a binding constant that is at least 103M-1, 104M-1, or 105M-1 greater than the binding constant of either of the two members of the binding pair to other molecules in a biological sample). Specific examples of specific binding sites include specific binding proteins (e.g., antibodies, lectins, avidins 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 described above, or nucleic acid probes.

[0038]

[0063] As used herein, the terms "stain", "staining" or the like generally refer to any treatment of a biological specimen to detect and / or differentiate the presence, location, and / or amount (e.g., concentration) of a particular molecule (e.g., lipid, protein, or nucleic acid) or a particular structure (e.g., normal or malignant cells, cytosol, nucleus, Golgi apparatus, or cytoskeleton) within the biological specimen. For example, staining can provide contrast between a particular molecule or a particular cellular structure and the surrounding portions of the biological specimen, and the intensity of the staining can provide a measure of the amount of a particular molecule within the specimen. Staining can be used to aid in the observation of molecules, cellular structures, and organisms using bright field microscopes as well as other observation instruments such as phase contrast microscopes, electron microscopes, and fluorescence microscopes. Staining performed by System 2 can be used to visualize the outline of a cell. Other staining performed by System 2 can rely on the staining of a particular cellular component (e.g., molecule or structure) without or with relatively little 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 reactions between molecules (including non-covalent interactions), such as hybridization reactions between nucleic acid molecules. Specific staining methods include, but are not limited to, primary staining methods (e.g., H&E staining, Pap staining, etc.), enzyme-linked immunohistological methods, and in situ RNA and DNA hybridization methods, such as fluorescent in situ hybridization (FISH).

[0039]

[0064] As used herein, the term "substantially" refers to a qualitative condition describing the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the art will understand that biological and chemical phenomena, when they exist, rarely proceed to completion and / or completeness 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" refers to any molecule whose presence, location, and / or concentration is or can be determined. 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 energy type, such as electrical energy, into acoustic, sonic, or vibrational energy, etc. Typically, the first energy type is electrical, electromagnetic, or electrostatic energy. The transducer may be of any type, including single-element, multi-element, array, mechanically focused, acoustically lensed, mechanically unfocused, mechanically collimated, mechanically defocused, mechanically scanned, electronically scanned, etc.

[0042]

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

[0068] Devices and Systems

[0069] Without wishing to be bound by any particular theory, it is believed that non-contact on-slide distribution, replenishment, and / or mixing of fluids on a microscope slide can improve staining quality, reduce total assay time, save costs by requiring lower concentrations of reagents (e.g., antibodies), and eliminate cross-contamination risks. With this in mind, one aspect of the present disclosure is a microscope slide holder comprising a slide support member and at least one acoustic source for introducing acoustic 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 including cells and / or tissues, and mixing using acoustic waves occurs without damaging the cells and / or tissues within the sample. In some embodiments, mixing occurs without substantially increasing the temperature of any fluids 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 fluids or sample by more than 10 degrees).

[0043]

[0070] In some embodiments, the acoustic 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 in 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 a slide. Without wishing to be bound by any particular theory, it is believed that low frequency acoustic 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., 0.1 mm to about 3 mm). More specifically, the surface transducer converts an input frequency and voltage into a vertical diaphragm displacement by an electromagnet. The oscillation of the transducer diaphragm propagates through the slide mounting 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 yet 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 a frequency ranging from about 0.5 Hz to about 2000 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 0.5 Hz to about 1000 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 1 Hz to about 1000 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 1 Hz to about 750 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 1 Hz to about 500 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 1 Hz to about 250 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 1 Hz to about 200 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 1 Hz to about 150 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 10 Hz to about 200 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 50 Hz to about 200 Hz. In some embodiments, the acoustic source operates at a frequency ranging from about 100 Hz to about 200 Hz, hi some embodiments, the acoustic source operates at a frequency of about 150 Hz.

[0047]

[0074] In some embodiments, the frequency used in certain embodiments of the present disclosure is influenced by the energy absorption characteristics of the sample or support member for a certain frequency. To the extent that a certain frequency is better or preferentially absorbed by the sample, it may be preferable to better influence the effect of the acoustic waves on the mixing. In some embodiments, the acoustic waves 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 bunched or regularly spaced.

[0048]

[0075] In some embodiments, the acoustic source operates at a first frequency for a first time interval, and then operates at a second frequency for a second time interval. In other embodiments, the acoustic source may initially operate at a first frequency, which may be increased or decreased over time (e.g., raised or lowered over time at predetermined intervals). For example, the first frequency may be 100 Hz, the second frequency may be 200 Hz, and the frequency may be raised from 100 Hz in 10 Hz intervals every 0.5 seconds until the 100 Hz to 200 Hz frequency is achieved. In other embodiments, the acoustic source employs frequency modulation, such that the frequency is deviated by a value that is + / - 20% of a predetermined frequency value. For example, an acoustic source operating at 100 Hz may have its frequency deviated by + / - 10 Hz. In other embodiments, the acoustic source may operate at multiple frequencies simultaneously, each of which may be modulated.

[0049]

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

[0050]

[0077] In embodiments in which 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 may be adjusted to operate in an out-of-phase manner such that the acoustic waves generated by the acoustic sources do not cancel each other out, thereby allowing for 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 may be modulated throughout their operation. It is understood that:

[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 may be modulated over time. For example, the acoustic source may initially operate at a first amplitude, which may be increased or ramped up over time until a final amplitude is reached. In some embodiments, the amplitude is increased by a predetermined amount for a predetermined amount of time. In some embodiments, the input amplitude is selected such that fluid present on the slide is retained, i.e., is 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 backside of a microscope slide 10. In some embodiments, such as that depicted in FIG. 1A, the slide support member 20 is substantially the same size as the microscope slide 10. Of course, one of ordinary skill 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, and as 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 (dotted 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 allows elastic waves from the acoustic source 30 to radiate, conduct, and / or transmit through the support surface 20 and to a specimen or fluid located on the slide 10. For example, the support member may be constructed of a metal (e.g., copper, aluminum, brass, chromium, silver, gold, platinum, and titanium, or an alloy including any of the metals identified 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 thermal restriction 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 includes one or more retaining members 22, such as springs, clips, or tabs, for releasably securing the microscope slide 10 to the slide support member 20. In some embodiments, the retaining members 22 limit 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 top 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 in motion. In some embodiments, the acoustic source moves up and down to contact the slide support when in motion. In other embodiments, the transducer is at least partially embedded within the slide support member 20, e.g., integrated within the slide support member 20 such that the slide support and the transducer constitute a single element.

[0057]

[0084] In another embodiment, the acoustic source (or a portion of the acoustic source) is 1B, the transducer is recessed within the slide support member 20 but still in communication with the bottom surface 26 of the support member 20 (see FIG. 1H). In yet other embodiments, the transducer is in direct communication with the slide. For example, the acoustic source (or a top portion of the acoustic source) may be recessed within the slide support member 20 and in direct communication with the bottom surface 28 of the microscope slide 10 (see FIG. 1I). FIG. 1K illustrates a support member 20 with a cavity 29 for inserting an appropriately sized acoustic source.

[0058]

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

[0059]

[0086] One skilled in the art will appreciate that the transducer 30 may be placed at any location 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 boundaries of the specimen-containing end 50. In some embodiments, the transducer 30 is placed at a location that approximates the center of the specimen-containing end 50 (see FIG. 1B). In other embodiments, the transducer is placed at a location along the slide 10 where the majority of the fluid will be dispensed. In some embodiments, the transducer is placed at least 1 cm from the edge of the microscope slide. In some embodiments, the transducer is placed at least 1.5 cm from the edge 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 may be placed at any position relative to the slide. In some embodiments, and as illustrated in FIG. 1C, transducers 30A and 30B may be placed at opposite ends of support member 20. In other embodiments, and as illustrated in FIG. 1D, the tops 31A and 31B of the two transducers are positioned at diagonal corners of the specimen-containing end 50 of slide 10. In embodiments in which multiple transducers are provided, the transducers may be spaced 0.1 cm to 10 cm apart from one another.

[0061]

[0088] Although Figures 1A and 1C illustrate a transducer 30 having an upper surface 31 in a plane that is substantially parallel to the plane of the support member 20 or slide 10, one skilled in the art will understand that the upper surface 31 may be offset at any angle relative to the support member 20 or slide 10.

[0062]

[0089] In some embodiments, the one or more acoustic sources in communication with the microscope slide are surface transducers and the surface area of ​​the transducer is, in total, at least about 5% of the surface area of ​​the microscope slide. In other embodiments, the one or more acoustic sources in communication with the microscope slide are surface transducers and the surface area of ​​the transducer is, in total, at least about 10% of the surface area of ​​the microscope slide. In yet other embodiments, the one or more acoustic sources in communication with the microscope slide are surface transducers and the surface area of ​​the transducer is, in total, at least about 15% of the surface area of ​​the microscope slide. In further embodiments, the one or more acoustic sources in communication with the microscope slide are surface transducers and the surface area of ​​the transducer is, in total, 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 acoustic waves are introduced along various portions of the microscope slide. Those skilled in the art will appreciate that the movement of the acoustic source during operation can promote uniform mixing of the fluid on the surface of the microscope slide. Those skilled in the art will also appreciate that the acoustic source can be repositioned depending on 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 to accommodate a different support member, an acoustically conductive substrate, to enhance contact with the support member, etc.

[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 to distribute, replenish, and / or mix the dispensed fluid or fluids (as further described herein).

[0065]

[0092] In other embodiments, the slide holder 5 may include multiple support members 21. Figures 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 Figure 2C, the microscope slide 10 may be supported by rails (not depicted) that extend substantially along the longitudinal edges of the slide. In these embodiments, the transducer 30, or its top surface 31, may be in direct communication with the bottom surface 28 of the microscope slide 10.

[0066]

[0093] Alternatively, the transducer 30, or its top surface 31, may be in indirect communication with the microscope slide 10. For example, a conductive material 23 may be placed between the microscope slide 10 and the transducer 30 to enhance or distribute the acoustic waves provided to the microscope slide (the transducer is thus in communication 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, or 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, and with reference to FIGS. 4A and 4B, the slide holder includes a support member 20 positioned between a microscope slide 10 and a cantilever 70. In these embodiments, the cantilever includes at least a distal portion 71 in communication with an acoustic source 30. In some embodiments, neither the support member 20 nor the microscope slide 10 is positioned above the distal portion 71. In some embodiments, a single acoustic source 30 is positioned within the confines of the distal portion 71. In other embodiments, multiple acoustic sources 30 are positioned within the confines of the distal portion 71, each of the multiple acoustic sources 30 may be spaced or positioned from one another in any manner. In still other embodiments, one or more acoustic sources 30 are positioned within the confines of the distal portion 71, and at least one additional acoustic source 30 is positioned in communication with the cantilever 70 at a location below the support member 20.

[0068]

[0095] In some embodiments, the distal portion 70 extends from the support member 20 by at least 1 inch. In still other embodiments, distal portion 70 extends from support member 20 by at least 2 inches. In other embodiments, distal portion 70 extends from support member 20 by at least 3 inches. In other embodiments, distal portion 70 extends from support member 20 by at least 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 (aluminum, copper, stainless steel, etc.).

[0070]

[0097] In other embodiments, and referring to FIG. 4C , the slide holder includes a support member 20 positioned between the microscope slide 10 and a cantilever 70, and a fluid (e.g., LCS (liquid cover glass), Ventana Medical Systems, Inc., Tucson, AZ, USA) is positioned between the support member 20 (identified as a heated plate) and the slide 10. In some embodiments, the surface of the microscope slide may include multiple separate pools of fluid that are separately dispensed onto the surface of the slide, and the acoustic source 30 allows for 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) that may be positioned such that the acoustic source coupled to the arm contacts a support member or slide, thereby being moved to introduce low frequency acoustic waves into the slide or a specimen on the slide.

[0072]

[0099] Slide Tray

[0100] Referring to FIG. 3, the present disclosure also provides a microscope slide holder adapted to hold a plurality of microscope slides 10. The present invention contemplates a slide tray 100 configured such that each individual microscope slide 10 within the tray 100 is held in a separate horizontal position (but in 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 those illustrated in any of Figures 1A-1G, 2A-2C, 4A-4C, 5A-5B, or 6. Indeed, each position within the slide tray may comprise a slide support member, one or more holding members, and at least one acoustic source, as described above. Suitable slide trays 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, e.g., 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 capacities are disclosed in US Pat. No. 7,468,161, which is incorporated herein by reference in its entirety.

[0073] In some embodiments, the slide tray is a generally rectangular tray configured to hold two rows of slides held side-by-side on either side of a central longitudinal axis of the slide tray such that the long sides of the slides are disposed outwardly from the long central axis of the tray. Each acoustic source 30 in the slide tray 100 may be communicatively coupled to the controller 60. In some embodiments, a substance may be placed between each adjacent microscope slide or adjacent support member in the slide tray to reduce the amount of acoustic energy transmitted between adjacent slide holders, i.e., to prevent or mitigate transmission of acoustic waves from a first transducer at a first location to a microscope slide at a second location adjacent the first location.

[0074]

[0101] Automated 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 as described herein, and a slide holder or slide tray comprising at least one acoustic source for non-contact mixing. Of course, one of ordinary skill in the art will appreciate that slide trays such as those described above incorporating an acoustic source may be utilized in the staining systems and specimen processing apparatus described in U.S. Patent Nos. 8,663,991, 8,048,373, and 7,468,161, and U.S. Patent Publication No. 2016 / 0282239, the disclosures of each of which are incorporated herein by reference in their entirety.

[0075]

[0103] In some embodiments, a specimen processing device incorporating a slide holder or slide tray as described herein is an automated device such as the BENCHMARK XT instrument, SYMPHONY instrument, BENCHMARK ULTRA instrument, etc., sold by Ventana Medical Systems, Inc., the assignee of numerous U.S. patents that disclose systems and methods for performing automated analyses, 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 Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively, specimens may 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 from Agilent Technologies, CoverStainer (batch stainer), Leica ST4020 Small Linear from Leica Biosystems Nussloch GmbH Stainer (batch stainer), Leica ST5020 Multistainer (batch stainer), and Leica ST5010 Autostainer XL Series (batch stainer) H&E stainers.

[0077]

[0105] The specimen processor can apply fixatives to the specimen. Fixatives can include crosslinkers (aldehydes, such as formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde crosslinkers), oxidants (metal ions and metal complexes, such as osmium tetroxide and chromate), protein denaturants (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fixative, B5 fixative, Rossman's solution, and Gendre's solution), microwaves, and miscellaneous fixatives (e.g., excluded volume fixative and vapor fixative). An acoustic source in communication with the microscope slide can be used to distribute any of these fixatives evenly within the slide or another fluid, as described in detail herein.

[0078]

[0106] If the specimen is a paraffin-embedded sample, the specimen may be deparaffinized by the specimen processor using an appropriate deparaffinization fluid. After the waste remover removes the deparaffinization fluid, any number of substances may be applied to the specimen in succession. The substances may be for pre-treatment (e.g., protein cross-linking, nucleic acid exposure, etc.), denaturation, hybridization, washing (e.g., Stringency Wash), detection (e.g., linking visual or marker molecules to probes), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, coverslipping, or the like. Again, any of these substances applied may be mixed or distributed by use of an acoustic source.

[0079]

[0107] The specimen processor 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, rinses, and / or conditioning agents. The substances can be fluids (e.g., gases, liquids, or gas / liquid mixtures), or the like. The fluids can be solvents (e.g., polar solvents, non-polar solvents, etc.), solutions (e.g., aqueous solutions, or other types of solutions), or the like. The reagents can include, but are not limited to, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen retrieval fluids (e.g., aqueous or non-aqueous based antigen retrieval solutions, antigen retrieval buffers, etc.), or the like. The probes can be isolated nucleic acids or isolated synthetic oligonucleotides attached to a detectable label. The labels can include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

[0080]

[0108] Automated IHC / ISH slide stainers typically include at least: reservoirs of various reagents used in the staining protocol, a reagent dispensing unit in fluid communication with the reservoirs for dispensing the reagents onto the slides, a waste removal system for removing used reagents and other waste from the slides, and a control system that coordinates the operation of the reagent dispensing unit and the waste removal system. In addition to performing the staining steps, many automated slide stainers may also perform steps that are ancillary to staining (or are compatible with separate systems that perform such ancillary steps), including slide baking (to attach the sample to the slide), dewaxing (also called 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 incorporated herein by reference in its entirety, describes several specific examples of automated IHC / ISH slide stainers and their various features, 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) automated slide stainers. Additionally, Ventana Medical Systems, Inc. is the assignee of several United States patents that disclose systems and methods for performing automated analyses, 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 Application Nos. 20030211630 and 20040052685, each of which is incorporated by reference herein in its entirety.

[0081]

[0109] Commercially available staining units typically operate on one of the following principles: (1) open individual slide staining (such as implemented in DAKO AUTOSTAINER Link 48 (Agilent Technologies) and intelliPATH (Biocare Medical) stainers), where the slide is positioned horizontally and the reagent is dispensed as a puddle onto the surface of the slide containing the tissue sample; (2) liquid overlay technique (such as implemented in VENTANA BenchMark and DISCOVERY stainers), where the reagent is either covered with or dispensed through an inert fluid layer deposited on top of the sample; (3) capillary gap staining (such as the staining principle used by DAKO TECHMATE, Leica BOND, and DAKO OMNIS stainers), where the slide surface is placed in close proximity to another surface (which may be another slide or a cover plate) to create a narrow gap that allows capillary forces to increase and keep the liquid reagent in contact with the sample. Some iterations of capillary gap staining do not mix the fluids 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 parallel surfaces are translated relative to each other to agitate the reagents during incubation to effectively mix the reagents (such as the staining principle implemented in the DAKO OMNIS slide stainer (Agilent)). In translating gap staining, a translatable head is positioned above the slide. The lower surface of the head is spaced from the slide by a first gap that is small enough to allow a meniscus of liquid 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 to define a second gap between the mixing extension and the slide that is smaller than the first gap. During translation of the head, the lateral dimension of the mixing extension is sufficient to generate lateral movement of liquid on the slide in a direction generally extending from the second gap to the first gap, see WO2011-139978 A1.Recently, it has been proposed to deposit reagents onto slides using inkjet technology. See WO2016-170008 A1. This list of staining techniques is not intended to be comprehensive, and either slide holders or slide trays incorporating acoustic mixing may be used in conjunction with such systems to achieve distribution and mixing of staining reagents.

[0082]

[0110] In some embodiments, an apparatus for automatically processing biological specimens comprises at least one slide tray (such as those described herein) that holds a plurality of slides in a generally horizontal position, where the biological specimens are located on the slides, each of the plurality of slides in communication with at least one acoustic source to enable the introduction of acoustic waves into the slides and / or samples; one or more workstations that receive the slide tray and perform one or more slide processing operations on the plurality of slides held in the slide tray; a transporter that moves the slide tray into and out of the one or more workstations; a fluidics module in fluid communication with the one or more workstations that supplies reagents to the one or more workstations; a pneumatic module in fluid communication with the one or more workstations and the fluidics module, where the pneumatic module supplies vacuum and / or pressurized gas to the one or more workstations and the fluidics module; and a control module in electrical communication with the transporter, the one or more workstations, the fluidics module, and the pneumatic module, where the control module coordinates the function of the components of the apparatus (including the acoustic source) during processing of the biological specimens.

[0083]

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

[0084]

[0112] In some embodiments, at least one of the workstations includes a movable nozzle assembly including one or more nozzles through which reagents are delivered to the slides. The nozzles may be dispense nozzles or lift nozzles.

[0085]

[0113] In some embodiments, the workstation can perform slide processing operations on one or more individual slides in the slide tray, e.g., at least two or four slides in the slide tray, or the workstation can perform slide processing operations (including mixing operations by an acoustic source) on all of the slides in the slide tray simultaneously. In some embodiments, the one or more workstations dispense reagents onto slides in the slide tray without a significant amount of reagent contacting a first slide contacting a second slide, thereby minimizing cross-contamination between slides. Such a workstation can include one or more directional nozzles that dispense reagents onto the slides, e.g., the one or more directional nozzles can include a pair of directional nozzles that dispense reagents in opposing 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 reagents toward the bottom surface of the slide. In other embodiments, one or more workstations can simultaneously dispense a reagent (e.g., the same reagent) to at least two slides held in a slide tray within a given workstation, or one or more workstations can simultaneously dispense a reagent (e.g., the same reagent) to all of the slides held in a slide tray within a given workstation. Following dispense of a fluid or reagent, an acoustic source can be enabled 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 includes performing slide processing operations on a plurality of slides at one or more workstations while the plurality of slides are held in a spatially coplanar, generally horizontal position in a slide tray, wherein each of the plurality of slides is in communication with at least one acoustic source to enable introduction of acoustic waves into the slide and / or the sample, and the set of slide processing operations includes at least flowing one or more stains from at least one reagent container, through a fluidics module and out of at least one dispensing nozzle positioned above the slide tray. performing slide processing operations including staining samples on slides and solvent exchange; transporting a slide tray holding the plurality of slides to an automated coverslipper workstation after performing a set of slide processing operations including at least staining and solvent exchange; coverslipping the plurality of slides held in the slide tray with separate respective coverslips using the automated coverslipper workstation while the plurality of slides are held in a spatially coplanar, generally horizontal position in the slide tray such that the coverslips on the slides are spaced apart from one another; and removing the slide tray holding the coverslipped slides from the automated coverslipper workstation.In some embodiments, the processing includes steps of (i) baking the sample under a radiant heating device, (ii) deparaffinizing the sample, (iii) staining the sample by delivering one or more staining agents through one or more fluidics components and out of one or more nozzles positioned generally above the slide tray, where the one or more fluidics components fluidly contact at least one reagent container holding the one or more staining agents with the one or more nozzles, (iv) solvent exchanging the sample, and (v) coverslipping the sample with a separate coverslip, wherein the foregoing steps are performed automatically by an apparatus comprising two or more workstations, and a slide tray holding the slides is moved between these two or more workstations during processing.

[0087]

[0115] In some embodiments, after the specimen is processed, the user can transport the slide containing the specimen to the 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 an automated embodiment, the imaging device is a digital pathology device such as that disclosed in International Patent No. PCT / US2010 / 002772 (Patent Publication No. WO / 2011 / 049608) entitled IMAGING SYSTEMS AND TECHNIQUES, or as disclosed in U.S. Patent Application Publication No. 2014 / 0178169, filed February 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 incorporated herein by reference in their entireties. In other embodiments, the imaging device includes a digital camera coupled to a microscope.

[0088]

[0116] Control System

[0117] With reference to FIG. 1A, the transducer 30 may be controlled by a controller 60. In some embodiments, and as depicted in FIG. 1E, the transducer may 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 circuit board assembly (PCBA) that includes 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 provided by the signal generator into acoustic vibrations. In some embodiments, the control system is included within a casing. In some embodiments, the control system may be communicatively coupled to a computer 64. In some embodiments, the control system for the transducer is a controller used to control an automated stainer.

[0089]

[0118] In some embodiments, the control system may further comprise one or more sensors for monitoring the acoustic waves provided to the microscope slide, hi other embodiments, the control system may 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 may be used to optimize mixing. As an example, optical or video detection may be used to detect a color change as a colored reagent mixes into a clear fluid. Other optical measurements such as spectral excitation, absorption, light scattering, fluorescence, luminescence, emission, polarized light microscopy, Raman scattering, and spectral analysis may also be used to monitor the mixing of fluids in contact with a sample on the surface of a microscope slide. Data may be acquired and analyzed by a computer or control system controlling the mixing process. For example, if sufficient mixing is achieved based on the received data, a transducer may be turned off. As another example, if mixing is insufficient based on the received data, the controller may increase the time the transducer is turned on, or the controller may 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 a surface of a microscope slide by introducing low frequency acoustic waves (e.g., frequencies that do not damage cells, such as frequencies below 2000Hs) to the microscope slide. In some embodiments, the introduction of acoustic waves promotes the formation of a substantially uniform distribution of fluid on a biological sample provided on the surface of the microscope slide. For example, a fluid may be dispensed into a predetermined area on the surface of the microscope slide, and upon activation of the acoustic source and introduction of acoustic waves, the fluid may be distributed beyond the initial area of ​​dispensing. In some embodiments, the distribution of fluid on the surface of the microscope slide by the introduction of acoustic waves may be used to promote the replenishment of fluid (e.g., reagent) onto a biological sample loaded on the surface of the slide. For example, the biological sample may absorb a reagent deposited on the surface (or may absorb the reagent unevenly), and eventually the amount of reagent in contact with the biological sample may be substantially depleted (or depleted from certain areas or portions of the sample). Activation of the acoustic source can facilitate distribution of another aliquot of reagent to the biological sample, thus replenishing the reagent in contact with the biological sample. Activation of the acoustic source can also facilitate redistribution of reagent from other areas of the slide to the biological sample faster than 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 the second fluid, e.g., a reagent, the second fluid may be substantially uniformly distributed within the first fluid following the introduction of the acoustic waves. In some embodiments, substantially uniformly distributed means that the reagent concentrations at two separate points on the slide differ by no more than 15% in magnitude. In some embodiments, substantially uniformly distributed means that the reagent concentrations at two separate points on the slide differ by no more than 10% in magnitude. In other embodiments, substantially uniformly distributed means that the reagent concentrations at two separate points on the slide differ by no more than 5% in magnitude. In yet other embodiments, substantially uniformly distributed means that the reagent concentrations at two separate points on the slide differ by no more than 2% in magnitude. Of course, one skilled in the art will understand that any number of fluids may be deposited on the surface of a microscope slide and each of these fluids may be mixed, i.e., substantially uniformly distributed, within one another upon the introduction of the acoustic waves.

[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 skilled in the art will be able to 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 entire volume present on the slide is substantially uniformly distributed and / or mixed as desired.

[0094]

[0124] Generally, the method includes (i) introducing a fluid to a surface of a slide, and (ii) introducing low frequency acoustic waves to the slide. In some embodiments, the method includes, but is not limited to, (a) a detection step for feedback control of an acoustic source, (b) a fluid removal step, and / or (c) a further fluid dispensing step. The method includes further 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) uniformly distributing the first reagent on the slide containing the specimen by introducing an acoustic wave to the slide containing the specimen. In some embodiments, the uniform distribution allows for the advancement of fluid to areas of the slide that are free of fluid. As described herein, the acoustic wave can be generated by a transducer in communication with the sample. In some embodiments, the reagent is introduced into a first fluid reservoir (e.g., a reservoir containing a buffer) on the surface of the slide or in close proximity to a first fluid reservoir on the slide. In some embodiments, the reagent is a detection probe. In some embodiments, the detection probe is a binding moiety specific for a particular target (e.g., an antibody, a nucleic acid) within a biological sample. In some embodiments, the detection probe utilized is a primary antibody, specifically a primary antibody that allows for the detection of a protein target (or an epitope of a protein target) within 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 allows for detection of a nucleic acid sequence target within a sample, hi other embodiments, the specific binding moiety is a nucleic acid probe that 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 for a time interval ranging from about 0.5 seconds to about 6 hours. In some embodiments, the elastic waves are introduced for a time interval ranging from about 0.5 seconds to about 240 seconds. In other embodiments, the elastic waves are introduced for a time interval ranging from about 1 second to about 180 seconds. In other embodiments, the elastic waves are introduced for a time interval ranging from about 1 second to about 120 seconds. In other embodiments, the elastic waves are introduced for a time interval ranging from about 1 second to about 60 seconds. In other embodiments, the elastic waves are introduced for a time interval ranging from about 1 second to about 30 seconds. In other embodiments, the elastic waves are introduced for a time interval ranging from about 1 second to about 15 seconds. In other embodiments, the elastic waves are introduced for 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 properly distributed or mixed (e.g., by using a feedback mechanism described herein). If the detecting step determines that the fluid and / or reagent is not properly mixed, the operating parameters of the acoustic source may be adjusted (e.g., frequency, amplitude, duration of operation, pulsed vs. continuous introduction of acoustic waves, or any combination thereof).

[0098]

[0128] In some embodiments, the sample is pulsed with acoustic waves. In some embodiments, the pulsing of the sample with acoustic waves may be at regular intervals. For example, the sample may be pulsed with acoustic waves for a certain predefined amount of time (e.g., about 0.5 second intervals), followed by a predefined amount of time (e.g., about 1 second intervals) where no acoustic waves are introduced. In other embodiments, the pulsing of the sample with acoustic waves may be at non-regular intervals. In other embodiments, the determination of whether the sample is pulsed with acoustic waves may be determined by using a detector that provides feedback on the degree of mixing, or a detector that can detect whether a portion of the slide or sample needs refilling.

[0099]

[0129] In some embodiments, the sample is diluted with water for the entire time interval that the reagent is in contact with the sample. The sample may be pulsed with elastic waves throughout (e.g., during the incubation period). For example, if an antibody is introduced to the sample and the protocol requires that the antibody remain in contact with the sample for a time interval (e.g., incubation period) of about 360 seconds, elastic waves may be introduced to the sample for a predefined amount of time at set intervals during the incubation period. For example, pulses of elastic waves may be introduced at 5 second time 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 introduction of the antibody. Of course, rather than pulsing the sample with elastic waves at predefined intervals or for a predefined amount of time, a feedback control device (such as those described herein) may be utilized to determine whether introduction of a pulse of elastic waves is necessary, including the duration of the pulse.

[0100]

[0130] In other embodiments, the slide or sample may be pulsed with acoustic 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 a protocol calls for adding a particular aliquot of fluid every two minutes, acoustic waves may be provided to the slide and / or sample for at least a predetermined amount of time each time an aliquot is added. Of course, additional pulses may be provided between dispense cycles as needed and as described above.

[0101]

[0131] Additionally, if a particular system or instrument calls for moving a slide between different stations or processing areas of the system or instrument (e.g., sample staining area, sample incubation area), acoustic waves can be pulsed into the sample before and / or after the slide movement to ensure that fluids are properly distributed and / or mixed before, during, and after such movement. Following mixing of the first reagent into the first fluid reservoir, the mixed first reagent / fluid reservoir can be removed from the surface of the slide. A first detection reagent can then be introduced and then distributed on the surface of the slide or mixed with a second fluid reservoir 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, a substrate for the enzyme (a detectable moiety, e.g., a chromogen moiety) can be introduced so that a colored precipitate can be detected. In yet other embodiments, an anti-label antibody (secondary antibody) is introduced to elicit detection, the anti-label antibody being specific to the label of the detection probe. For example, if the label is a hapten, an anti-hapten antibody specific for the hapten label is introduced, which anti-hapten antibody contains a detectable moiety. In some embodiments, the detectable moiety of the anti-hapten antibody is an enzyme, and a substrate for 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 of skill in the art. The introduction of the detection probe and / or detection reagent can be repeated "n" times to represent any desired number of targets in the sample.

[0102]

[0132] The methods disclosed herein are also suitable for multiplex assays. For example, a first detection probe specific to a first target and a second detection probe specific to a second target can be introduced simultaneously or sequentially. Once both the first and second detection probes are introduced to the sample, an elastic wave can be introduced to 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 promote uniform detection probe concentration during staining and / or reduced staining artifacts. Those skilled in the art will understand that any number of detection probes can be introduced to the sample on the surface of a 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 generated at a frequency of about 0. The acoustic waves are introduced for a time interval ranging from about 5 seconds to about 6 hours. In some embodiments, the acoustic waves are introduced for a time interval ranging from about 0.5 seconds to 240 seconds. Following introduction of the detection probe, one or more detection reagents may be introduced, again either simultaneously and / or sequentially, and again mixed with the acoustic waves upon introduction.

[0103]

[0133] In some embodiments, a method of replenishing a fluid or reagent includes (i) contacting a sample on a slide containing the specimen with a first reagent, (ii) allowing time for the reagent to react with or be absorbed by the sample, and (iii) uniformly distributing the first reagent on the slide containing the specimen by introducing low frequency acoustic waves to the slide containing the specimen, thereby replenishing the reagent in areas where the reagent has been 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 introducing acoustic waves. As described herein, the acoustic waves may be generated by a transducer in communication with the sample. In some embodiments, the reagent is introduced into a first fluid reservoir (e.g., a reservoir 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 moiety specific for a particular target in the biological sample. In some embodiments, the detection probe utilized is a primary antibody, specifically a primary antibody that allows for the detection of a protein target (or an epitope of a protein target) in a 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 allows for the detection of a nucleic acid sequence target in a sample. In other embodiments, the specific binding site is a nucleic acid probe, which is conjugated to a detectable label or reporter, such as a chromogen, a fluorophore, a hapten, or an enzyme.

[0104]

[0134] In some embodiments, the method of processing a slide containing a specimen includes (i) dispensing a first fluid onto a first portion of a microscope slide, and (ii) distributing the first fluid onto the specimen by introducing elastic waves 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 for a particular target within the biological sample. In some embodiments, the method further includes the step of introducing an additional aliquot of fluid to the slide (in any region) and then distributing the fluid by introducing elastic waves.

[0105]

[0135] Working Example

[0136] General Experimental Protocol

[0137] A 45mm diameter transducer (4ohm, 5W, Adafruit.com product ID=1784) was used to mix the fluids on the slide. An amplifier IC board (Adafruit product ID=1552, TPA2012) was needed to power the transducer. The input to the amplifier is a signal generator sine wave and the amplifier output to the transducer is a PWM wave. The amplifier IC, TPA2012D2, is a stereo class D audio amplifier with 2x2.1 output, shown in Figure 3. The board comes with pins to adjust the gain of the amplifier and all experiments were run at 24dB gain. Two shutdown pins (SDR and SDL) were used to turn the transducer on / off via the PRISM board and Atlas software. This gave the advantage of identifying the length of time to mix and understanding how the transducer on:off ratio and period time length affected the mixing. The signal generator used was an Agilent 33512B. Channel 1 of the signal generator was used to output a sine wave with a specific frequency and amplitude, the frequency being the resonance of the assembly.

[0106]

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

[0107]

[0139] The transducer is screwed to the Slide Processing Station (SPS) heating plate and the bottom of the transducer is fixed to the lab bench. The wet slide is placed on the SPS heating plate. The label edge of the slide is taped to the slide alignment plate and the SPS heating plate.

[0108]

[0140] A diagram of the setup is shown in Figure 6. EXAMPLES

[0109]

[0141] Mixing fluorescein into 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 actuate the transducer vertically up and down, thereby allowing mixing of the fluids on the slide.

[0110]

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

[0111]

[0145] General Procedure

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

[0112]

[0147] 2. Wetting the slide - Dip the slide into the reagent and flick the slide.

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

[0113]

[0149] 4. Add 50 μL of salt buffer and, to visualize mixing, add fluorescein to the four corners and five central spots at 10 μL per spot.

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

[0114]

[0151] 6. Turn off the transducer, remove the slide, and clean the SPS heating platen with alcohol.

[0152] result

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

[0115] [Table 1]

[0116]

[0154] The results of mixing are illustrated in FIG. 7. Each frame in FIG. 7 illustrates the fluorescein dye spreading throughout the fluid volume on the slide. The dye at the four corners of the free edge of the slide and at the center of the slide mixed for approximately 1 second before mixing. The dye at the four corners closest to the tape end of the slide started to mix, but such mixing at those locations took longer, which is believed to be due to the edges being more mechanically stiffer since they are held down by the tape. Qualitatively, the dye estimated that it took approximately 7 seconds for the fluid to spread evenly across the slide. Applicants have shown that the non-contact mixing means disclosed herein advantageously allows for mixing of fluids without contacting the sample with any physical device or gas stream. It is believed that this will eliminate concerns of potential cross-contamination between slides if this technology is incorporated into a staining platform. Since the frequency for mixing here is relatively low, approximately 150 Hz, tissue damage is unlikely. Without wishing to be bound by any particular theory, it is believed that cell damage occurs in the ultrasonic range (above about 18,000 Hz) and that in commercially available ultrasonic devices, cell damage is caused by shear forces generated by bubble collapse. No cavitation or bubble collapse was observed in the system provided herein. EXAMPLES

[0117]

[0155] Quantifying Mixture

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

[0118]

[0157] The experimental setup was similar to that presented in Example 1 herein. To quantify, data from individual frames of video data (see FIG. 8A) were extracted and the standard deviation of pixels within the region of interest was determined (see FIG. 8B). More specifically, FIG. 8A (a) provides an image of the fluid volume on the slide before mixing. The white box designates the region of interest (ROI). The ROI is 398×181 pixels, which equates to 70% of the slide stained area or approximately 50×20 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, FIG. 8B (a) illustrates that the standard deviation of the green values ​​of the pixels within the region of interest (ROI) was calculated for each frame and (b) provides a plot of the green value standard deviation within the ROI during the mixing process. In essence, as fluorescein was mixed throughout a predefined region of interest, the individual green values ​​of each pixel began to trend toward neighboring green pixels of the same value. Ideally, a uniformly mixed solution was assumed to have a standard deviation of zero. EXAMPLES

[0119]

[0158] Input Voltage Sweep

[0159] The motivation for this experiment was to visualize and observe wave propagation in the fluid on the slide at different input voltages from 45 to 90 mVpp. The transducer was turned on and the voltage was swept consecutively at five different input values: 45 mVpp, 50 mVpp, 55 mVpp, 60 mVpp, and 90 mVpp. It was observed that as the voltage increased, the area of ​​the standing wave across the slide increased (see FIG. 9). At 45 mVpp, the standing wave was confined to one corner of the slide, whereas at 90 mVpp, the standing wave was present across the entire slide. At 50 mVpp, the standing wave was seen at the edge of the slide that had progressed most freely vertically. This suggested that the slide could function as a cantilever (see FIG. 4 herein for an embodiment employing a cantilever design). EXAMPLES

[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 herein. The input to the transducer was 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 300 μL on-slide fluid volume demonstrated coating and mixing, shown in FIG. 10A. More specifically, FIG. 10A illustrates (a) 30 μL fluorescein dye and 270 μL Apollo buffer volume on the slide before mixing, (b) 6 seconds of transducer on. The fluid front is driven toward the label end of the slide, spreading the fluid across the slide. (c) 20 seconds of mixing (d) 2 minutes 30 seconds of mixing - the fluid has coated nearly the entire slide and the dye has mixed with the solution. A 100 μL on-slide fluid volume demonstrated mixing but not coating, shown in 10B. More specifically, FIG. 10B illustrates (a) 10 μL fluorescein dye and 90 μL Apollo buffer solution before mixing, (b) 20 seconds of mixing. The fluid collects at one end of the slide due to the SPS heater and transducer being uneven. (c) 1 min 20 sec mixing. The fluids have mixed, but the fluids do not spread across the slide. (d) Visible light image of the mixed fluids. 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] EXAMPLES

[0123]

[0163] Transducer Position

[0164] The purpose of this set of experiments was to sweep the transducer position along the SPS hot plate to qualitatively determine the transducer location that demonstrated the most rapid mixing. Four locations were tested, such as the one illustrated in FIG. 5B. The setup and procedures for these experiments are as described in Example 1. To visualize mixing, fluorescein was used as a dye, and a total of 50 μL of fluorescein was distributed in 10 μL aliquots at the four corners and center of the slide within a 450 μL salt buffer volume.

[0124]

[0165] The results are summarized in Table 2 below. Each setting was evaluated for time to qualitatively determine if the fluid on the slide was homogenously mixed. Mixing fluids with the transducer at both ends, the free end, or the label end of the SPS heating plate required higher input voltage and time to completely mix the solution on the slide. The transducer appeared to mix the fluid on the slide most quickly (at about 7 seconds) when positioned in the center of the slide.

[0125] [Table 3] EXAMPLES

[0126]

[0166] Transducer period vs. on:off ratio

[0167] In previous mixing experiments using a transducer, the transducer was turned on and off. They observed that turning the switch on and off seemed to spread (push outward) and contract (relax) the fluid on the slide, respectively. These experiments investigated whether this spreading and contraction could enhance mixing.

[0127]

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

[0128]

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

[0129]

[0170] Table 3 summarizes the results of the study and the approximate time to mixing is the time for the fluid to mix uniformly on the slide. This was determined qualitatively. Uniform mixing on the slide was achieved in 7 seconds with this setup, so 5 seconds was chosen as the maximum duration time.

[0130] [Table 4]

[0131]

[0171] The results showed that longer transducer on times promoted mixing. Because this was a qualitative observation, and because the transducer could have caused ripples in the fluid while on, it was difficult to know with absolute certainty when the solution was homogenously mixed. However, a transducer period of about 5 seconds with a roughly 90% "on" ratio was predicted to likely mix the fluid as quickly as when the transducer was "on" 100% of the time. Therefore, turning the transducer "off" does not appear to have any effect on hastening 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 referenced herein and / or listed in the Application Data Sheets are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified if necessary to employ concepts from the various patents, applications, and publications to still provide further embodiments.

[0133]

[0173] Although the present disclosure has been described with reference to certain illustrative 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 particularly, reasonable variations and modifications are possible in the arrangement of component parts and / or object combination arrangements within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the present disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses shall also be apparent to those skilled in the art.

Claims

1. 1. A method for delivering a reagent to a microscope slide containing a specimen, comprising: (i) contacting a sample on a microscope slide containing said specimen with a first reagent; (ii) allowing time for the first reagent to react with or be absorbed by the sample on the microscope slide containing the specimen; (iii) uniformly distributing the first reagent on a microscope slide containing the specimen by introducing low frequency acoustic waves by at least two acoustic sources onto the microscope slide containing the specimen to supply reagent to areas at least partially depleted of reagent; (iv) performing step (iii) in a feedback manner by using one or more detectors configured to monitor the first reagent on a microscope slide containing the specimen and determine whether the first reagent is properly distributed; The method includes:

2. 10. The method of claim 1, further comprising the step of introducing an additional aliquot of the first reagent onto the microscope slide containing the specimen prior to uniformly distributing the first reagent by the introduction of acoustic waves.

3. The method of claim 1 or 2, wherein the low frequency acoustic waves are generated by a transducer in communication with a slide support that supports the microscope slide.

4. 4. The method of claim 1, further comprising the step of: (v) adjusting operating parameters of the at least two acoustic sources when it is determined that reagents are not properly mixed on the microscope slide containing the specimen.

5. The method of claim 4 , wherein the operating parameters include frequency, amplitude, duration of operation, pulsed versus continuous introduction of elastic waves, or combinations thereof.

6. 4. The method of claim 1, further comprising the step of pulsating the sample on a microscope slide containing the specimen with the acoustic waves.

7. 4. The method of claim 1, further comprising optimizing the distribution of the sample on a microscope slide containing the specimen using optical detection and analysis.

8. The method of claim 7, wherein the optical detection and analysis detects a color change on a microscope slide containing the specimen.

9. the step of uniformly distributing the first reagent is performed by a slide holder; The slide holder comprises: (i) a slide support having a support surface configured to support at least a portion of a backside of the microscope slide containing the specimen, the backside of the microscope slide containing the specimen being opposite a surface of the microscope slide containing the specimen that includes the specimen; (ii) at least one acoustic source fitted into the slide support and at least partially contacting a bottom surface of the slide support, the support surface and the bottom surface being on opposite sides of the slide support; The method of claim 1 , comprising:

10. The method of claim 9 , wherein the slide support comprises a cavity for inserting the at least one acoustic source.

11. The method of claim 9 or 10, wherein the at least one acoustic source is controlled by a controller.

12. The method of claim 11 , wherein the controller comprises one or more sensors configured to monitor elastic waves provided to a microscope slide containing the specimen.

13. The method of claim 11 , wherein the controller comprises one or more feedback mechanisms configured to monitor mixing of one or more liquids disposed on a surface of the microscope slide containing the specimen.

14. The method of claim 9 , wherein the slide support is constructed of metal.

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