Contactless mixing using modulation-type air-jet

Abstract

To realize an efficient mixing of fluids on a slide.SOLUTION: There is disclosed a device for contactlessly mixing fluids on an upper surface of a slide. The device has a first nozzle array and a second nozzle array. The first nozzle array is adapted to provide a bulk fluid flow to the fluids on the upper surface of the slide. The second nozzle array is adapted to provide a first local fluid flow to at least a part of the fluids in the upper surface of the slide.SELECTED DRAWING: Figure 9B

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 589,234, filed on November 21, 2017, the disclosure of which is incorporated herein by reference in its entirety.

Background Art

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

[0003] Devices for automated staining, particularly for high-volume staining with traditional reagents such as hematoxylin and eosin (H&E), are mainly 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 sequentially immersed in a number of chemical solution containers. The slides are mounted vertically in a slide holder basket, and a clamp that engages and disengages the basket is used to move the slides from solution to solution. The clamp can include a mechanism for tilting the basket, which assists in removing excess solution before the basket is submerged in the next solution. Further automated staining devices of the "dip and dunk" type are described in U.S. Patent No. 5,573,727 to Keef, U.S. Patent No. 6,080,363 to Takahasi et al., U.S. Patent No. 6,436,348 to Ljungmann et al., and U.S. Patent Application Publication No. 2001 / 0019703 listing Thiem et al. as inventors.

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

[0005] Efficient mixing of fluids is an important step in many industrial, chemical, and pharmaceutical processes, as well as in biotechnological applications. Mixing on a small scale is often a difficult task. In some embodiments, molecular diffusion becomes the main mixing mechanism that slows down the overall process. The integration of active mixers is often difficult, increasing the cost of any such device and introducing cross-contamination between samples. SUMMARY OF THE INVENTION

[0006] Disclosed herein are systems and methods for non-contact dispersion, replenishment, and / or mixing (collectively referred to herein as "mixing") of one or more fluids on a substrate (e.g., a microscope slide). The use of a plurality of gas flows directed at a fluid (e.g., a fluid paddle) present on the surface of the substrate (e.g., a flat support surface) provides movement and direction of the fluid with respect to a predetermined area of the fluid, and thus enables mixing, dispensing, and / or replenishment of the fluid or multiple components within the fluid. The Applicant has discovered that the introduction of such directed gas flows (such as separate or discrete streams) achieves bulk mixing of the fluid, in contrast to mere agitation or local mixing. In fact, the Applicant has discovered that the non-contact mixer of the present disclosure enables improved mixing compared to simply agitating the fluid on the substrate with the induction of vortices (e.g., mixing by the non-contact mixer of the present disclosure enables faster and more thorough mixing). Further, the non-contact mixer of the present disclosure enables fluid movement at multiple locations across the slide such that bubbles, if formed, are displaced and / or removed. In contrast, mere vortex mixing may move bubbles to the center of the fluid.

[0007] Mixing a fluid according to the methods described herein reduces the risk of (i) staining artifacts; (ii) enables a uniform reagent concentration across cells and / or tissue during staining to reduce, for example, the formation of brightly stained areas (e.g., bright spots) or darkly stained areas (e.g., dark spots); (iii) enables an increase in mixing frequency; (iv) increases mixing efficacy; and / or (v) reduces or eliminates the presence of air bubbles in the fluid, Applicants have similarly found. It is also believed that the use of the devices and methods disclosed herein can enable the use of lower concentrations of detection probes (e.g., antibodies) or other reagents in any histological or cytological staining procedure. Further, the use of the devices and methods enables low manufacturing and maintenance costs, reduced process time, and / or reduced contamination. These and other advantages are further described herein.

[0008] One aspect of the disclosure is a device that enables non-contact mixing of a fluid on a substrate, the device being configured to direct a plurality of gas flows in and / or on the surface of the substrate such that at least two discrete fluid motions are imparted to a predetermined portion of the fluid at different times, the two discrete fluid motions enabling cross-mixing. In some embodiments, the device is configured such that the gas flows reduce the loss of fluid from the substrate or the splashing of fluid from the substrate to an adjacent substrate while properly mixing the fluid present on the substrate surface.

[0009] Another aspect of the present disclosure is an automated slide processing apparatus comprising at least one non-contact mixer for mixing fluids present on an upper surface of a slide, the non-contact mixer including a first nozzle set and a second nozzle set, the first nozzle set configured to impart a first motion to a first portion of the fluid present on the upper surface of the slide, and the second nozzle set configured to impart a second motion to at least a second portion of the fluid present on the upper surface of the slide. In some embodiments, the second motion induces cross-mixing of the fluid. In some embodiments, the first and second motions are opposing motions. In some embodiments, the second nozzle set imparts a second motion and a third motion, the second and third motions being the same or different, however, at least one of the second or third motions is opposite to the first motion. In some embodiments, a gas flow enables the fluid or any portion of the fluid to move within a substantially circular path.

[0010] In some embodiments, the first set of nozzles operates independently of and at a different time than the second set of nozzles, i.e., the first set of nozzles operates exclusively from the second set of nozzles. Thus, the first nozzle set can impart a first motion, at least during operation of the first nozzle set, and the second nozzle set can impart at least a second motion, at least during operation of the second nozzle set. One of ordinary skill in the art will understand that the first nozzle set can comprise a plurality of nozzles, and each of the plurality of nozzles of the first nozzle set operates simultaneously to effect a first fluid motion. By way of example, the first nozzle set can comprise a first and a second nozzle that direct a gas flow towards one area of the fluid, and a third and a fourth nozzle that simultaneously direct the gas flow towards another area of the fluid, where the first and second nozzles can direct the gas in a first direction, such that the third and fourth nozzles can direct the gas in a second, opposite direction. Of course, the second nozzle operates in a similar manner and can be similarly configured to effect at least a second motion. In some embodiments, the automated slide processing apparatus further comprises a second non-contact mixer, e.g., one or more discrete nozzle sets, each discrete nozzle set being capable of independent operation.

[0011] In some embodiments, the first portion is a majority of the fluid present on the slide. In some embodiments, the first motion is applied to the entire fluid. In some embodiments, the first motion is one of clockwise or counterclockwise agitation. In yet other embodiments, the first motion is imparted to at least 60% of the fluid. In still further embodiments, the first motion is imparted to at least 70% of the fluid. In still further embodiments, the first motion is imparted to at least 80% of the fluid.

[0012] In some embodiments, the second motion is imparted to at least two discrete portions of the fluid present on the upper surface of the slide. In some embodiments, the second motion is imparted to at least three discrete portions of the fluid present on the upper surface of the slide. In some embodiments, the second motion is imparted to the central portion of the slide (e.g., the central one-third of the entire fluid). In some embodiments, the second nozzle set is further adapted to impart a third motion to two ends of the fluid (e.g., the first one-third and the last one-third of the entire fluid), the two ends of the fluid are adjacent to the central portion respectively, and the third motion is opposite to the second motion. In some embodiments, the first motion is counterclockwise agitation, the second motion is clockwise agitation, and the third motion is counterclockwise agitation.

[0013] In some embodiments, the second nozzle set is configured such that the second motion is imparted to at least two different portions of the fluid, e.g., non-adjacent portions of the fluid. In some embodiments, the second nozzle set is further adapted to impart a third motion to the central portion of the fluid. In some embodiments, the first motion is counterclockwise motion, the second motion is clockwise motion, the third motion is counterclockwise motion, and the second motion is imparted to two ends of the fluid located on both sides of the central portion.

[0014] In some embodiments, at least one non-contact mixer is positioned on the upper surface of the slide. In some embodiments, at least one non-contact mixer is positioned between about 0.3 inches (7.62 mm) and about 1.5 inches (38.1 mm) above the slide. In some embodiments, at least one non-contact mixer is positioned substantially parallel to the upper surface of the slide. In some embodiments, the body of at least one non-contact mixer is offset at a predetermined angle with respect to the upper surface of the slide (e.g., offset at an angle between about 1 degree and about 20 degrees).

[0015] In some embodiments, the first and second nozzle sets are configured to direct a gas flow to a predetermined position on the upper surface of the slide. In some embodiments, the first and second nozzle sets are configured to direct a gas flow to a predetermined position on the upper surface of the slide at a predetermined angle of incidence with respect to the slide surface. In some embodiments, the angle of incidence of the gas flow with respect to the upper surface of the slide (e.g., the angle from a line perpendicular to the surface) is between about 15 degrees and about 90 degrees. In some embodiments, the gas flow can be directed to a position between the fluid on the slide and the edge of the upper surface of the slide.

[0016] In some embodiments, the first nozzle set includes from 2 to 6 nozzles. In some embodiments, the first nozzle set includes from 4 to 6 nozzles. In some embodiments, the nozzles of the first nozzle set are grouped into two parallel rows along the longitudinal axis of the non-contact mixer. In some embodiments, each nozzle of the first nozzle set independently has a specific tilt angle and offset angle as those terms are defined herein. In some embodiments, the second nozzle set includes from 2 to 4 nozzles. In some embodiments, the nozzles of the second nozzle set are grouped into two substantially parallel rows along the longitudinal axis of the non-contact mixer. In some embodiments, each nozzle of the second nozzle set independently has a specific tilt angle and offset angle.

[0017] In some embodiments, the automated slide processing apparatus is a staining apparatus. In some embodiments, the automated slide processing apparatus further includes a control system, and the control system is adapted to independently operate the first and second nozzle sets (e.g., pulse drive at a predetermined interval and / or at a predetermined frequency) to provide the first and second movements and any additional movements if the non-contact mixer is so configured. In some embodiments, the automated slide processing apparatus further includes a third nozzle set to provide further directional movement to the fluid present on the upper surface of the slide.

[0018] Another aspect of the present disclosure is a method of operating a non-contact mixer, the non-contact mixer including a first nozzle set and a second nozzle set, the first nozzle set being adapted to impart a first motion to a fluid present on an upper surface of a slide, the second nozzle set being adapted to impart a second motion to at least a portion of the fluid present on the upper surface of the slide, the method including operating the first nozzle set during a first period and then operating the second nozzle set during a second period, provided that the first and second nozzle arrays do not operate simultaneously. In some embodiments, the first and second nozzle sets operate together at least once. In some embodiments, the first and second nozzle sets operate at least twice. In some embodiments, the first and second nozzle sets are communicatively coupled to a control system having one or more sensors, the sensors being adapted to determine the degree of mixing during or after operation of the first and / or second nozzle sets.

[0019] Another aspect of the present disclosure is an automated slide processing apparatus, the automated slide processing apparatus comprising: (i) at least one fluid dispenser configured to dispense a fluid onto an upper surface of a specimen support slide; and (ii) a non-contact mixer for mixing the fluid present on the upper surface of the slide, the non-contact mixer comprising a first nozzle array and a second nozzle array, the first nozzle array being adapted to impart a bulk fluid flow to the fluid present on the upper surface of the slide, the second nozzle array being adapted to impart at least a first local fluid flow to at least a portion of the fluid present on the upper surface of the slide. In some embodiments, the first local fluid flow induces cross-mixing within the fluid. In some embodiments, the automated slide processing apparatus is a staining apparatus, the fluid present on the upper surface of the slide includes a reagent, non-limiting examples of the reagent including a staining reagent, a counterstaining reagent, or a washing reagent. Other reagents and / or fluids that can be present on the surface of the slide are known to those skilled in the art.

[0020] In some embodiments, the bulk fluid flow is a movement in one direction of clockwise or counterclockwise movement, e.g., agitation or other rotational movement. In some embodiments, the first local fluid flow is the other movement of clockwise or counterclockwise movement. In some embodiments, the first local fluid flow is provided to the central portion of the fluid present on the upper surface of the slide. In some embodiments, the first local fluid flow is provided to the central portion of the fluid present on the upper surface of the slide, and the central portion represents about one-third of the fluid present on the upper surface of the slide. One of ordinary skill in the art will understand that the first local fluid flow can be provided to any portion of the fluid on the slide if the first local fluid flow allows for cross-mixing with any additional local fluid flow provided.

[0021] In some embodiments, the second nozzle array is further adapted to provide a second local fluid flow and a third local fluid flow, and the second and third local fluid flows occur at two different ends of the fluid on the slide, the two ends are adjacent to the central portion respectively, and the second and third local fluid flows are opposite to the first local fluid flow. In some embodiments, the second local fluid flow and the third local fluid flow are provided substantially simultaneously from the second nozzle array.

[0022] In some embodiments, the automated slide processing apparatus is a staining apparatus, and the fluid present on the upper surface of the slide includes, but is not limited to, a staining reagent, a counterstaining reagent, or a washing reagent.

[0023] Another aspect of the present disclosure is a method of operating a non-contact mixer, the non-contact mixer comprising a first nozzle array and a second nozzle array, the first nozzle array being adapted to provide a bulk fluid flow to a fluid (or a predetermined portion of the fluid) present on an upper surface of a slide, the second nozzle array being adapted to provide at least a first local fluid flow to at least a portion of the fluid present on the upper surface of the slide, the method comprising operating the first nozzle array during a first period (e.g., to induce a bulk fluid flow for at least a portion of the time the first nozzle array operates), and then operating the second nozzle array during a second period (e.g., to induce at least one local flow for at least a portion of the time the second nozzle operates), provided that the first and second nozzle arrays do not operate simultaneously. In some embodiments, one of the first or second periods is a predetermined period. In some embodiments, one of the first or second periods is determined in real time using a feedback mechanism configured to interpret a level or degree of mixing achieved during operation of the first and / or second nozzle arrays. In some embodiments, the feedback mechanism includes one or more sensors communicatively coupled to a control system. In some embodiments, the first and second nozzle arrays operate sequentially at least twice. In some embodiments, the operation of the first nozzle array always follows the operation of the second nozzle array, even if the operation of the second nozzle array occurs during a portion of the period during which the first nozzle array operated. In other embodiments, the operation of the non-contact mixer can include (i) operating the first nozzle array; (ii) then operating the second nozzle array; and (iii) then operating the first nozzle array. In some embodiments, the second nozzle array is configured such that at least second and third local motions are induced, the second and third local fluid motions being induced during operation of the second nozzle array, the second and third local fluid motions occurring substantially simultaneously with each other.

[0024] Another aspect of the present disclosure is an automated slide processing apparatus, the automated slide processing apparatus comprising: (a) at least one fluid dispenser configured to dispense fluid onto an upper surface of a specimen support slide; and (ii) a non-contact mixer for dispensing fluid present on the upper surface of the slide, the non-contact mixer comprising a first nozzle array in fluid communication with a first plenum, the first nozzle array including a set of first primary nozzles directing a gas flow in a first direction and a set of second primary nozzles directing a gas flow in a second direction, and a second nozzle array in fluid communication with a second plenum. In some embodiments, the second nozzle array includes a set of first secondary nozzles directing a gas flow in a third direction and a set of second secondary nozzles directing a gas flow in a fourth direction. In some embodiments, the second nozzle array further includes a further set of secondary nozzles, each further set of nozzles being adapted to direct a gas flow in another direction.

[0025] In some embodiments, the first and second directions are opposite to each other. In some embodiments, the gas flows emitted from the sets of first and second primary nozzles are directed along substantially the periphery of the fluid present on the upper surface of the slide and at any angle of incidence with respect to the upper surface of the slide (or the fluid positioned on the slide). In some embodiments, the gas flows emitted from the sets of first and second primary nozzles are directed to a portion of the slide adjacent to the fluid and at any angle of incidence with respect to the upper surface of the slide (or the fluid positioned on the slide).

[0026] In some embodiments, the gas flow exiting from the set of first primary nozzles (with respect to the fluid, with respect to the area of the slide that does not contain the fluid, or both) is directed substantially along the first longitudinal axis of the slide, and the gas flow exiting from the set of second primary nozzles is directed substantially along the second longitudinal axis of the slide. In some embodiments, the gas flows exiting from the sets of first and second primary nozzles form an incident angle with the surface of the slide that is in the range from about 20 degrees to about 80 degrees. In some embodiments, the gas flows exiting from the sets of first and second primary nozzles are independently offset by up to + / - 15 degrees with respect to the longitudinal axis of the slide.

[0027] In some embodiments, the first nozzle array provides bulk fluid motion to the fluid present on the upper surface of the slide. In some embodiments, each nozzle in the set of first secondary nozzles directs the gas flow to a different location on the upper surface of the slide, and each nozzle in the set of second secondary nozzles directs the gas flow to yet another different location on the upper surface of the slide. In some embodiments, the second nozzle array establishes at least two local fluid flows. In some embodiments, the second nozzle array establishes at least three local fluid flows.

[0028] Another aspect of the present disclosure is a method of processing a specimen support slide, the method comprising: (i) depositing a first fluid onto the specimen support slide; and (ii) uniformly distributing the deposited first fluid over the specimen support slide, the deposited first fluid being distributed by introducing a first set of gas flows into the deposited first fluid, thereby imparting a first fluid motion to a first portion of the deposited first fluid during a predetermined first period, and introducing a second set of gas flows into the deposited first fluid, thereby imparting at least a second fluid motion to at least a second portion of the deposited first fluid during a predetermined second period. In some embodiments, the first and second motions provide cross mixing. In some embodiments, the first fluid motion is provided by a first nozzle array and at least the second fluid motion is provided by a second nozzle array. In some embodiments, the first portion is a majority of the first fluid on the slide and the first set of gas flows induces bulk fluid motion. In some embodiments, the second portion is a central portion of the first fluid on the slide (e.g., the central portion of the paddle of the fluid). In some embodiments, at least the second motion is a local fluid motion imparted to the central portion of the fluid on the slide. In some embodiments, the second motion induces cross mixing. In some embodiments, the first fluid is a reagent. In some embodiments, the first fluid is a mixture having a plurality of components.

[0029] Another aspect of the present disclosure is a method of processing a specimen support slide, the method including: (i) depositing a first reagent onto the specimen support slide; and (ii) uniformly distributing the deposited first reagent over the specimen support slide, the deposited first reagent being distributed by introducing a set of first pulse-driven gas jets into the deposited first reagent, thereby imparting a first fluid motion to a first portion of the deposited first reagent during a predetermined first period, and introducing a set of second pulse-driven gas jets into the deposited first reagent, thereby imparting at least a second fluid motion to at least a second portion of the deposited first reagent during a predetermined second period. In some embodiments, the first portion of the deposited first reagent includes a majority of the deposited first reagent. In some embodiments, the first fluid flow is a bulk fluid flow. In some embodiments, at least the second portion of the deposited first reagent is a central portion of the deposited first reagent. In some embodiments, the first and second motions are in the same direction. In some embodiments, the first and second motions are in opposite directions. In some embodiments, the first predetermined period ranges from about 2 seconds to about 10 seconds. In some embodiments, the second predetermined period ranges from about 2 seconds to about 10 seconds. In some embodiments, the set of first gas jets and the set of second gas jets each act sequentially at least twice. In some embodiments, the set of first gas jets and the set of second gas jets each act sequentially at least four times. In some embodiments, the pulse driving by the set of first or second gas jets occurs at a frequency ranging from about 4 Hz to about 20 Hz. In some embodiments, a further reagent is dispensed onto the upper surface of the slide and mixing with the gas jets is performed again to mix the second reagent.

[0030] Another aspect of the present disclosure is a non-contact mixer having a first nozzle array and a second nozzle array, the first nozzle array being adapted to provide one or more gas flows to the fluid and / or the substrate such that substantially all of the fluid on the substrate moves along a first substantially circular path (a first "swirl" motion), and the second nozzle array being adapted to provide one or more gas flows to the fluid and / or the substrate such that three different portions of the fluid on the substrate move along second, third, and fourth substantially circular paths (first, second, and third "swirl" motions). In some embodiments, the second nozzle array is adapted to provide cross-mixing of the fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031]

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DETAILED DESCRIPTION OF THE INVENTION

[0032] Specified

[0033] Conversely, unless explicitly stated otherwise, in any method claimed in this specification that includes two or more steps or acts, it should be similarly understood that the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0034] As used herein, the singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "include" is defined inclusively such that "includes A or B" means including A, B, or both A and B.

[0035] 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" is inclusive, i.e., at least one is included, but is construed to include one or more of a number or list of elements and optionally further non-enumerated items. Only terms that are clearly indicated to the contrary, such as "only one of" or "exactly one of", or when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall be construed simply to indicate exclusive alternatives (i.e., "one or the other but not both") when preceded by exclusive terms such as "either", "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.

[0036] As used herein and in the claims, the phrase "at least one" refers to a list of one or more elements and means at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically recited in the list of elements, and is to be understood not to exclude any combination of elements in the list of elements. This provision similarly allows elements other than those specifically identified in the list of elements referred to by the phrase "at least one" to be optionally present, whether or not they are related to the specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") means, in one embodiment, at least one A, optionally including two or more, with B being absent altogether (and, optionally, including elements other than B); in another embodiment, at least one B, optionally including two or more, with A being absent altogether (and, optionally, including elements other than A); in yet another embodiment, at least one A, optionally including two or more, and at least one B, optionally including two or more, (and, optionally, including other elements); and so on.

[0037] As used herein, the terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. In particular, each of the terms is defined without contradiction to the general provisions of U.S. patent law for "comprising," and thus is construed as an open term meaning "at least the following," and is similarly construed as not excluding additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Further, while steps and processes can be outlined herein in a particular order, those skilled in the art will recognize that the ordering of steps and processes can vary.

[0038] As used herein, the term "biological sample" or "tissue sample" refers to any sample containing biomolecules (proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (humans; domestic animals such as cats, dogs, horses, cows, and pigs; and laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears like Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, cell fragments, or organelles (such as those obtained by lysing cells and separating the components of the cells by centrifugation or other methods). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (such as obtained by visual biopsy or needle biopsy), nipple aspirate fluid, earwax, milk, vaginal fluid, saliva, swabs (such as oral swabs), or any substance containing biomolecules derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized sample or a liquefied sample) prepared from a tumor or a portion of a tumor obtained from a subject.

[0039] As used herein, the phrase "bulk fluid flow" refers to the motion imparted to the majority of the fluid on a substrate. The bulk fluid flow can follow a circular path. The bulk fluid flow can be in a particular direction, for example, clockwise or counterclockwise.

[0040] As used herein, the term "fluid" refers to any liquid including water, solvents, solutions (e.g., buffers), etc. The term "fluid" similarly refers to any mixture, colloid, suspension, etc. The term "fluid" similarly encompasses reagents, stains, and other specimen processing agents (e.g., adhesives, fixatives, etc.) that can be applied to microscope slides and / or specimens. The fluid can 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 dye or stain molecules (e.g., H&E staining solution, Pap staining solution, etc.). Still further examples of fluids include paraffin-embedded biological specimens, aqueous washing solutions, and solvents and / or solutions for deparaffinizing hydrocarbons (e.g., aromatic compounds such as alkanes, isoalkanes, xylene, etc.). Still further examples of fluids include solvents (and mixtures thereof) used to dehydrate or rehydrate biological specimens.

[0041] As used herein, the terms "nozzle array", "nozzle set", and "set of nozzles" each refer to a series of nozzles that are adapted together to provide a fluid flow, such as a bulk fluid flow or a local fluid flow. In some embodiments, a "nozzle array" can itself include a plurality of "sets of nozzles" or at least two series of nozzles.

[0042] As used herein, the term "plurality" refers to two or more, e.g., three or more, four or more, five or more, etc.

[0043] As used herein, the term "reagent" refers to any liquid or liquid composition used in a specimen processing operation that includes adding a liquid or liquid composition to a slide. Examples of reagents and processing fluids include solutions, emulsions, suspensions, and solvents (pure or mixtures thereof). These and other examples can be aqueous or non-aqueous. Further examples include solutions or suspensions of specific binding entities, antibodies, nucleic acid probes, and solutions or suspensions of dye or stain molecules (e.g., H&E staining solution, Pap staining solution, etc.). Still further examples include paraffin-embedded biological specimens, aqueous wash solutions, and solvents and / or solutions for deparaffinizing hydrocarbons (e.g., aromatic compounds such as alkanes, isoalkanes, xylene, etc.).

[0044] As used herein, the phrase "regional fluid flow" refers to a flow of fluid that is within a particular portion or area of the fluid on a substrate but less than the majority of the fluid on the substrate. The regional fluid flow can be in a particular direction, e.g., clockwise or counterclockwise. One of ordinary skill in the art will understand that any fluid present on the substrate can be divided into several areas, regions, or portions, and regional fluid flow can be imparted to any of these areas, regions, or portions.

[0045] As used herein, the term "slide" refers to any suitable sized substrate (e.g., made of glass, quartz, plastic, silicon, etc., either wholly or in part) on which a biological specimen is placed for analysis, and more particularly, to a "microscope slide", such as a standard 3-inch by 1-inch microscope slide or a standard 75 mm by 25 mm microscope slide. Examples of biological specimens that can be placed on a slide include, without limitation, 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 at specific locations on a slide. In some embodiments, the term "slide" can refer to SELDI and MALDI chips as well as silicon wafers.

[0046] As used herein, the terms "stain", "staining", or the like as used herein generally refer to any treatment of a biological specimen to detect and / or discriminate the presence, location, and / or amount (such as concentration) of a particular molecule (such as a lipid, protein, or nucleic acid) or a particular structure (such as a normal or malignant cell, 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 portion 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 assist in the observation of molecules, cellular structures, and organisms not only by bright-field microscopy but also by other observation tools such as phase-contrast microscopy, electron microscopy, and fluorescence microscopy. Some of the staining performed by System 2 can be used to visualize the outline of cells. Other staining performed by System 2 can rely on a particular cellular component (such as a molecule or structure) being stained while other cellular components are not stained or are stained relatively less. Examples of types of staining methods performed by System 2 include, without limitation, histochemical methods, immunohistochemical methods, and other methods based on intermolecular reactions (including non-covalent interactions) such as hybridization reactions between nucleic acid molecules. Particular staining methods include, but are not limited to, primary staining methods (such as H&E staining, Pap staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods such as fluorescence in situ hybridization (FISH).

[0047] As used herein, the term "substantially" means a qualitative state indicating the entire or nearly entire range or degree of a characteristic or property of interest. 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%.

[0048] Devices and Systems

[0049] One aspect of the present disclosure is a non-contact mixer device configured to introduce a gas, such as a pulse of gas, into a fluid on a substrate, e.g., a fluid on the surface of a specimen-supporting microscope slide, to effect mixing of one or more components within the fluid (a paddle of fluid on the upper surface of the microscope slide). In some embodiments, the substrate or microscope slide includes a biological sample containing cells and / or tissue, and mixing by the pulse of gas occurs without damaging the cells and / or tissue within the sample and / or with minimal waste, i.e., the pulse of gas acts to minimize the amount of fluid directed away from the surface of the substrate.

[0050] In some embodiments, the gas flow or pulse of gas is generated by a non-contact mixer device, the non-contact mixer including a plurality of nozzles adapted to direct the gas flow or pulse of gas to different areas (e.g., at a predetermined angle, a predetermined area, a predetermined position) on the surface of the substrate. When doing so, the directed gas flow enables any fluid present on the slide to vibrate, move, and / or agitate. As further described herein, the plurality of nozzles are fluidly connected to a plenum, the plenum being in further communication with an inlet. The non-contact mixer can include one or more plenums, each plenum being in fluid communication with a different inlet and each plenum being in further communication with a different set of nozzles.

[0051] Referring to FIGS. 1A-1B and FIGS. 2A-2B, the non-contact mixer 10 can include one or more plenums 11A and 11B, and each of the one or more plenums can have a plurality of nozzles 12A or 12B that are in fluid communication with the respective plenum. In some embodiments, each of the one or more plenums 11A and 11B is in fluid communication with an inlet 13A and 13B, respectively, independently. Thus, a gas stream entering through inlets 13A and 13B can flow into plenums 11A or 11B, respectively, where the gas stream is distributed to each of the individual gas nozzles 12A or 12B and ejected as a gas stream or jet. FIGS. 1A and 1B show that the non-contact mixer can comprise a set of two nozzles, each set of nozzles being in fluid communication with a plenum 11A or 11B, and the plenum 11A or 11B being in communication with an inlet 13A or 13B, respectively, although the non-contact mixer can include only a single plenum or can comprise three or more plenums.

[0052] In practice, the non-contact mixer shown in FIGS. 2A and 2B is shown as a monolithic device, but one of ordinary skill in the art will understand that two or more non-contact mixers can be positioned over a substrate, each non-contact mixer including one or more plenums and / or a plurality of nozzles in fluid communication with an inlet. Thus, one or more non-contact mixers can be appropriately configured to direct a gas stream or a pulse of gas to different areas on the surface of the substrate, as detailed herein. For example, the system herein can include two or more non-contact mixers, each non-contact mixer having a single inlet in communication with a single plenum, and the single plenum being in communication with a plurality of nozzles.

[0053] As shown in FIGS. 3A and 3B, in some embodiments, the nozzle, plenum, and inlet are housed within a body 14, such as a monolithic body or a body composed of multiple components, and the body 14 can be configured for incorporation into a larger structure, such as a dyeing apparatus. The body 14 can include one or more mounting or attachment points 15 so that the non-contact mixer can be removably incorporated into a dyeing apparatus or other equipment. In some embodiments, also, as shown in FIGS. 4A and 4B, the mounting or attachment points can open at both its upper and lower portions, enabling a plurality of points for connection to another device or equipment. The body 14 can be composed of metal, alloy, polymer, or copolymer. In some embodiments, the body 14, plenum, inlet, and nozzle can be produced by machining a solid block, molding, or otherwise fastening multiple components together to form the non-contact mixer 10. In some embodiments, the non-contact mixer and all of its components can be produced using 3D printing.

[0054] In some embodiments, and referring at least to FIGS. 1A and 1B, a plurality of nozzles 12A or 12B are disposed along the lower surface 16 of the non-contact mixer 10. In some embodiments, the plurality of nozzles are arranged in two or more rows. In some embodiments, the nozzles can be arranged in rows parallel to each other, for example, parallel to the longitudinal axis 20 of the non-contact mixer. In other embodiments, the nozzles can be arranged in a staggered configuration. In other embodiments, the nozzles can be configured randomly with respect to each other. In embodiments where there are sets of multiple nozzles, each set of nozzles can be configured independently. For example, FIG. 7 shows a first set of nozzles parallel to the longitudinal axis 20 (each individual nozzle is labeled 12B); and a second set of nozzles substantially parallel to the longitudinal axis 20 (each individual nozzle is labeled 12A).

[0055] There are many variables that can determine the gas flow rate through any individual gas nozzle, including, in addition to the gas pressure supplied to the plenum through the inlet, the size and shape of the nozzle, and the number of nozzles in fluid communication with any individual plenum. One of ordinary skill in the art will understand that there are many variables.

[0056] In some embodiments, each inlet is configured to receive pressurized gas from an external gas source (e.g., a pump, a pneumatic compressor, a blower, a fan, or, if not yet sufficiently pressurized, some other means sufficient to pressurize the gas from the gas source). In some embodiments, the gas flow rate through each inlet, independently, is in the range of about 1 L / min to about 5 L / min. In other embodiments, the gas flow rate through each inlet, independently, ranges from about 2 L / min to about 25 L / min. Without being bound by any particular theory, it is believed that the flow rate through each nozzle in communication with the inlet is approximately the same.

[0057] The nozzles can have any size or shape. For example, the nozzles can be conical (or substantially conical), ports with helical striations, small arrays of angled orifices with small internal conical openings, or arrays with uniform round internal profiles and external bosses. Such further non-limiting examples of nozzle shapes are shown in FIG. 8. In some embodiments, also, regardless of the size or shape of the nozzles, the nozzles can be grouped together, and in some embodiments, the simultaneous operation of a group of nozzles can enable the individual gas streams emanating from each individual nozzle to merge.

[0058] In some embodiments, the nozzle has an opening with a diameter in the range of from about 0.025 inches (0.635 mm) to about 0.035 inches (0.889 mm). In other embodiments, the nozzle has an opening with a diameter in the range of from about 0.010 inches (0.254 mm) to about 0.030 inches (0.792 mm). In still other embodiments, the nozzle has an opening with a diameter of about 0.050 inches (1.27 mm). One of ordinary skill in the art will understand that the nozzles can each have openings of the same or different diameters.

[0059] In some embodiments, the plurality of nozzles are equally spaced from each other. For example, considering nozzles having circular openings, nozzle 12 can be spaced from each other by from about 0.150 inches (3.81 mm) to about 0.300 inches (7.96 mm) as measured from the center of one circular opening to the center of another circular opening. In another example, again assuming circular openings, the nozzles can be spaced from each other by from about 0.300 inches (7.96 mm) to about 0.500 inches (12.7 mm). In still another example, again assuming circular openings, the nozzles can be spaced from each other by about 1.00 inches (25.4 mm). Of course, one of ordinary skill in the art will understand that the nozzles can have any geometric shape, such as circular, oval, triangular, square, and the like.

[0060] The nozzles can be provided at various angles such that the gas flow from each nozzle can be directed as desired (e.g., at a specific angle in any of the coordinate directions towards the surface of the substrate) (see, for example, FIGS. 4A and 4B). When any nozzle is considered as a single point, one of ordinary skill in the art will understand that the nozzle can be formed at any angle and in any direction (x, y, or z) within the body of the non-contact mixer such that the gas flow or jet emanates from the nozzle (12A or 12B) and aims at the substrate surface (70) at a predetermined incident angle at a predetermined position. By combining nozzles having different angles in any of the x, y, or z dimensions into one array, one of ordinary skill in the art will further understand that various angles along various coordinates will enable the movement of the fluid on the substrate in a direction governed by the set of nozzles.

[0061] Figures 5A and 5B show two nozzle angles shown as "slant angle" and "offset angle". Those skilled in the art will understand that by independently adjusting the slant angle and offset angle of each nozzle, the gas jet can be directed (and at a specific entry angle) to different areas or positions on the substrate positioned under the non-contact mixer from the nozzle. Generally, the slant angle refers to the angle with respect to the flat surface of the substrate (i.e., a slant angle of 90 degrees is directly below (perpendicular to the surface); a slant of 0 degrees is parallel to the substrate surface). In some embodiments, the slant angle enables the gas flow emitted from the nozzle to impart motion to the fluid on the substrate. In some embodiments, the slant angle can be defined by considering the incident angle formed between the gas flow emitted from the nozzle and the surface of the substrate positioned under the non-contact mixer. In some embodiments, it is believed that the jets will also interact fluidly with each other such that the actual entry point on the substrate is governed not by "unpredictable" but by the combination of compressible hydrodynamics and nozzle orientation. For example, in Figure 5A, the gas flow 30 has an incident angle of 55 degrees with the substrate. On the other hand, also as shown in Figure 5B, the gas flow 31 has an incident angle of 60 degrees with the substrate.

[0062] In some embodiments, the slant angle ranges from about 5 degrees to about 90 degrees. In other embodiments, the slant angle ranges from about 10 degrees to about 70 degrees. In other embodiments, the slant angle ranges from about 15 degrees to about 65 degrees. In still other embodiments, the slant angle ranges from about 20 degrees to about 60 degrees. In further embodiments, the slant angle ranges from about 25 degrees to about 55 degrees. In yet further embodiments, the slant angle ranges from about 30 degrees to about 55 degrees.

[0063] In addition to the tilt angle, the nozzle can be defined by an offset angle, which refers to the angle by which the gas nozzle deviates from an axis parallel to the longitudinal axis 20 or an axis parallel to the horizontal axis 21 (see FIGS. 5A and 5B). In some embodiments, the offset angle enables the gas flow exiting the nozzle to impart directivity to the fluid on the substrate. In some embodiments, the offset angle ranges from about 0 degrees to about 25 degrees. In other embodiments, the offset angle ranges from about 5 degrees to about 15 degrees. In still other embodiments, the offset angle ranges from about 10 degrees to about 15 degrees.

[0064] According to another example, FIG. 5B shows a gas flow 31 having a tilt angle of 60 degrees with respect to a plane parallel to the horizontal axis 21, and the gas flow also has an offset angle of 10 degrees with respect to an axis parallel to the horizontal axis 21. Similarly, FIG. 5A shows a gas flow 30 having a tilt angle of 55 degrees with respect to a plane perpendicular to the longitudinal axis 20, and the gas flow is offset by 5 degrees with respect to an axis parallel to the longitudinal axis 20.

[0065] In the context of a microscope slide positioned beneath the non-contact mixer 10, in some embodiments, a set of nozzles (e.g., 4 to 12 nozzles) is arranged to enable a bulk fluid flow to the fluid positioned on the surface of the slide. In some embodiments, the jets of gas exiting some of the nozzles within the set of nozzles are directed towards the longitudinal edge of the slide or towards the fluid near the longitudinal edge of the slide.

[0066] As an example, and with reference to FIGS. 6C and 6D, the first nozzle array can be adapted to impart a bulk fluid motion 50 to the fluid present on the surface of the substrate. Here, the nozzles within the first nozzle array are arranged such that the gas flow is directed substantially towards the edge 60 of the microscope slide, but not completely parallel to the edge 60. The resulting bulk fluid motion shown in FIGS. 6C and 6D is in the counterclockwise direction, but one of ordinary skill in the art will understand that the nozzle array can be configured such that the bulk fluid motion is in the clockwise direction. FIGS. 6A and 6B similarly show three discrete local fluid flows 51, 52, and 53, or 54, 55, and 56, and each of the discrete local fluid flows is provided by a second nozzle array configured to direct the gas flow at a predetermined incident angle to a predetermined position on the surface of the slide. In FIG. 6A, the local fluid flows 51 and 53 are shown in the counterclockwise direction, while the local fluid flow 52 is in the clockwise direction. In FIG. 6B, the local fluid flows 55 and 56 are shown in the clockwise direction, while the local fluid flow 54 is in the counterclockwise direction. One of ordinary skill in the art will similarly understand that the nozzles within the second nozzle array can be further adapted such that four or more local fluid flows are provided and each adjacent local fluid flow is in a different direction.

[0067] Automated Slide Processing System

[0068] Another aspect of the disclosure is an automated slide processing apparatus comprising at least one non-contact mixer configured to introduce a gas pulse into a fluid or paddle on the surface of a specimen support microscope slide to effect mixing of one or more components within the fluid or paddle. In some embodiments, the automated slide processing system includes at least one non-contact mixer having two discrete nozzle arrays, each nozzle array being adapted to impart a flow to at least a portion of the fluid on the surface of the slide. In some embodiments, the automated slide processing system includes a non-contact mixer having two discrete nozzle arrays, the first nozzle array being adapted to impart a bulk fluid flow in a first direction and the second nozzle array being adapted to impart at least one local fluid flow to at least a portion of the fluid, the at least one local fluid flow being in a direction opposite to the bulk fluid flow imparted. In some embodiments, the automated slide processing apparatus includes at least two non-contact mixers.

[0069] In some embodiments, the specimen processing apparatus is an automated apparatus such as the BENCHMARK XT instrument, the SYMPHONY instrument, the BENCHMARK ULTRA instrument, sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of a plurality of U.S. patents disclosing systems and methods for performing automated analysis, the plurality of U.S. patents including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Published Patent Applications Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively, the specimen may be processed manually.

[0070] Examples of commercially available H&E stainers include the VENTANA SYMPHONY (individual slide stainer) and VENTANA HE 600 (individual slide stainer) series H&E stainers from Roche; the Dako CoverStainer (batch stainer) from Agilent Technologies; the Leica ST4020 small linear stainer (batch stainer), Leica ST5020 multi-stainer (batch stainer), and Leica ST5010 automated stainer XL series (batch stainer) H&E stainers from Leica Biosystems Nussloch GmbH. The non-contact mixer described herein can be added to any of the above specimen processing systems.

[0071] The specimen processing apparatus can apply a fixative to the specimen. Fixatives can include crosslinking agents (aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde crosslinking agents), oxidizing agents (such as metal ions and complexes such as osmium tetroxide and chromic acid), protein denaturants (such as acetic acid, methanol, and ethanol), fixatives of unknown mechanism (such as mercuric chloride, acetone, and picric acid), combination reagents (such as Carnoy's fixative, Methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Zenker's fluid), microwaves, and various miscellaneous fixatives (such as exclusion volume fixation and vapor fixation). The non-contact mixer in fluid communication with the microscope slide can be used to uniformly distribute any of these fixatives onto the slide or into another fluid, as detailed herein.

[0072] When the specimen is a sample embedded in paraffin, the sample can be deparaffinized by a specimen processing apparatus using an appropriate deparaffinizing liquid(s). After the waste remover removes the deparaffinizing liquid(s), any number of substances can be successively applied to the specimen. The substances can be for pretreatment (e.g., protein cross-linking, nucleic acid exposure, etc.), denaturation, hybridization, washing (e.g., stringent washing), detection (e.g., visual or ligation of a marker molecule to a probe), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, coverslipping, or the like. Again, any of these substances being applied can be mixed or dispensed through the use of the non-contact mixer described herein.

[0073] The specimen processing apparatus can apply a wide range of substances to the specimen, and the wide range of substances can then be uniformly dispensed and / or mixed using a non-contact mixer in fluid communication with a slide holder. The substances include, without limitation, stains, probes, reagents, rinses, and / or conditioners. The substances can be a fluid (e.g., a gas, a liquid, or a gas / liquid mixture) or the like. The fluid can be a solvent (e.g., a polar solvent, a nonpolar solvent, etc.), a solution (e.g., an aqueous solution or another type of solution), or the like. The reagents can include, without limitation, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen retrieval solutions (e.g., light source-activating solutions based on water or non-water, antigen retrieval buffers, etc.), or the like. The probe can be an isolated nucleic acid or an isolated synthetic oligonucleotide attached to a detectable label. The label can include a radioisotope, an enzyme substrate, a cofactor, a ligand, a chemiluminescent or fluorescent agent, a hapten, and an enzyme.

[0074] Automated IHC / ISH slide stainers typically include at least reservoirs for the 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 for coordinating the operation of the reagent dispensing unit and the waste removal system. In addition to performing the staining steps, many automated slide stainers can also perform (or be compatible with a separate system for performing) the steps associated with staining, including slide baking (for adhering 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 some specific examples of automated IHC / ISH slide stainers, including the intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND, and Lab Vision Autostainer (Thermo Scientific), and their various features. Further, Ventana Medical Systems, Inc. is the assignee of a plurality of U.S. patents disclosing systems and methods for performing automated analysis, the plurality of U.S. patents 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. Patent Application Publication Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety.

[0075] Commercially available staining units typically operate based on one of the following principles. The following principles are: (1) Open-type individual slide staining, where the slide is positioned horizontally and the reagent is dispensed as a paddle on the surface of the slide containing the tissue sample, such as that implemented on the DAKO AUTOSTAINER LINK 48 (Agilent technologies) and intelliPATH (Biocare Medical) stainers; (2) Liquid overlay technology, where the reagent is covered by an inert fluid layer deposited over the sample or dispensed through the inert fluid layer, such as that implemented on the BENTANA BenchMark and DISCOVERY stainers; (3) Capillary gap staining, where the slide surface is placed in proximity to another surface (which can be another slide or a cover plate) to create a narrow gap, and through that narrow gap, capillary forces draw up the liquid reagent and maintain the liquid reagent in contact with the sample, such as the staining principle used by the DAKO TECHMATE, Leica BOND, and DAKO OMNIS stainers. Several repetitions of capillary gap staining do not mix the fluid within the gap (such as on the DAKO TECHMATE and Leica BOND). In a variation of capillary gap staining called dynamic gap staining, capillary forces are used to apply the sample to the slide, and then the parallel surfaces translate relative to each other to agitate the reagent during incubation, thereby resulting in reagent mixing (such as the staining principle implemented on the DAKO OMNIS slide stainer (Agilent)). In translational gap staining, the translatable head is positioned over the slide. The lower surface of the head is spaced from the head by only a first gap that is small enough for a liquid meniscus to form on the slide from the liquid 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 and defines a second gap that is smaller than the first gap between the mixing extension and the slide.During the translation of the head, the lateral dimension of the mixing and extending portion is sufficient to generate lateral movement of the liquid on the slide in a direction that substantially extends from the second gap to the first gap. See WO2011-139978A1. It has recently been proposed to use inkjet technology to deposit reagents on the slide. This list of staining techniques is not intended to be exhaustive, and any of the non-contact mixers described herein can be used in conjunction with such a system to effect the dispensing and mixing of any fluid present on a specimen support microscope slide, including staining reagents.

[0076] Some embodiments are devices for automatically processing biological specimens, the device comprising at least one slide tray (such as those described herein) for holding a plurality of slides in a substantially horizontal position, wherein a biological specimen is located on the slide; one or more workstations for receiving the slide tray and performing one or more slide processing operations on the plurality of slides held within the slide tray; a transporter for moving the slide tray into and out of one or more of the workstations; a fluidics module in fluid communication with one or more of the workstations for supplying reagents to the one or more workstations; a pneumatic module in fluid communication with the one or more workstations and the fluidics module for supplying negative pressure and / or pressurized gas to the one or more workstations and the fluidics module; a non-contact mixer positioned over the slides within the slide tray to effect mixing; and a control module in electrical communication with the transporter, the one or more workstations, the fluidics module, and the pneumatic module for coordinating the functions of the components of the device (including the non-contact mixer and its various components) during the processing of the biological specimen.

[0077] In some embodiments, an apparatus for automatically processing biological specimens further includes a control system for independently controlling each non-contact mixer such that mixing is adjusted for each slide. In some embodiments, an apparatus for automatically processing biological specimens further includes one or more sensors or other feedback mechanisms to enable monitoring of the mixing and / or dispensing of fluid dispensed onto the surface of the slide. In some embodiments, the control system includes a microprocessor and one or more microcontrollers, and the one or more microcontrollers receive instructions from the microprocessor and separately control one or more of the one or more workstations, the fluidics module, the non-contact mixer, and / or the transporter. In some embodiments, at least one of the workstations includes a movable nozzle assembly, and the nozzle assembly includes one or more nozzles through which reagents are delivered to the slide. The nozzles can be dispense nozzles.

[0078] In some embodiments, the workstation can perform slide processing operations on one or more slides within the slide tray, e.g., at least two or four slides within the slide tray, or the workstation can simultaneously perform slide processing operations (including mixing operations by a non-contact mixer) on all slides within the slide tray. In some embodiments, one or more workstations dispense reagents to the slides within the slide tray such that a significant amount of reagent that contacts the first slide does not contact the second slide, thereby minimizing cross-contamination between slides. Such a workstation can include one or more directional nozzles for dispensing reagents onto the slides, e.g., the one or more directional nozzles can include a pair of directional nozzles that dispense reagents in opposite directions across the surface of the slide. In more particular embodiments, the one or more directional nozzles can further include a directional nozzle that dispenses reagent toward the lower surface of the slide. In other embodiments, one or more workstations can simultaneously dispense reagents (e.g., the same reagent) to at least two slides held within the slide tray within a given workstation, or one or more workstations can simultaneously dispense reagents (e.g., the same reagent) to all slides held within the slide tray within a given workstation. Following the dispensing of fluids and / or reagents, the non-contact mixer can be activated independently to distribute and / or mix the fluids on the surface of the slide.

[0079] Some embodiments are automated methods for processing a plurality of slides that support biological tissue samples. The method includes performing a set of slide processing operations on a plurality of slides within one or more workstations while the plurality of slides are held in a substantially horizontal position in a spatially co-planar manner within a slide tray, wherein each of the plurality of slides is positioned proximate to at least one non-contact mixer; the set of slide processing operations includes staining and solvent exchange of the samples on the slides in a substantially horizontal position in a spatially co-planar manner by flowing one or more stains from at least one reagent container through a fluidics module and out of at least one dispensing nozzle positioned over the slide tray; after performing the set of slide processing operations including at least staining and solvent exchange, transporting the slide tray holding the plurality of slides to an automated coverslipper workstation; coverslipping the plurality of slides held within the slide tray by respective separate coverslips using the automated coverslipper workstation while the plurality of slides are held in a substantially horizontal position in a spatially co-planar manner within the slide tray, whereby the coverslips on the slides are spaced apart from each other; and removing the slide tray holding the coverslipped slides from the automated coverslipper workstation.In some embodiments, processing comprises: (i) baking the sample under a radiative heater; (ii) deparaffinizing the sample; (iii) staining the sample by delivering one or more stains through one or more fluidic components and out of one or more nozzles positioned substantially over the slide tray, wherein the one or more fluidic components fluidically connect at least one reagent container holding the one or more stains to the one or more nozzles; (iv) solvent-exchanging the sample; and (v) coverslipping the sample with a separate coverslip, and the steps are automatically performed by an apparatus having two or more workstations between which a slide tray holding the slides moves during processing.

[0080] In some embodiments, after the specimen has been processed, the user may transport the specimen support slide to an imaging device. In some embodiments, the imaging device is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the imaging device is a digital pathology device disclosed in International Patent Application No. PCT / US2010 / 002772, entitled “IMAGING SYSTEM AND TECHNIQUES” (published as Patent Publication No. WO / 2011 / 049608), or in U.S. Patent Application Publication No. 2014 / 0178169, filed Feb. 3, 2014, entitled “IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME”. International Patent Application No. PCT / US2010 / 002772 and U.S. Patent Application Publication No. 2014 / 0178169 are hereby incorporated by reference in their entireties. In other embodiments, the imaging device includes a digital camera coupled to a microscope.

[0081] Control system

[0082] In some embodiments, the non-contact mixer can be controlled by a controller. In some embodiments, the control system is in communication with an actuator, a valve, and / or a solenoid and can control the air entering the inlet of the non-contact mixer or enable pulsed driving of the gas flow from each of the nozzles in fluid communication with the inlet. In some embodiments, the control system further includes a computer (at least one processor and a non-tangible memory containing instructions and recording information), and the computer controls the opening and closing of the actuator, the valve, and / or the solenoid such that an array of nozzles or a set of nozzles forming part of the non-contact mixer operates (e.g., is pulsed at regular time intervals or at a regular frequency). The control system enables dynamic adjustment of the actuator, the valve, and / or the solenoid such that the flow and pressure are independently controlled along each plenum.

[0083] In some embodiments, the control system can further include one or more sensors for monitoring the gas jets directed towards the microscope slide. In other embodiments, the control system can include one or more feedback mechanisms for monitoring the mixing of one or more fluids present on the surface of the slide.

[0084] In some embodiments, the control system can further include one or more sensors for monitoring surface tension, fluid volume, fluid temperature, and other mechanical properties of the fluid on the microscope slide. In other embodiments, the control system monitors the integrity of the fluid on the slide and includes one or more feedback mechanisms to dynamically adjust the actuator, the valve, and / or the solenoid to prevent or reduce the forced removal of one or more fluids from the slide such that the flow and pressure are independently controlled along each plenum.

[0085] Optical or video detection and analysis can be used to optimize mixing. As an example, optical or video detection can be used to detect a change in color when a colored reagent is mixed to form a clear fluid. Other optical measurements, such as spectral excitation, absorption, light scattering, fluorescence, luminescence, radiation, polarized microscopy, Raman scattering, and spectral analysis can be used to monitor the mixing of a fluid in contact with a sample on the surface of a microscope slide. Data can be collected and analyzed by a computer or control system that controls the mixing process. For example, if sufficient mixing is achieved based on the received data, the non-contact mixer can be turned off. As another example, if the mixing is insufficient based on the received data, the controller can increase the time that the non-contact mixer is turned off, or the controller can change one or more of the parameters associated with non-contact mixing, such as the gas jet pulse frequency, the gas pressure, or the set of nozzles through which the gas is delivered.

[0086] Method

[0087] The present disclosure similarly provides a method of mixing fluids present on a substrate, such as a slide, using the non-contact mixer described herein. By "mixed with the contactless mixer," it is meant that the non-contact mixer operates to effect mixing, dispensing, or replenishing of fluids within paddles on the surface of a specimen support slide. As noted herein, the non-contact mixer is configured such that a pulse of gas (e.g., a jet of gas or a gas stream) emitted from a plurality of nozzles or a series of sets of nozzles of the non-contact mixer provides a fluid flow (e.g., agitation) to the fluid(s), causing movement of the fluid in at least one direction within at least one area of the slide. As used herein, a "pulse" of gas can mean that the gas flow is "turned on" for a particular period, e.g., for one second, and then "turned off." This can be considered a continuous stream. Similarly, "pulse" can also mean "turning on" the gas flow for a set period, during which the gas is modulated at a particular frequency, e.g., a 1 Hz frequency is applied to the gas flow for a period of five seconds, (i.e., the pulse can be a series of "turning on" and "turning off" for a set period).

[0088] Accordingly, another aspect of the present disclosure provides a method of dispensing and / or mixing a fluid on the surface of a microscope slide by introducing a gas pulse into a paddle on the surface of a specimen support slide. In some embodiments, the introduction of the pulsed-driven gas jet into the fluid causes movement and / or vibration of the fluid, thus providing a bulk fluid flow and / or a local fluid flow, and ultimately enabling a substantially uniform distribution of the fluid across the biological sample. For example, also in some embodiments, the fluid can be dispensed into a predetermined area on the surface of the microscope slide, and by activating the non-contact mixer and introducing the pulsed-driven gas jet, the fluid can be dispensed beyond the initial area of dispensing. In some embodiments, the dispensing of the fluid on the surface of the microscope slide by the introduction of the pulsed-driven gas jet can be used to facilitate the replenishment of the fluid (e.g., reagent) to the biological sample mounted on the surface of the slide. For example, the biological sample can absorb (or can absorb unevenly) the reagent deposited on its surface, and ultimately, a certain amount of the reagent in contact with the biological sample can be substantially depleted (or depleted from a particular region or portion of the sample). Activation of the non-contact mixer (or an even individual nozzle array within the non-contact mixer) facilitates the dispensing of another aliquot of the same reagent to the biological sample, thus replenishing the reagent in contact with the biological sample. Activation of the non-contact mixer (or individual nozzle array) also facilitates the redistribution of the reagent from other areas of the slide to the biological sample faster than by diffusion means, thus replenishing the fluid or reagent in contact with the biological sample.

[0089] In other embodiments, the first fluid can already be present on the surface of a microscope slide (e.g., a fluid paddle), and following the introduction of a second fluid, e.g., a reagent introduced via a dispenser, the second fluid can be substantially uniformly distributed within the first fluid following the introduction of a pulsed-driven gas jet from a non-contact mixer. In some embodiments, by "substantially uniformly distributed," it is meant that the reagent concentration between two distinct points on the slide differs by no more than 10% in magnitude. In other embodiments, by substantially uniformly distributed, it is meant that the reagent concentration between two distinct points on the slide differs by no more than 5% in magnitude. In yet another embodiment, by substantially uniformly distributed, it is meant that the reagent concentration between two distinct points on the slide differs by no more than 2% in magnitude. Of course, any number of fluids can be deposited on the surface of a microscope slide, and one of ordinary skill in the art will understand that each of those fluids can be mixed, i.e., substantially uniformly distributed within each other's fluids, by the introduction of a pulsed-driven air jet.

[0090] In some embodiments, the methods disclosed herein are suitable for dispensing and / or mixing any volume of fluid on the surface of a slide. In some embodiments, the volume of fluid that can be dispensed 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 dispensed 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 dispensed and / or mixed according to the methods disclosed herein ranges from about 50 μL to about 750 μL. In still other embodiments, the volume of fluid that can be dispensed and / or mixed according to the methods disclosed herein ranges from about 50 μL to about 500 μL. In still other embodiments, the volume of fluid that can be dispensed and / or mixed according to the methods disclosed herein ranges from about 100 μL to about 500 μL. One of ordinary skill in the art will be able to select a properly configured non-contact mixing nozzle array (e.g., the use of specific nozzles having a specific configuration, air jet pulse frequency, time, pressure, and / or flow rate) including all operating parameters.

[0091] Generally, the method includes: (i) introducing a fluid onto the surface of a slide; and (ii) introducing a pulse of a gas jet onto the slide that introduces fluid motion and / or vibration into the slide. In some embodiments, the method includes additional steps, the additional steps including, but not limited to: (a) a detection step for feedback control of a non-contact mixer, (b) a fluid removal step; and (c) an additional fluid dispense step.

[0092] In some embodiments, a method of processing a specimen support slide includes (i) contacting a sample on the specimen support slide with a first reagent; and (ii) uniformly dispensing the first reagent on the specimen support slide by introducing a pulsed air jet onto the specimen support slide. As noted herein, the pulsed air jet can be delivered to a paddle through a properly configured non-contact mixer, e.g., a non-contact mixer described herein that promotes at least bulk mixing of fluids or reagents within the paddle and / or local mixing of fluids or reagents. In some embodiments, the pulsed air jet of air emanates from a first nozzle array and a second nozzle array of the non-contact mixer. In some embodiments, the pulsed air jets of air from the first and second nozzle arrays are offset in timing, thus allowing alternating periods of bulk fluid flow and local fluid flow. In some embodiments, the uniform dispensing allows the fluid to advance into areas of the slide that are fluid-free.

[0093] In some embodiments, the reagent is introduced into or proximate to a first fluid paddle (e.g., a paddle containing 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 a biological sample. In some embodiments, the detection probe utilized is a primary antibody, i.e., a primary antibody that enables detection of a protein target (or an epitope of a protein target) in the sample. In some embodiments, the primary antibody is conjugated to a detectable label such as a fluorophore, hapten, or enzyme. In other embodiments, the detection probe is a nucleic acid probe that enables detection of a nucleic acid sequence target in the sample. In other embodiments, the specific binding moiety is a nucleic acid probe, and the nucleic acid probe is conjugated to a detectable label such as a fluorophore, hapten, or enzyme.

[0094] In some embodiments, the pulsed drive by any nozzle of the nozzle occurs at a frequency in the range from about 0.5 Hz to about 15 Hz. In some embodiments, the acoustic source operates at a frequency in the range from about 0.5 Hz to about 10 Hz. In some embodiments, the pulsed drive by any nozzle of the nozzle occurs at a frequency in the range from about 1 Hz to about 10 Hz. In some embodiments, the pulsed drive by any nozzle of the nozzle occurs at a frequency in the range from about 1 Hz to about 20 Hz.

[0095] In some embodiments, the first set of nozzles can operate at a first frequency or range of first frequencies; while the second set of nozzles can operate at a second frequency or range of second frequencies. Similarly, any set of nozzles of the non-contact mixer can operate at a first frequency for a first period of time and then at a second frequency for a second period of time. In other embodiments, a particular set of nozzles of the non-contact mixer can first operate at a first frequency, and the frequency can increase or decrease over time (e.g., ramp up or ramp down over time at a predetermined interval). For example, the first frequency can be 10 Hz, the second frequency can be 20 Hz, and the frequency can be ramped from 10 Hz in 1 Hz increments every 0.5 seconds until the 20 Hz frequency is achieved. In other embodiments, the pneumatic source uses frequency modulation, whereby the frequency is offset by an amount that is + / −20% of a predetermined frequency value.

[0096] In some embodiments, following the introduction of the reagent, a gas jet pulse is delivered from at least one nozzle array of the non-contact mixer to the paddle, resulting in at least bulk fluid mixing within the paddle. In some embodiments, the pulse-driven gas jet is introduced for a total duration ranging from about 0.5 seconds to about 30 seconds. In some embodiments, the pulse-driven gas jet is introduced for a total duration ranging from about 0.5 seconds to about 20 seconds. In other embodiments, the pulse-driven gas jet is introduced for a total duration ranging from about 1 second to about 15 seconds. In other embodiments, the pulse-driven gas jet is introduced for a total duration ranging from about 5 seconds to about 15 seconds. In other embodiments, the pulse-driven gas jet is introduced for a total duration of about 10 seconds.

[0097] In some embodiments, the pulse driving of the sample by the gas jet can be at regular intervals. For example, the sample is pulse-driven by the gas jet for a certain predetermined time (e.g., 0.5 second intervals), followed by a predetermined time (e.g., 1 second intervals) during which no gas jet is introduced at all. In other embodiments, the pulse driving of the sample by the gas jet can be at irregular intervals. In other embodiments, the determination of whether to pulse-drive the sample by the gas jet can be made by using a detector that provides feedback about the degree of mixing, or a detector that can detect whether a slide or a portion of the sample needs replenishment.

[0098] In some embodiments, during a set period of time (e.g., during a culture period) when the reagent is in contact with the sample, the sample can be pulse-driven by a pulsed gas jet. For example, if an antibody is introduced into the sample and the protocol requires that the antibody remain in contact with the sample for a period of 360 seconds (e.g., the culture period), the gas jet can be introduced into the sample for a predetermined time at set intervals during the culture period. For example, gas pulses can 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 following the introduction of the antibody. Of course, instead of pulse-driving the sample with a gas jet at a predetermined interval or for a predetermined time, a feedback control device (such as those described herein) can be utilized to determine whether pulse introduction is necessary, including the length of time during which the non-contact mixer can operate therebetween.

[0099] In embodiments where the non-contact mixer includes a plurality of nozzle arrays, the gas jet can be pulse-driven sequentially from each nozzle array. For example, the gas jet can be pulse-driven from the first nozzle array during a first period, and then followed by pulse-driving by the gas jet from the second nozzle array during a second period. The sequential operation of the first and second nozzle arrays can occur one or more times, e.g., from 1 to 20 times. As an example, the gas jet within the first nozzle array can be pulse-driven for a period of 5 seconds to impart a bulk fluid flow to paddles present on the upper surface of the slide. Subsequently, pulse-driving by the gas jet from the second nozzle array for 5 seconds can impart a local fluid flow to a predetermined portion of the fluid present on the slide. In this example, the process of sequential operation can be repeated three or more times. Those skilled in the art will understand that each nozzle array can operate independently and that each array can be pulse-driven for any period of time to effect sufficient mixing.

[0100] In other embodiments, the slide or sample can be pulsed-driven by gas from a non-contact mixer (from any one or more sets of nozzles or an array of nozzles) each time a fluid is dispensed onto the slide or sample for any purpose (i.e., fluid replenishment, fluid dispensing, and / or fluid mixing). For example, if a protocol requires adding a fixed aliquot of fluid every two minutes, the gas pulse can be supplied to the slide and / or sample for at least a predetermined time each time an aliquot is added. Of course, additional pulses can be supplied during the dispense cycle as needed.

[0101] In some embodiments, the method further includes detecting whether the fluid and / or reagent is properly dispensed or mixed (e.g., by using a feedback mechanism described herein). If the detection step determines that the fluid and / or reagent is not properly mixed, the operating parameters of the non-contact mixer, such as pulse frequency, time, pressure, flow rate, and / or nozzle selection, can be adjusted.

[0102] Furthermore, when a particular system or device is required to move slides between different stations or processing areas (e.g., sample staining area, sample culture area) of the system or device, a non-contact mixer is utilized to deliver a gas pulsed jet before and / or after the movement of the slide, thereby ensuring that the fluid is properly dispensed and / or mixed before, during, and after any such movement. Following the mixing of the first reagent into the first fluid paddle, the mixed first reagent / fluid paddle can be removed from the surface of the slide. Thereafter, the first detection reagent can be introduced and then dispensed onto the surface of the slide or mixed with a second fluid paddle present on the slide. In some embodiments, the first detection reagent is specific for the label of the detection probe. For example, if the label is an enzyme, a substrate for the enzyme (detectable moiety, e.g., chromogenic moiety) can be introduced such that a colored precipitate is detected. In yet other embodiments, an anti-label antibody (secondary antibody) is introduced to elicit detection, and the anti-label antibody is specific for the label of the conjugate. For example, if the label is a hapten, an anti-hapten antibody specific for the hapten label is introduced, and the 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 conjugate and the target. The detectable moiety can then be detected according to processes known to those skilled in the art. The introduction of the detection probe and / or the detection reagent can be repeated "n" times to reflect any desired number of targets within the sample.

[0103] The methods disclosed herein are also suitable for multiplex assays. For example, a first detection probe specific for a first target and a second detection probe specific for a second target can be introduced simultaneously or sequentially. When both the first and second detection probes are introduced into the sample, a non-contact mixer can be utilized to introduce a gas pulse into the sample such that the first and second detection probes are mixed and uniformly distributed. Without being bound by any particular theory, it is believed that the uniform distribution of the first and second detection probes can facilitate a uniform detection probe concentration during staining and / or a reduction in staining artifacts. One of ordinary skill in the art will understand that any number of detection probes can be introduced into the sample on the surface of the slide simultaneously or sequentially and that a number “n” of detection probes can be mixed by the non-contact mixer of the present disclosure. Following the introduction of the detection probes, one or more detection reagents can also be introduced, again either simultaneously and / or sequentially, and again mixed by the non-contact mixer.

[0104] In some embodiments, a method of replenishing a fluid or reagent includes (i) contacting a sample on a specimen support slide with a first reagent; allowing time for the reagent to react with or be absorbed by the sample; and (iii) uniformly distributing the first reagent on the specimen support slide by introducing a gas pulse from a non-contact mixing device to the specimen support slide, thereby replenishing the reagent to areas where the reagent has at least partially depleted. In some embodiments, the method optionally includes the step of introducing a further aliquot of the first reagent before uniformly distributing the first reagent through the introduction of the gas pulse. In some embodiments, the reagent is introduced into a first fluid paddle (e.g., a paddle comprising 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 a biological sample. In some embodiments, the detection probe utilized is a primary antibody, i.e., a primary antibody that enables the detection of a protein target (or an epitope of a protein target) in the sample. In some embodiments, the primary antibody is conjugated to a detectable label such as a fluorophore, hapten, or enzyme. In other embodiments, the detection probe is a nucleic acid probe that enables the detection of a nucleic acid sequence target in the sample. In other embodiments, the specific binding moiety is a nucleic acid probe, and the nucleic acid probe is conjugated to a detectable label such as a fluorophore, hapten, or enzyme.

[0105] In some embodiments, a method of processing a specimen support slide includes (i) dispensing a first fluid onto a first portion of a microscope slide; and (ii) distributing the first fluid on the microscope slide by introducing a gas pulse into the specimen support slide by a non-contact mixer device. 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 moiety specific for a particular target within the biological sample. In some embodiments, the method further includes introducing a further aliquot of the fluid (in any region) onto the slide, and then distributing the fluid through the introduction of a gas-pulsed jet.

[0106] Examples

[0107] Example 1 - Non-Contact Mixer

[0108] The nozzle array (see FIG. 10A) was developed to enable mixing in two intentional stages. During the first stage, the entire slide is to be mixed in a single swirl, and then, during the second stage, the slide is to be split into three swirls for "cross-mixing" as a result of the single swirl. Only two jets are used for each stage, simplifying the design and reducing the chance of interference between the jets. This array demonstrated the concept for the overall slide and cross-mixing. FIGS. 10B and 10C show the results of the dye being mixed with a fluid (e.g., reaction buffer) after the operation of the non-contact mixer.

[0109] Example 2 - Non-Contact Mixer

[0110] The nozzle array (see FIG. 11A) was developed to enable mixing in two intentional stages. During the first stage, the entire slide is to be mixed with a single swirl, and then, during the second stage, the slide is to be split into three swirls for "cross-mixing" as a result of the single swirl. This array similarly shows the concept for the overall slide and cross-mixing. FIG. 11B shows the result of the dye being mixed with the fluid (e.g., reaction buffer) after the operation of the non-contact mixer.

[0111] Example 3 - Pulse Driving Method

[0112] The nozzle array of the non-contact mixer (e.g., the non-contact mixer of FIG. 10A or FIG. 11A) is pulse-driven using a function generator that uses a high-speed (2 ms response time) valve and a modulation technique called "pulse width modulation (PWM)" for the set frequency, and during each cycle, the amount of "on time" versus "off time" varies. This ratio is called the duty cycle. Thus, a 20% duty cycle at any frequency means that 20% of the time is "on" and 80% of the time is "off". The frequency only determines how often the on-off switching occurs. For example, at 1 Hz and 80% duty cycle, the valve turns on for 800 ms and turns off for 200 ms and repeats indefinitely. At 50% duty cycle and 1 Hz, 500 ms is on and 500 ms is off. In any case, the period is independent of the duty cycle at 1000 ms, and the ratio of the "on" to "off" time indicates the duty cycle. Thus, for example, at 20% duty cycle and 10 Hz, the valve turns on for 20 ms and turns off for 80 ms and repeats (100 ms period).

[0113] At a 20% duty cycle, increasing the frequency up to 8 or 16 Hz "evens out" the small pulses, so that the small pulses generate smaller waves (less agitation), and the short 20% duty cycle simply means that not much air is being emitted. As the PWM frequency increases, it approaches a similarity to the basic (non-PWM) air stream with 20% less air supply. At a 50% duty cycle and any frequency, the results are generally less impressive than at 20% or 80% duty cycles. 50% is low enough to significantly reduce the overall air flow, but does not seem to be low enough to allow standing wave agitation nor high enough to provide good bulk fluid motion.

[0114] At an 80% duty cycle and any frequency, the mixing is usually better than at 20% or 50%. An 80% duty cycle and a 4 Hz startup frequency sometimes provide a combination of agitation and bulk fluid motion, but at 16 Hz, the mixing is usually best for almost all nozzles.

[0115] Based on these tests, it seems that bulk fluid motion enables sufficient mixing across the slide - much more important than local agitation - and that steady non - PWM nozzles provide most of the bulk fluid motion.

[0116] Further embodiments and / or components

[0117] One aspect of the present disclosure is an automated slide processing apparatus, the automated slide processing apparatus comprising: (a) at least one fluid dispenser configured to dispense fluid onto an upper surface of a specimen support slide; and (b) at least one non-contact mixer for mixing fluid present on the upper surface of the slide, the non-contact mixer comprising a first nozzle set and a second nozzle set, the first nozzle set being configured to impart a first motion to the fluid present on the upper surface of the slide, and the second nozzle set being configured to impart a second motion to at least a portion of the fluid present on the upper surface of the slide (e.g., including, but not limited to, a portion of the fluid in which the first motion is induced). In some embodiments, the second motion induces cross-mixing of the fluid. In some embodiments, the first nozzle set operates independently of and at a different time than the second nozzle set, i.e., the first nozzle set operates exclusively from the second nozzle set. In some embodiments, the automated slide processing apparatus includes a second non-contact mixer. In some embodiments, the first motion is clockwise or counterclockwise. In some embodiments, the second motion is the other of clockwise or counterclockwise.

[0118] Another aspect of the present disclosure is an automated slide processing apparatus, (i) the automated slide processing apparatus comprising a non-contact mixer for mixing fluid present on the upper surface of the slide, the non-contact mixer comprising a first nozzle array and a second nozzle array, the first nozzle array being adapted to impart a bulk fluid flow to the fluid present on the upper surface of the slide, and the second nozzle array being adapted to impart at least a first local fluid flow to at least a portion of the fluid present on the upper surface of the slide. In some embodiments, the first local fluid flow induces cross-mixing within the fluid. In some embodiments, the automated slide processing apparatus is a staining apparatus, the fluid present on the upper surface of the slide includes a reagent, and non-limiting examples of the reagent include a staining reagent, a counterstaining reagent, or a washing reagent.

[0119] As described herein, an automated slide processing apparatus can be tied to an imaging device. In some embodiments, the imaging system or device can be a multispectral imaging (MSI) system or a fluorescence microscopy system. The imaging system used herein is MSI. MSI generally equips a computerized microscope-based imaging system for the analysis of pathological specimens by providing access to the spectral distribution of images at the pixel level. There are various multispectral imaging systems, but an operational aspect common to all of these systems is the ability to form multispectral images. A multispectral image is an image that captures image data at specific wavelengths or within specific spectral bandwidths across the electromagnetic spectrum. These wavelengths can be selected by optical filters or by the use of other devices capable of selecting a predetermined spectral component including electromagnetic radiation at wavelengths beyond the visible light range, such as, for example, infrared (IR).

[0120] The MSI system can include an optical imaging system, a portion of which includes a spectral selectivity system that is adjustable to define a predetermined number N of discrete optical bands. The optical system can be adapted to image a tissue sample illuminated in a transmissive state by a broadband light source on an optical detector. The optical imaging system, in one embodiment, can include a magnification system, such as a microscope, and has a single optical axis that is substantially spatially aligned with a single optical output of the optical system. The system forms a sequence of images of the tissue because the spectral selectivity system is adjusted or regulated (e.g., by a computer processor) to ensure that images are acquired in different discrete spectral bands. The apparatus can further include a display on which at least one visually recognizable image of the tissue appears from the sequence of acquired images. The spectral selectivity system can include a light dispersive element, such as a diffraction grating, an assembly of optical filters, such as thin film interference filters, or any other system adapted to select a particular passband from the spectrum of light transmitted from the light source through the sample and toward the detector in response to user input or commands of a pre-programmed processor.

[0121] In an alternative implementation, the spectral selectivity system defines several optical outputs corresponding to N discrete spectral bands. This type of system captures the transmitted light output from the optical system and spatially redirects at least a portion of this light output along N spatially distinct optical paths to image an image of a specified spectral band on a detector system along an optical path corresponding to this specified spectral band.

[0122] Embodiments of the subject matter and the operations described in this specification can be implemented in digital electronic circuitry, or in computer software, firmware, hardware, or in combinations of one or more of them that include the structures disclosed in this specification and their structural equivalents. For example, a control system can comprise computer hardware and / or software that includes any of the components described in this specification. Embodiments of the subject matter described in this specification can be implemented as one or more computer programs, i.e., as one or more modules of computer program instructions encoded on a computer storage medium for execution by, or to control the operation of, a data processing apparatus. Any of the modules described in this specification can include logic executed by a processor(s). As used in this specification, "logic" refers to any information having the form of an instruction signal and / or data that can be applied to affect the operation of a processor. Software is an example of logic.

[0123] A computer storage medium can be, or can include, a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or a combination of one or more of them. Further, a computer storage medium is not a propagated signal but can be the source or destination of computer program instructions encoded in an artificially generated propagated signal. A computer storage medium can also be, or can include, one or more discrete physical components or media (such as multiple CDs, disks, or other storage devices), or can be included in such. The operations described in this specification can be implemented as operations performed by a data processing apparatus on data stored on one or more computer-readable storage devices or received from other sources.

[0124] The term "programmed processor" encompasses all types of devices, apparatuses, and machines for processing data, including, by way of example, programmable microprocessors, computers, system-on-chips, or a plurality or combination of the foregoing. The apparatus may include special purpose logic circuitry, such as a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In addition to hardware, the apparatus may also include code for creating an execution environment for a computer program of interest, such as processor firmware, a protocol stack, a database management system, an operating system, a cross-platform runtime environment, a virtual machine, or code constituting one or more combinations thereof. The apparatus and the execution environment may implement various different computing model infrastructures, such as web services, distributed computing, and grid computing infrastructures.

[0125] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiler-type or interpreter-type languages, declarative or procedural languages, and can be deployed as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program can correspond to a file in a file system, but it does not have to. The program can be stored as part of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, subprograms, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers that are distributed across one site or multiple sites and interconnected by a communication network.

[0126] The processes and logic flows described herein can be performed by one or more programmable processors executing one or more computer programs to perform actions by operating on input data and generating output. The processes and logic flows can also be performed by, or implemented as, special purpose logic circuitry, e.g., an FPGA (Field Programmable Gate Array) or an ASIC (Application Specific Integrated Circuit).

[0127] Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, as well as any one or more processors of any kind of digital computer. In general, a processor will receive instructions and data from a read only memory or a random access memory or both. Essential elements of a computer are a processor for performing actions in accordance with instructions, and one or more memory devices for storing the instructions and data. In general, a computer will be operatively coupled to one or more mass storage devices for storing data, such as magnetic disks, magneto - optical disks, or optical disks, or to receive data from them, or transfer data to them, or both. However, a computer need not necessarily have such devices. Further, a computer may be embedded in another device, to name but a few examples, such as a mobile phone, a personal digital assistant (PDA), a portable audio or video player, a game console, a global positioning system (GPS) receiver, or a portable storage device (e.g., a universal serial bus (USB) flash drive). Devices suitable for storing computer program instructions and data include, by way of example, semiconductor memory devices, such as, EPROM, EEPROM, and flash memory devices; magnetic disks, such as internal hard disks or removable disks; magneto - optical disks; and all forms of non - volatile memory, media, and memory devices including CD - ROM and DVD - ROM disks. The processor and the memory may be supplemented by, or incorporated in, special purpose logic circuitry.

[0128] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non - patent publications, referred to herein and / or listed in the application data sheet, are hereby incorporated by reference in their entirety. Aspects of the embodiments may be modified, as appropriate, to use the concepts of various patents, applications, and publications thereby providing still further embodiments.

[0129] Although the present disclosure has been described with reference to a plurality of exemplary embodiments, it should be understood that numerous other modifications and embodiments may be devised by those skilled in the art that will fall within the spirit and scope of the principles of the present disclosure. More specifically, suitable variations and modifications are possible in the component parts and / or arrangements of the combination of subject matter within the scope of the above disclosure, drawings, and 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 will be apparent to those skilled in the art as well.

Claims

1. 1. An automated slide processing apparatus comprising: at least one fluid dispenser configured to dispense fluid onto an upper surface of the specimen support slide; and a non-contact mixer for mixing fluids present on the upper surface of the slide, the non-contact mixer comprising a first nozzle set and a second nozzle set, the first nozzle set adapted to impart a first motion to the fluids present on the upper surface of the slide and the second nozzle set adapted to impart at least a second motion to at least a portion of the fluids present on the upper surface of the slide, the at least second motion enabling cross-mixing of the fluids.

2. 10. The automated slide processing device of claim 1, wherein the first motion is one of a clockwise or counterclockwise agitation.

3. 3. The automated slide processing apparatus of claim 1 or 2, wherein the second motion is the other of the clockwise or counterclockwise agitation.

4. The automated slide processing apparatus of claim 3 , wherein the second motion is imparted to a central portion of the fluid.

5. 5. The automated slide processing apparatus of claim 4, wherein the second nozzle set is further adapted to impart a third motion to two ends of the fluid, the two ends of the fluid each adjacent the central portion, and the third motion being opposite to the second motion.

6. 6. The automated slide processing device of claim 5, wherein said first motion is a counterclockwise agitation, said second motion is a clockwise agitation, and said third motion is a counterclockwise agitation.

7. The automated slide processing device of claim 3 , wherein the second motion is imparted to at least two portions of the fluid.

8. 8. The automated slide processing device of claim 7, wherein the second nozzle set is further adapted to impart a third motion to the central portion of the fluid.

9. 9. The automated slide processing apparatus of claim 8, wherein the first motion is a counterclockwise motion, the second motion is a clockwise motion, and the third motion is a counterclockwise motion, and the second motion is imparted to two ends of the fluid located on either side of the central portion.

10. The automated slide processing apparatus of claim 1 , wherein the non-contact mixer is positioned above the upper surface of the slide.

11. The automated slide processing apparatus of claim 2 , wherein the non-contact mixer is positioned substantially parallel to the upper surface of the slide.

12. 12. The automated slide processing device of claim 1, wherein the first and second nozzle sets are configured to direct a stream of gas to a predetermined location on the upper surface of the slide.

13. 13. The automated slide processing device of claim 1, wherein the first nozzle set comprises from 2 to 6 nozzles.

14. The automated slide processing device of claim 1 , wherein the second set of nozzles comprises from 2 to 4 nozzles.

15. 6. The automated slide processing apparatus of claim 5, wherein the nozzles of the first nozzle set are grouped into two parallel rows along a longitudinal axis of the non-contact mixer.

16. 6. The automated slide processing apparatus of claim 5, wherein the nozzles of the second nozzle set are grouped into two substantially parallel rows along a longitudinal axis of the non-contact mixer.

17. 17. The automated slide processing apparatus of claim 1, wherein the first motion is imparted to a majority of the fluid on the upper surface of the slide.

18. The automated slide processing apparatus of claim 1 , wherein the second motion is imparted to at least two discrete portions of the fluid present on the upper surface of the slide.

19. The automated slide processing apparatus of claim 1 , wherein the second motion is imparted to at least three discrete portions of the fluid present on the upper surface of the slide.

20. 2. The automated slide processing device of claim 1, wherein said first motion is one of a clockwise or counterclockwise motion and said second motion is opposite to said first motion.

21. 21. The automated slide processing device of claim 1, wherein the automated slide processing device is a staining device and the fluid present on the upper surface of the slide comprises a reagent selected from the group consisting of a staining reagent, a counterstain reagent, or a washing reagent.

22. 22. The automated slide processing apparatus of claim 1, further comprising a control system, said control system adapted to independently operate said first and second nozzle sets to impart said first and second motions.

23. 23. The automated slide processing device of claim 22, wherein the control system comprises one or more sensors for monitoring surface tension, volume of the at least one fluid, temperature of the at least one fluid, or a mechanical property of the at least one fluid on a microscope slide.

24. 24. The automated slide processing apparatus of claim 22 or 23, wherein the control system includes one or more sensors for monitoring multiple streams of gas emanating from the first and second sets of nozzles.

25. 25. The automated slide processing apparatus of any one of claims 22 to 24, wherein the control system includes one or more feedback mechanisms for monitoring mixing.

26. 26. The automated slide processing apparatus of any one of claims 22 to 25, wherein the control system is adapted to dynamically adjust actuators, valves, and / or solenoids to control gas flow.

27. 27. The automated slide processing apparatus of claim 1, further comprising a third nozzle for imparting further motion to the fluid present on the upper surface of the slide.

28. 1. An automated slide processing apparatus comprising: at least one fluid dispenser configured to dispense fluid onto an upper surface of the specimen support slide; and a non-contact mixer for mixing fluids present on the upper surface of the slide, the non-contact mixer comprising a first nozzle array and a second nozzle array, the first nozzle array adapted to impart a bulk fluid flow to the fluids present on the upper surface of the slide and the second nozzle array adapted to impart at least a first localized fluid flow to at least a portion of the fluids present on the upper surface of the slide.

29. 30. The automated slide processing apparatus of claim 28, wherein the bulk fluid flow is in a clockwise or counterclockwise direction.

30. 30. The automated slide processing device of claim 28 or 29, wherein the first localized fluid flow is in the other of the clockwise or counterclockwise directions.

31. 31. The automated slide processing apparatus of claim 30, wherein the first localized fluid flow is applied to a central portion of the fluid present on the upper surface of the slide.

32. 32. The automated slide processing apparatus of claim 31, wherein the second nozzle array is further adapted to provide a second localized fluid flow and a third localized fluid flow, the second and third localized fluid flows occurring at two different ends of the fluid on the slide, the two ends each adjacent the center portion, and the second and third localized fluid flows being opposite the first localized fluid flow.

33. 33. The automated slide processing device of any one of claims 28 to 32, wherein the automated slide processing device is a staining device and the fluid present on the upper surface of the slide comprises a reagent selected from the group consisting of a staining reagent, a counterstain reagent, or a washing reagent.

34. 1. An automated slide processing apparatus comprising: at least one fluid dispenser configured to dispense fluid onto an upper surface of the specimen support slide; and a non-contact mixer for distributing fluid present on the upper surface of the slide, the non-contact mixer comprising: a first nozzle array in fluid communication with a first plenum, the first nozzle array comprising a first set of primary nozzles directing a flow of gas in a first direction and a second set of primary nozzles directing the flow of gas in a second direction; and a second nozzle array in fluid communication with a second plenum, the second nozzle array comprising a first set of secondary nozzles directing the flow of gas in a third direction and a second set of secondary nozzles directing the flow of gas in a fourth direction.

35. 35. The automated slide processing device of claim 34, wherein the first and second directions are opposite to one another.

36. 36. The automated slide processing device of claim 35, wherein gas streams emanating from the first and second sets of primary nozzles are directed substantially along a periphery of the fluid present on the upper surface of the slide.

37. 36. The automated slide processing device of claim 35, wherein the gas flows emanating from the first set of primary nozzles are substantially along a first longitudinal axis of the slide and the gas flows emanating from the second set of primary nozzles are substantially along a second longitudinal axis of the slide.

38. 38. The automated slide processing device of claim 37, wherein gas streams from said first and second sets of primary nozzles form an angle of incidence with said surface of said slide that ranges from about 5 degrees to about 90 degrees.

39. 38. The automated slide processing apparatus of claim 37, wherein gas flows emanating from said first and second sets of primary nozzles are independently offset by up to + / - 15 degrees relative to said longitudinal axis of said slide.

40. 37. The automated slide processing device of claim 36, wherein the first nozzle array imparts bulk fluid motion to the fluid present on the upper surface of the slide.

41. 35. The automated slide processing device of claim 34, wherein each nozzle in the first set of secondary nozzles directs a stream of gas to a different location on the upper surface of the slide and each nozzle in the second set of secondary nozzles directs a stream of gas to a different location on the upper surface of the slide.

42. 42. The automated slide processing device of claim 41, wherein said second nozzle array establishes at least two localized fluid streams.

43. 1. A method of processing a specimen support slide comprising: (i) depositing a first reagent onto the specimen support slide; and (ii) uniformly distributing the deposited first reagent onto the specimen support slide, wherein the deposited first reagent is distributed by introducing a first set of pulsed gas jets into the deposited first reagent, thereby causing a first fluid motion to a first portion of the deposited first reagent for a first predetermined time period, and introducing a second set of pulsed gas jets into the deposited first reagent, thereby causing a second fluid motion to a second portion of the deposited first reagent for a second predetermined time period.

44. 44. The method of claim 43, wherein the first portion of the deposited first reagent comprises a majority of the deposited first reagent.

45. 45. The method of claim 43 or 44, wherein the first fluid flow is a bulk fluid motion.

46. 46. ​​The method of any one of claims 43 to 45, wherein at least the second portion of the deposited first reagent is a central portion of the deposited first reagent.

47. 47. The method of any one of claims 43 to 46, wherein the first and second movements are in the same direction.

48. 47. The method of any one of claims 43 to 46, wherein the first and second movements are in opposite directions.

49. 47. The method of any one of claims 43 to 46, wherein the total period for pulsing is in the range of from about 4 seconds to about 120 seconds.

50. 47. The method of any one of claims 43 to 46, wherein the first predetermined period of time is in a range from about 2 seconds to about 10 seconds.

51. 47. The method of any one of claims 43 to 46, wherein the second predetermined period of time is in a range from about 2 seconds to about 10 seconds.

52. 47. The method of any one of claims 43 to 46, wherein the first set of gas jets and the second set of gas jets each act sequentially at least twice.

53. 47. The method of any one of claims 43 to 46, wherein pulsing the first or second set of gas jets occurs at a frequency ranging from about 4 Hz to about 20 Hz.

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