WO2025024557A1

WO2025024557A1 - Staining methods for automated systems

Info

Publication number: WO2025024557A1
Application number: PCT/US2024/039349
Authority: WO
Inventors: Jeffrey SCHLOSSER; Kyler CONNELLY; Bradley Leong
Original assignee: Ruby Robotics Inc
Current assignee: Ruby Robotics Inc
Priority date: 2023-07-25
Filing date: 2024-07-24
Publication date: 2025-01-30
Anticipated expiration: 2026-01-25

Abstract

Staining devices and methods for use with automated systems are provided herein. Staining methods include disposing a substrate in proximity to a staining surface to establish an overlap between the staining surface and the substrate. Staining methods further include maintaining a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, wherein a staining volume has one or more meniscus edges. Staining methods further include disposing a portion of the staining liquid on the staining surface and distributing the staining liquid on the substrate.

Description

STAINING METHODS FOR AUTOMATED SYSTEMS

TECHNICAL FIELD

[0001] The present invention is directed to systems, methods, and devices configured to facilitate sample processing, including automated sample processing and rapid on-site sample evaluation.

BACKGROUND

[0002] Rapid On-Site Evaluation (ROSE) is an important aspect of biopsy procedures that can decrease the number of needle passes, increase patient safety, and increase diagnostic yield. A purpose of ROSE is to determine whether a biopsy sample has adequate cellular and tissue content to produce a definitive diagnosis when analyzed in a pathology lab. Other uses for ROSE include informing tissue collection and triage, as well as providing a preliminary diagnosis to the interventionalist.

[0003] Traditionally in ROSE, a biopsy sample is smeared or touch imprinted onto a glass slide by a cytotechnologist or cytopathologist to create a thin layer of cellular material. The slide is then manually stained to increase contrast between different biological elements of the slide using a rapid stain such as Diff Quik, Toludine Blue, or others. Finally, the slide is examined by cytology personnel under a light microscope and analyzed for adequacy, triage, and/or preliminary diagnosis.

[0004] ROSE has proven its utility in a wide range of biopsy sites including thyroid, liver, pancreas, lung, breast, sentinel lymph nodes, bone marrow, and more. ROSE is recommended by many leading clinical societies such as the Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, and American Thyroid Association. Furthermore, ROSE may become increasingly important in procedures such as bronchoscopic lung biopsy in order to facilitate delivery of therapeutics (cryotherapy, microwave ablation, drug delivery', etc.) immediately following ROSE-facilitated confirmation of positive biopsy results.

[0005] However, ROSE is only used in roughly half of non-dermatological biopsy procedures due to cytology staff shortages, logistical challenges, and pathologist bandwidth. These challenges are amplified in procedures such as bronchoscopic lung biopsy, EndoBronchial UltraSound (EBUS) lymph node staging, and percutaneous CT-guided biopsy, since long procedure times put additional strain on the cytology department’s resources. Furthermore, ROSE adequacy and diagnosis is subject to interoperator variability in both slide preparation and interpretation- no two cytologists will produce an equivalent slide or make the same adequacy call- which is often a source of frustration and time delays for the interventional staff. For complex procedures such as bronchoscopic lung biopsy, 20-40% or more of biopsies are unsuccessful in providing an ultimate diagnosis in the pathology lab — requiring repeat biopsy procedures — even with the use of traditional ROSE methods.

[0006] Given the shortcomings of traditional ROSE, it is not surprising that other approaches have been tried to improve the process of adequacy assessment and on-site diagnosis. There are several devices that provide ROSE slide digitization and transmission, or telerobotic microscopes that enable cytopathologists to view ROSE slides remotely. However, such devices still rely on cytology personnel in the operating room to prepare slides, which does not overcome the associated logistical and interoperator variability problems with some ROSE implementations.

[0007] Some potential solutions, such as raman scattering microscopy of excised tissue and needle-based confocal laser microscopy are being pursued. While attractive in principle, these approaches do not match the resolution and fidelity of tissue imaged on glass slides. Furthermore, these approaches create unfamiliar images which requires new training for interventionalists and pathologists.

[0008] Accordingly, there is a need for improved sample processing methods and technology to reduce sample processing times, increase uniformity of sample processing, and reduce operator hours required for sample processing.

SUMMARY

[0009] In an embodiment, a method of distributing a staining liquid over a substrate. The method includes disposing a substrate in proximity to the staining surface to establish an overlap between the staining surface and the substrate; maintaining a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, the staining volume having one or more meniscus edges; disposing a portion of the staining liquid on the staining surface; and distributing the staining liquid on the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying figures, which are incorporated herein, form part of the specification and illustrate embodiments of systems, methods, and devices for rapid on- site cytological evaluation. Together with the description, the figures further explain the principles of and enable a person skilled in the relevantart(s) to make and use the methods, systems, and devices described herein. The drawings are provided to illustrate various features of the embodiments described herein and are not necessarily drawn to scale. In the drawings, like reference numbers indicate identical or functionally similar elements.

[0011] FIG. 1 illustrates a system for performing rapid on site evaluation system (ROSE) for sample analysis.

[0012] FIG. 2A illustrates a method of distributing a staining liquid over a substrate consistent with embodiments hereof.

[0013] FIG. 2B and FIG. 2C provides a schematic illustrating a method of distributing a staining liquid consistent with embodiments hereof.

[0014] FIGS. 3A-3K illustrate stages in a process of distributing staining liquid over a substrate consistent with embodiments hereof.

[0015] FIGS. 4A-4J illustrate stages in a process of distributing staining liquid over a substrate consistent with embodiments hereof.

[0016] FIGS. 5 A-5V illustrate stages in a process of distributing staining liquid over a substrate consistent with embodiments hereof.

[0017] FIGS. 6A-6N illustrate stages in a process of distributing staining liquid over a substrate consistent with embodiments hereof.

[0018] FIGS. 7A-7D illustrate stages in a process of distributing staining liquid over a substrate consistent with embodiments hereof.

[0019] FIGS. 8A-8B illustrate a staining cartridge including a staining reserv oir consistent with embodiments hereof.

[0020] FIG. 9 illustrates a staining structure consistent with embodiments hereof.

DETAILED DESCRIPTION

[0021] The following detailed description is merely illustrative in nature and is not intended to limit the invention or the application and uses of the invention. Although the description of the invention is in the context of systems, methods, and devices for facilitating automated ROSE techniques, the disclosure should not be considered so limiting. For example, although systems, methods, and devices may be discussed herein with respect to ROSE of biopsy samples, any biological samples may be suitable for analysis by embodiments hereof. Modifications may be made to the embodiments described herein without departing from the spirit and scope of the present invention. Therefore, the following detailed description is not meant to be limiting. Furthermore, there is no intention to be bound by any expressed or implied theory presented in the preceding technical field, background, summary, or the following detailed description.

[0022] The present disclosure addresses issues outlined above. The disclosure relates to a robotic automated staining and sample processing device that rapidly produces consistent high- resolution cytologic images using raw biopsy samples harvested by an interventional staff, and is therefore suitable for ROSE. Images may be displayed directly to the interventionalist on the machine, and also available to be transmitted for remote consultation by a cytopathologist. Al algorithms may assist the cytopathologist by identifying important regions of interest and cell types. As enabled by highly repeatable sample preparation and imaging, fully automated adequacy and diagnosis (using Al) may be available directly to the interventionalist without the need for remote consultation. With the approach described herein, the interventionalist and remotely located cytopathologist may view high resolution smear images that are prepared and stained in a familiar manner, without the need for on-site cytology' personnel. Accordingly, embodiments of the disclosure provided herein may serve to reduce operator hours performing ROSE, increase throughput, reduce time to result, and increase result consistency.

[0023] Embodiments disclosed herein may facilitate rapid staining and evaluation of samples by sample separation techniques that use a minimum or reduced amount of sample as compared to other methods. In embodiments, the systems, methods, and devices described herein may be used in conjunction with other systems, methods and devices to assess, measure, or evaluate therapeutic effects on sample tissue. For example, after a therapy has been delivered (e.g., drug, ablation, etc.), tissue may be harvested and stained to determine the efficacy of therapy. For example, tissue sampled from an area in the periphery of a tumor after an ablation may be stained according to the systems, methods, and devices described herein and evaluated to ensure or determine that the area was sufficiently ablated on a cellular level. Similarly, tissue sampled from an area in the periphery of a tumor after a drug is injected intratumorally may be stained according to embodiments described herein and evaluated to ensure or determine that the drug sufficiently penetrated the tissue on a cellular level.

[0024] Automated sample staining may provide substantial benefits over traditional manual staining techniques, specifically by providing at least faster staining and more economical use of staining fluids. Samples may be stained faster and less staining fluid may be wasted. Further, when automated sample staining is used as part of an automated processing pipeline, it eliminates the need for pausing the pipeline to permit manual intervention. Automated sample staining techniques discussed herein take advantage of fluid properties and behaviors including surface tension, adhesion, capillary action, wicking, etc. Taking advantage of these properties and behaviors may be facilitated by employing automated staining techniques, as discussed herein, that minimize the generation of bubbles while eliminating bubbles that do form. The formation of bubbles may be particularly disadvantageous to automated systems because any breaks in a fluid may reduce or prevent effects such as siphons, capillary' action, surface tension, adhesion, wicking, etc. Further, staining techniques as discussed herein may increase and/or optimize the fluid properties and behaviors discussed above.

[0025] The present disclosure describes devices, systems, and methods for facilitating staining and sample processing. Sample staining methods described herein involve the deposition of staining fluids and the staining of samples (e g., a tissue sample or other biological sample). Sample staining devices and methods described herein facilitate rapid, repeatable, and automated sample staining with a minimum of operator intervention. Each of the described methods and devices may be compatible with a variety or array⁷ of actuators and systems configured to facilitate the described methods to be performed with the disclosed device structures. The presented combinations of features are discussed by way of example only, and the sample staining methods, staining devices, and sample staining systems described herein may each be used or employed with alternate methods, systems, and devices, as the case may be. Further, individual aspects of each of the methods, systems, and devices described herein may be employed individually or in any suitable combination with any other individual aspects of the methods, systems, and devices described herein. Some of these individual combinations may be discussed herein for example or illustrative purposes. Although the extensive variety of all such combinations prevents an individual description of each combination herein, it is understood that all such combinations fall within the scope of this disclosure.

[0026] For example, any of the methods or portions of the methods described herein may be carried out with suitable portions of the devices and systems described herein without requiring the entirety⁷ of the devices and systems described herein and/or may be employed with alternate devices and systems. Some methods or portions of methods may also be carried out manually with suitable portions of the devices described herein and or with systems described herein using different devices. Devices described herein may be carried out with suitable portions of the methods and systems described herein without requiring the entirety' of the methods and systems described herein and/or may be employed with alternate methods and systems. Systems described herein may be carried out with suitable portions of the methods and devices described herein without requiring the entirety of the methods and devices described herein and/or may be employed with alternate methods and devices.

[0027] In embodiments, the integrated sample staining and processing system 100 may be configured to work with common glass slides (or other suitable substrates) as sample substrates. This arrangement may place certain demands on the integrated sample staining and processing system 100 for precise manipulators, grippers, and other positioning actuators. In embodiments, as described herein, the sample staining and processing system 100 may include a staining structure configured to facilitate the staining methods discussed herein. A staining structure as described herein may be permanent or disposable and may be integral or removable with respect to the sample staining and processing system. Embodiments of the staining structure include, for example, a staining cartridge 303, as described below. The various features of the staining cartridge 303, as described herein, may be included in a staining structure that is permanent or disposable and integral or removable. The sample staining and processing system 100 may be configured to accept or to include one or more purpose built cartridges or staining structures, such as staining cartridge 303, described below. Such a staining cartridge 303 may include some or all of the sample staining components needed for a particular sample staining sequence. Sample staining components may include, but are not limited to a staining plate or multiple staining plates, sample slide or multiple sample slides, one or more staining liquids, one or more fixatives, one or more washing liquids, and one or more stain or other liquid storage units. As used herein, “staining liquid’' may refer to a liquid based stain (for example, including immunohistochemistry (IHC) stains, fluorescent stains, antibody stains, biologic stains, any stains that select for a biological entity such as a gene or protein, and/or any other stain or combination of staining liquids known in the art), and/or to any other liquid used in the staining process, such as fixatives and washing liquids (e.g., water, alcohol, etc.). The staining cartridge may hold or guide each element with sufficient relative position as needed for the sample staining steps. By incorporating suitable precision within the staining cartridge, the precision demands on the processing system may be reduced accordingly. Further, the staining cartridge may also be configured to contain all of the contaminated elements for safe disposal and prevention of contaminating the processing svstem. [0028] In further embodiments, the integrated sample staining and processing system 100 may be configured to work with common glass slides (or other suitable substrates) as sample substrates without the use of cartridges. In such embodiments, the sample substrates may be handled, maneuvered, manipulated, etc., with actuators of the system, including manipulators, grippers, rollers, pinchers, and any other suitable actuator.

[0029] The present disclosure may include references to relative terms such as “top,” “bottom,” “up,” and "down." These terms are used for clarity and ease of reference. For example, a “top” of a structure or device may refer to the portion of that structure or device that faces up during usage as described herein. Relative directional terms as used herein are not limiting and do not limit the orientations, positions, angles, or functionality of the structures and devices discussed herein. Furthermore, the methods, systems, and devices as discussed herein are not limited to use in the orientations as described herein. For example, although the staining cartridge is described with a staining plate sliding “above” the staining cartridge, the disclosure is not limited to this arrangement. One or more aspects of the system may be inverted with respect to the orientations disclosed herein without departing from the scope of this disclosure.

[0030] FIG. 1 : illustrates a rapid onsite evaluation system consistent with embodiments hereof. The rapid onsite evaluation system 100 may include various components. The components may be housed on a moveable cart 101 or as a benchtop system. Components may include a cart chassis with wheels, an integrated sample staining and processing system 100 that prepares, stains and images the biopsy samples, display 102 for viewing a prepared and imaged sample, and a user interface 103, including one or more user input devices, storage for disposable cartridges (into which the biopsy samples may be loaded) and one or more consumable repositories. A waste fluid bin may also be included. The integrated sample staining and processing system 100 may include one or more input slots into which disposable cartridges with sample may be loaded, one or more output slots for retrieving stained slides for future analysis, and an area to retrieve used cartridges that are ready for disposal. Additionally, there may be controls, such as one or more user input devices of a user interface 103, on the cart that facilitate inputting system parameters and performing operations such as manipulating the microscope image on the screen by panning, zooming, and jumping to various regions of interest.

[0031] The integrated sample staining and processing system 100 may include the necessary hardware and software components to carry out the methods and techniques discussed herein. For example, the integrated sample staining and processing system 100 may include actuators such as pumps, grippers, manipulators, robots, etc. to facilitate the methods and techniques discussed herein. The integrated sample staining and processing system 100 may further include the necessary computer components, e.g., processors, memory devices, input/output devices, displays, etc., to control the actuators and to carry out the various system control processes discussed herein. The integrated sample staining and processing system 100 may further include sensors or data gathering devices, such as cameras, optical devices, temperature measurement devices, pressure sensors, force sensors, etc. required to carry out the various systems and control processes discussed herein.

[0032] FIG. 2A illustrates a sample staining method consistent with embodiments hereof. FIGS. 2B-2C provide a schematic illustration of some of the aspects of the staining method 1000. The staining method 1000 may be performed according to the systems, techniques, and devices disclosed herein. The systems, techniques, and devices discussed herein provide examples that illustrate the operation and performance of elements of the staining method 1000 and are not exclusive. Accordingly, while the staining method 1000 may be performed by employing a staining cartridge 303 (or any other staining cartridge discussed herein) in conjunction with the integrated sample processing system 100 to cany' out all of the steps described herein, this disclosure is not limited to such a combination. The steps and operations of the staining method 1000 described herein may be carried out in any suitable order and in any suitable combination. In embodiments, some or all of the steps and operations of the staining method 1000 may be carried with the staining cartridge 303 and manual processing techniques. In other examples, the integrated sample processing system 100 may perform any selection of the steps and operations of the staining method 1000 alone or in any combination, with or without the use of the staining cartridge. The steps and operations of the staining method 1000 may each stand alone or may be performed in any suitable combination with other steps and operations of the staining method 1000 and with any suitable combination of manual and automated processing techniques.

[0033] The staining method 1000 operates to distribute staining liquid on a substrate through use of a staining surface. These are illustrated in FIGS. 2B and 2C. The substrate 201 is the substrate that an operator intends to stain and typically will include a sample (e.g., biological sample) disposed thereon for staining. The substrate 201 may be, for example, a specimen slide of suitable material (e.g., glass, etc.). The substrate 201 is not limited to specimen slides and may include any structure suitable for staining a sample including a cell culture dish, microwell plate, etc. The staining surface 204 is positioned opposite a side of the substrate 201 on which a sample is disposed. The substrate 201 and staining surface 204 may be flat or substantially flat (e.g., having an average deviation from the mean profile of less than 50 microns) and/or may have surface features, such as concavities, convexities, ridges, wells, bumps, etc., to promote stain distribution and/or sample disposition. The staining surface 204 may be located on any suitable structure, such as a flat surface (e.g., a coverslip), a disposable cartridge, a durable manifold, etc. During staining, the substrate 201 and the staining surface 204 are separated by a gap 290, which may be referred to herein as a vertical gap and/or a capillary gap. The term “vertical” as used with respect to these two structures does not require a specific orientation and is selected to indicate the relative spacing between the two. For example, as used herein, in the substrate 201 and the staining surface 204 were arranged side by side, the gap 290 would still be considered and referred to as a “vertical gap.” During staining, the gap 290 may be “maintained,” by suitable mechanical or electromechanical means, as discussed herein. In embodiments, the spacing of the gap 290 may vary in one or more dimensions across the overlap 291. For example, as discussed below with respect to FIGS. 4A-4J, the gap 290 may have a relatively smaller value at a center of the overlap 291 and have a relatively larger value at an edge of the overlap 291. This may be referred to as a gap profile. “Maintaining” the gap may include maintaining the gap at a specific predetermined distance, within tolerances, e.g., 1%, 5%, 10%, or with a specific gap profile of distances that vary across the length and width of the overlap 291. “Maintaining” the gap may further include maintaining the gap at a distance sufficient to maintain capillary action without breaking surface tension of a liquid disposed therein. Thus, maintaining the gap may be accomplished while permitting changes to the size of the gap that do not cause a loss of capillary action or surface tension. The staining surface 204 and the substrate 201 may be arranged in proximity to one another to establish an overlap 291, also referred to as a lateral or horizontal overlap. Use of the terms “lateral” and “horizontal” to refer to the overlap 291 are intended for relative illustration only. The nature of the overlap 291 is not altered by different orientations. The overlap 291 represents mutual areas of the substrate 201 and the staining surface 204 that overlap, or occupy the same area (albeit on different planes) when viewed along an axis that is orthogonal to the substrate 201 and the staining surface 204. The overlap 291 may also be understood as the area within which orthogonal vectors originating from the substrate 201 and extending towards the staining surface 204 will intersect with the staining surface 204. The overlap 291 (which is an area) and the gap 290 (which is a distance) together establish a staining volume 299, representing the volume between the overlapping areas of the substrate 201 and the staining surface 204.

[0034] During staining, the staining volume 299 is filled with a staining liquid. In embodiments disclosed herein, the staining liquid within the staining volume 299 is constrained only by the substrate 201, the staining surface 204 and the combination of adhesion between the staining liquid and the substrate 201 and staining surface 204 and surface tension of the staining liquid. Thus, the staining volume 299 may have one or more meniscus edges 293. The meniscus edges 293 are defined by a perimeter of the area of the overlap 291 and are unconstrained by walls or other physical structure. It is not required that the entire perimeter of the staining volume 299 be bounded by meniscus edges 293. In embodiments, at least a portion of the staining volume 299 may be bounded by walls or other physical structure while a remaining portion is bounded by one or more meniscus edges 293. As such, the staining volume 299 may be larger (e.g., by 1%, 5%, 10%, etc.) than the rectangular prism defined by the overlap 291 and the gap 290 by the volume of the liquid menisci or bulges that occur at the meniscus edges 293 of the staining volume 299.

[0035] During stain distribution, the overlap 291 is increased, as shown in FIG. 2C, thereby increasing the staining volume 299. As the overlap 291 is increased, additional staining liquid is provided to the staining volume 299, commensurate with the volumetric increase of the staining volume 299. In embodiments, additional staining liquid is received at the staining volume 299 through one or more of the meniscus edges 293. The additional staining liquid may already be present (e.g., predeposited) on the staining surface 204 and/or may be deposited on the staining surface 204 in a continuous fashion at a meniscus edge 293.

[0036] The operations of the method 1000 are described in further details below with respect to FIGS. 2A and 2B, which provide general examples of some of the structures and features and with respect to other figures providing specific examples of structures and features.

[0037] In an operation 1002, the staining method 1000 includes disposing staining liquid or a portion of staining liquid on a staining surface. Staining liquid deposition may include disposing at least one staining liquid on a staining surface. As described herein, the staining surface may be the staining surface 204, staining surface 304 (e.g., shown in FIG. 3 A) of a staining cartridge (e.g., the staining cartridge 303), or any other suitable staining surface. The staining surface may also include other structures not part of a staining cartridge, such as a coverslip or any other surface. Disposing the staining liquid on a staining surface includes steps and techniques adapted for applying fluid (e.g., a staining fluid) to a staining surface. Such steps and techniques may include, but are not limited to, disposing, dropping, dripping, spraying or otherwise depositing a disposed staining liquid on a staining surface in preparation for staining. Moreover, staining liquid disposition may include use of a stain delivery device, and/or a port, and/or any other suitable device. As described herein, the stain delivery device may include stain delivery device 302 (e.g, shown in FIG. 3 A), the port may include port 406 (e.g, shown in FIG. 4A). Staining liquid disposition may be performed manually, by an operator, or by an integrated sample processing system, as discussed herein. The staining surface and staining cartridge, as described herein, represents an illustrative w ay of facilitating staining liquid disposition, but are not required. The staining liquid disposition operation may be performed by any structure or device, manually or automatically operated, that is capable of disposing a liquid. For example, the stain delivery device 302 (e.g., shown in FIG. 3 A) may dispose the staining liquid by a physical force (e.g., exerting sufficient pressure within the stain deliver^' device 302 to dispose a predetermined amount of staining liquid on the staining surface) or a natural force (e.g., allowing the stain delivery device 302 to gravity feed the staining liquid onto the staining surface). In further embodiments, the stain may be delivered via capillary action. Further methods and techniques for staining liquid disposition are described below.

[0038] In an operation 1004, the staining method includes disposing a substrate in proximity to the staining surface. The substrate (e.g., substrate 201) may be disposed so as to establish an overlap (e g., overlap 291) with the staining surface (e.g., staining surface 204) As described herein, the substrate may be a staining plate (e.g, shown in FIG. 3A), which may include, for example, a specimen slide. Staining plate disposition may be performed manually, by an operator, or by an integrated sample processing system (e.g., by actuators thereof), as discussed herein. The staining plate, staining surface, and staining cartridge, as described herein, represent an illustrative way of facilitating staining plate disposition, but are not required. The staining plate disposition operation may be performed by any structure or device, manually or automatically operated, that is capable of arranging the staining plate and the staining surface 304 in a position having a vertical gap and a horizontal overlap (as shown, for example, in FIGS. 3A, 4A, 5A and 6A). The staining plate disposition operation may further be performed by any structure or device, manually or automatically operated, that is capable of controlling or maintaining the horizontal overlap between the staining plate and the staining surface, and may further include controlling the speed of change in horizontal overlap between the staining plate and the staining surface. Further methods and techniques for staining plate disposing are described below.

[0039] In an operation 1006, the staining method 1000 includes maintaining a gap between the substrate and the staining surface. The gap (e.g., gap 290) and the overlap (e.g., overlap 291) establish or define a staining volume (e.g., staining volume 299). The established staining volume may have one or more meniscus edges (e.g., meniscus edges 293). Maintaining the gap may be performed manually, by an operator, or by an integrated sample processing system, as discussed herein. As described herein, structures for maintaining the gap between the staining plate and the staining surface may include a gap maintenance structure. As used herein, “gap maintenance structure’⁷ refers to a structural element configured to maintain a specific gap between a staining plate (e.g., a substrate) and a staining surface. In an embodiment, one or more pair of ledges 308 (e.g., shown in FIG. 3 A) may provide a gap maintenance structure. The ledges 308 are configured to support a staining plate from underneath and to hold the staining plate at a fixed or predetermined distance above the staining surface. In a further embodiment, a gap maintenance structure may be configured to support a staining plate from underneath and to hold the staining plate at a fixed or predetermined distance below a staining surface. Such a gap maintenance structure is illustrated in FIG. 9, which shows a staining structure 900 including a staining surface 904 and a gap maintenance structure defined by one or more ledges 908. The ledges 908 are configured to support a staining plate (e.g., substrate) 901 and to position the staining plate 901 a predetermined or fixed distance below the staining surface 904 to establish a gap 290. It will be understood that features of the staining structure 900 may be combined with any other embodiments disclosed herein. Although described as “ledges” with respect to FIG. 9 and FIG. 3A, gap maintenance structures are not limited to such and may include any physical structure capable of supporting a staining plate. In still further embodiments, the gap may be maintained by a gap maintenance device configured to maintain a staining plate at a predetermined or fixed distance from a staining surface. Gap maintenance devices include, but are not limited to, robotic actuators and other mechanical devices such as lead screws, levers, etc. In still further embodiments, the gap may be maintained by combinations of gap maintenance structures and devices, for example, a gap maintenance structure may include a gantry that is moveable via action of a robotic actuator. Maintaining the gap between the staining plate and the staining surface includes steps and techniques as well as structures and devices adapted for maintaining the gap between the staining plate and the staining surface to avoid contact between the staining plate and staining surface and maintaining a sufficient gap (e.g., not too large and not too small) to maintain capillary action between the staining plate and staining surface (e.g., creating and maintaining a capillary gap). Such steps and techniques may include, but are not limited to, controlling or maintaining the horizontal overlap between the staining plate and the staining surface, controlling the speed of change in horizontal overlap between the staining plate and the staining surface, controlling or maintaining a vertical gap between the staining plate and the staining surface, or otherwise controlling the staining plate and staining surface and controlling or maintaining a horizontal overlap and a vertical gap between the two. The staining plate, staining surface, ledges and staining cartridge, as described herein, represents an illustrative way of facilitating the operation of maintaining the gap, but are not required. The gap maintenance operation may be performed by any structure or device, manually or automatically operated, that is capable of maintaining the relative positioning and/or controlling the relative movement between the staining plate and the staining surface. For example, in addition to the structures described herein that are configured to maintain the gap between the staining surface and the staining plate, one or more robotic actuators may be configured to maintain the gap between the staining surface and the staining plate. Further methods and techniques for maintaining the staining plate proximity to the staining surface are described below.

[0040] In an operation 1008, the staining method includes distributing the staining liquid on the substrate. In embodiments, distributing the staining liquid on the substrate may be performed by increasing the overlap (and thereby the staining volume) between the staining surface and the substrate and filling the staining volume with the staining liquid as the overlap is increased. Distributing the staining liquid is illustrated in FIG. 2C. As described herein, distributing the staining liquid includes steps and techniques described herein adapted for distributing fluid or liquid (e.g., a staining liquid) to the substrate. Such steps and techniques may include, but are not limited to, sliding, slipping, spreading, spraying, smearing, or otherwise distributing the disposed staining liquid on the substrate. Moreover, staining liquid distribution may include use of the stain delivery device 302 (e.g., shown in FIG. 3 A), the port 406 (e.g., shown in FIG. 4A), and any other suitable distribution device. The staining liquid distribution operation 1008 may also include controlling the flow of disposed staining liquid on the substrate.

[0041] According to embodiments herein, controlling the flow of disposed staining liquid on the substrate may use, for example, troughs in the staining cartridge (e.g., trough 307 surrounding the staining surface 304 of the staining cartridge 303 shown in FIG. 3A) situated such that a staining liquid will stay on a staining surface (e.g, staining surface 304 shown in FIG. 3A) and avoid the trough due to adhesion to the substrate (staining plate), adhesion to the staining surface, and the surface tension of the staining liquid. Moreover, according to embodiments herein, controlling the flow of disposed staining liquid on the substrate may also use hydrophobic and/or hydrophilic coatings to various surfaces including, but not limited to, the substrate (the staining plate), the staining surface, and/or the troughs in the staining cartridge. Staining liquid distribution may be performed manually, by an operator, or by an integrated sample processing system, as discussed herein. The staining plate, staining surface and staining cartridge, as described herein, represents an illustrative way of facilitating staining liquid distribution, but are not required. The staining liquid distribution operation may be performed by any structure or device, manually or automatically operated, that is capable of distributing a liquid. Further methods and techniques for distributing the staining liquid are described below.

[0042] FIGS. 3A-3K illustrate stages in a process of distributing staining liquid over a staining plate. According to the embodiment shown. FIGS. 3A-3K illustrate a staining plate 301, a stain delivery⁷ device 302, and a staining cartridge 303. As described herein, the staining plate 301 receives a sample. Disposing a sample on staining plate 301 includes steps and techniques adapted for spreading, smearing, or otherwise distributing a deposited sample across the staining plate 301 in preparation for staining. Moreover, as described herein, the stain delivery device 302 may include a tube shaped structure for delivering the staining liquid. The stain delivery device 302 is not limited to the shape illustrated in FIGS. 3A-3K, and any suitable structure for disposing a staining liquid onto a staining surface (e.g.. as described above with respect to operation 1002 of method 1000) is within the scope of the claimed invention.

[0043] The staining cartridge 303 may include a base 391 and any or all of a pair of ledges 308 forming a ledge structure, a staining surface 304 (e.g., a surface that receives the disposed staining liquid), a port 306, a trough 307 and a drain 305. The base 391 is a structure upon which or within which the other features of the staining cartridge 303 are disposed. As described herein, the ledges 308 are sufficiently dimensioned (e.g., in height and width) to create a gap 312 and maintain the gap 312 betw een the staining surface 304 and the staining plate 301 when the staining plate 301 is slid across the ledges 308 (e.g., as described above with respect to operation 1004 of method 1000) to create a horizontal overlap 311. It is not required that the staining plate 301 contact the ledges 308 directly, as the staining plate 301 may be in a holder or other device. Thus, the tops of the ledges 308 are disposed a predetermined distance above at least a portion of staining surface 304. As discussed below, the staining surface 304 may include one or more features to facilitate distribution of a staining liquid and thus may be characterized by a gap profile. The predetermined distance sets the height of the vertical gap 312. which may be a capillary gap. The vertical gap 312 (and thus the height of the tops of the ledges 308 above the staining surface 304) may be maintained at a height of less than 350 microns, less than 300 microns, less than 250 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns. The ledges 308 are not limited to the shape illustrated in FIGS. 3A-3K, and any suitable structure for maintaining the gap 312 between the staining surface 304 and the staining plate 301 (e.g., as described above with respect to operation 1006 of method 1000) is within the scope of the claimed invention. In some embodiments, the vertical gap 312 may be substantially the same height (e g., within 5%) across the overlap 311 and the staining surface 304 and the staining plate 301 may be substantially parallel. In further embodiments, as described below, the staining surface 304 may include raised features, such as the raised structure 310 where the height of the vertical gap 312 varies. The overlap 311 and the gap 312, together, define the staining volume 399.

[0044] As described herein, the trough 307 may be disposed within the staining cartridge 303. According to embodiments, the trough 307 is sufficiently dimensioned (e.g, in height and width) and situated such that a staining liquid will stay on the staining surface 304 and avoid the trough 307 due to adhesion to the substrate (staining plate), adhesion to the staining surface, and the surface tension of the staining liquid. As illustrated in FIG. 3 A. the trough 307 may be configured to surround or partially surround the staining surface 304 with a surface that is at lower height than the staining surface 304. The trough 307 is not limited to the shape illustrated in FIGS. 3A-3K, and any suitable structure for controlling the distribution of a staining liquid onto the staining surface 304 (e.g, as described above with respect to operation 1008 of method 1000) is within the scope of the claimed invention.

[0045] In embodiments, the cartridge 303 does not require a trough 307. In embodiments, the substrate 301 may not extend to the edges of the staining surface 304. Thus, a meniscus edge of the staining volume 399 may be established at the edge of the substrate 301 while the staining surface 304 continues to extend. In some embodiments, the staining surface 304 may extend to the ledges 308, and the meniscus edges (e.g., two edges) of the staining volume 399 may be established only at the ends of the substrate 301 while the lateral edges of the staining volume 399 are bounded by the ledges 308. In further embodiments, as discussed throughout, the cartridge 303 may include no ledges 308, and the gap 312 may be maintained by an actuator, e.g. a robotic actuator.

[0046] According to embodiments described herein, the drain 305 may either actively (e.g., via pump creating a vacuum sufficient to actively pull staining liquid away from the staining surface 304 through the drain 305) or passively (e.g., the drain 305 passively wicks staining liquid away from the staining surface 304) draw a fluid (e.g., staining liquid) away from the staining surface 304 via the duct 309 connected to the port 306. According to other embodiments herein, an absorbent pad (not shown) may remove the staining liquid from the staining surface 304. The absorbent pad may be located within the cartridge 303, e.g., in a compartment connected to the duct 309. Suction or vacuum pulled from the drain 305 may act to pull excess staining liquid into the compartment in the duct 309, where it may be absorbed by the absorbent pad. In this manner, all excess staining liquid may remain within the cartridge 303, keeping the system clean.

[0047] The staining cartridge 303 may further include a raised structure 310 disposed on the staining surface 304. As show n, the raised structure 310 creates a conical, tapered, or volcanolike surface around the port 306 (e.g., either an inlet or outlet port) of the staining surface 304 sufficient to draw a fluid (e.g., staining liquid) towards the port 306 due to increased capillary action as the capillary gap decreases. In embodiments, the raised structure 310 may be configured to extend away from the port 306 a sufficient distance so as to prevent air from entering the port 306 when staining liquid is drained through the port. A sufficient distance may include distances greater than 2 mm, greater than 3 mm, greater than 4 mm, and/or greater than 5 mm. A gap profile of the gap 312 may be defined by a constant height portion and a raised structure portion, wherein the constant height portion provides a constant height of the gap profile and the raised structure portion provides a decreasing height of the gap profile at the raised structure 310. The gap 312 may narrow' from the majority portion of the staining surface 304 towards the apex of the raised structure 310 where the port 306 is located. When the port 306 is an inlet port, the raised structure 310 may assists in pushing bubbles out of the staining surface area 304. Moreover, when the port 306 is an outlet port, the raised structure 310 assists in keeping liquid surrounding and flowing into the port 306 and preventing the port 306 from being exposed to air (e.g., when the port 306 is exposed to air, the air stops liquid from getting sucked into the port 306, preventing further draining). Furthermore, the design of the port 306 is not limited to what is showai in the figures, and embodiments described herein include more complex shapes than a simple circle shape. For example, a slit shape may direct the flowing staining liquid across a larger distance. In embodiments, the port 306 may include a series of apertures arranged linearly, connected to the duct 309 by a manifold. A port 306 that is slit shaped or includes a series of apertures may extend across the staining surface 304 and may extend at least 40%, at least 50%, at least 60%, at least 70%. or at least 80% of the width of the staining surface 304.

[0048] As shown in FIGS. 3A-3E, a staining liquid is placed, by the stain delivery device 302, on the exposed staining surface 304 in accordance w ith operation 1002 of method 1000. Placement on the exposed staining surface 304 may provide an opportunity for any bubbles that may exist in the staining liquid within the stain delivery device 302 to escape to the environment. The staining liquid may be disposed such that it is open to an unconstrained environment to which the bubble may freely escape. An unconstrained environment may include any environment that borders the staining liquid that does not prevent a bubble from escaping the liquid. Examples of unconstrained environments may include atmosphere or a local gaseous environment having a pressure and composition that does not limit the movement of bubbles from the liquid. . Once a bubble or air gap has entered the gap 312, it may be difficult to remove, resulting in portions of the substrate 301 receiving either no stain contact or reduced stain contact.

[0049] According to the embodiment shown, the staining plate 301. the stain delivery device 302 and the staining cartridge 303 move relative to one another such that the staining plate 301 is disposed in proximity to the staining surface 304 of staining cartridge 303 in accordance with operation 1004 of method 1000, and the ledges 308 maintain the gap 312 (e g., a capillary gap) between the staining plate 301 and the staining cartridge 303 in accordance with operation 1006 of method 1000. Moreover, according to embodiments, the stain delivery device 302 may remain stationary as the staining cartridge 303 is mobile (e.g., the staining cartridge 303 moves beneath the stationary stain delivery device 302). According to other embodiments, the staining cartridge 303 is stationary’ and the stain delivery device 302 is mobile (e.g.. the stain delivery device 302 moves above the stationary staining cartridge 303). In still other embodiments, both the stain delivery device 302 and the staining cartridge 303 may move. FIGS. 3F-3I illustrate the same movement shown in FIGS. 3A-3E, but as a cross-sectioned view and from a different angle to better illustrate the raised structure 310 and the duct 309 in relation to the staining surface 304, the port 306 and the staining plate 301. [0050] As shown in FIGS. 3J and 3K, the stain delivery device 302 is distributing the staining liquid on the staining surface 304 in accordance with operation 1008 of method 1000. As shown, the staining plate 301 (e.g., with a sample, not shown) is swiped or moved over the staining surface 304. According to embodiments described herein, the staining liquid fdls into the gap 312 (e.g., capillary gap) between the staining plate 301 and the staining surface 304 by entering the staining volume 399. In embodiments, the staining liquid enters the staining volume 399 at a meniscus edge (not shown) thereof.

[0051] As shown in FIGS. 3A-3E, the stain delivery⁷ device 302 may deposit a predetermined amount of staining liquid across the staining surface 304 prior to staining plate 301 swiping. The predetermined amount of staining liquid may be selected according to the volume of the gap 312 when the staining plate 301 fully covers the staining surface 304. The predetermined amount of staining liquid may be equal to that volume. In embodiments, the stain delivery⁷ device 302 may deposit the predetermined amount of staining liquid during the staining plate 301 swiping and may deposit staining liquid to the staining surface 304 in front of the moving staining plate 301. In still further embodiments, the stain delivery device 302 may deposit the predetermined amount of staining liquid to the staining plate 301 at the junction of the staining surface 304 and the staining plate 301, e.g., directly into the gap 312 at a meniscus edge thereof. [0052] Embodiments described herein may further include multiple stains. For example, according to embodiments shown in FIGS. 3A-3K, embodiments that include multiple stains include removing the staining liquid from the staining surface (e.g. , via the port 306, through the duct 309 and out the drain 305) and returning the staining cartridge 303 and the stain delivery⁷ device 302 to the position shown in FIG. 3A. Once the position between the staining cartridge 303 and the stain delivery device 302 has been reset, a second liquid (e.g., staining liquid) is added (e.g., as described above and shown according to the operations of method 1000). In this manner, any number of staining, fixing, and/or w ashing liquids may be introduced. In further embodiments, the port 306 may be used as an input/output port. A first staining liquid may be removed via the port 306 and a second staining liquid may be introduced to the gap 312 via the port 306. Potentially, the second staining liquid may be removed via the port 306 to introduce additional liquids (staining, washing, fixing, etc.) In this manner, any number of liquids may be introduced and removed.

[0053] FIGS. 4A-4J illustrate stages in a process of distributing staining liquid over a staining plate. According to an embodiment, FIGS. 4A-4J illustrate the staining plate 301 and a staining cartridge 403. As described herein, the staining plate 301 may receive a sample. Disposing a sample on the staining plate 301 may include steps and techniques adapted for spreading, smearing, or otherwise distributing a deposited sample across the staining plate 301 in preparation for staining.

[0054] According to embodiments, the staining cartridge 403 may include any or all of a pair of ledges 408 forming a ledge structure, a staining surface 404 (e.g, a surface that receives the disposed staining liquid), a port 406, a trough 407 and a drain 405. As described herein, the ledges 408 are dimensioned (e g., in height and width) to create a gap 412 and maintain the gap 412 between the staining surface 404 and the staining plate 301 when the staining plate 301 is slid across the ledges 408 (e.g., as described above with respect to operation 1004 of method 1000). In embodiments, the staining surface 404 may be parallel to the staining plate 301 and thus have a constant gap 412 across its entire surface. In such embodiments, the tops of the ledges 408 may be disposed at a predetermined distance above the staining surface 404. The predetermined distance sets the height of the vertical gap 412. The vertical gap 412 (and thus the height of the tops of the ledges 408 above the staining surface 404) may be maintained at a height of less than 350 microns, less than 300 microns, less than 250 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns, and may be a capillary gap. In further embodiments, as discussed below, a slope 410 disposed on the staining surface 404 may result in a variable gap 412 or gap profile. The gap profile of the gap 312 may be defined by the slope 410. which provides a variable gap. The ledges 408 are not limited to the shape illustrated in FIGS. 4A-4J, and any suitable structure for maintaining the gap 412 between the staining surface 404 and the staining plate 301 (e.g., as described above with respect to operation 1006 of method 1000) is within the scope of the claimed invention. Moreover, as described herein, the trough 407 is disposed within the staining cartridge 403. According to embodiments, the trough 407 is sufficiently dimensioned (e.g, in height and width) and situated such that a staining liquid 420 will stay on the staining surface 404 and avoid the trough 407 due to the surface tension of the staining liquid 420 and it’s adhesion to the surfaces it is in contact with. The trough 407 may be configured to surround or partially surround the staining surface 404 with a surface at a lower height. The trough 407 is not limited to the shape illustrated in FIGS. 4A-4J, and any suitable structure for controlling the distribution of a staining liquid onto the staining surface 404 (e.g., as described above with respect to operation 1008 of method 1000) is within the scope of the claimed invention.

[0055] In embodiments, the cartridge 403 does not require a trough 407. In embodiments, the substrate 401 may not extend to the edges of the staining surface 404. Thus, a meniscus edge of the staining volume 499 may be established at the edge of the substrate 401 while the staining surface 404 continues to extend. In some embodiments, the staining surface 404 may extend to the ledges 408, and the meniscus edges (e.g., two edges) of the staining volume 499 may be established only at the ends of the substrate 401 while the lateral edges of the staining volume 499 are bounded by the ledges 408. In further embodiments, as discussed throughout, the cartridge 403 may include no ledges 408, and the gap 412 may be maintained by an actuator, e.g. a robotic actuator.

[0056] According to embodiments described herein, the drain 405 may either actively (e.g., via pump creating a vacuum sufficient to actively pull the staining liquid 420 away from the staining surface 404 through the drain 405) or passively (e.g., the drain 405 passively wicks the staining liquid 420 away from the staining surface 404) draw a fluid (e.g., the staining liquid 420) away from the staining surface 404 via the duct 409. According to other embodiments herein, an absorbent pad (not shown) may remove the staining liquid 420 from the staining surface 404. The absorbent pad may be located within the cartridge 403, e.g., in a compartment connected to the duct 409. Suction or vacuum pulled from the drain 405 may act to pull excess staining liquid into the compartment in the duct 409, where it may be absorbed by the absorbent pad. In this manner, all excess staining liquid may remain within the cartridge 403, keeping the system clean.

[0057] The staining surface 404 may further include a slope 410. The slope 410 may provide a conical or tapered surface around the port 406 (e.g., either an inlet or outlet port) of the staining surface 404 sufficient to draw a fluid (e.g., the staining liquid 420) towards the port 406 via capillary action. The slope 410 may cover the entirety of the staining surface 404 and/or may cover only a portion of the staining surface 404. The slope 410 may serve to create a variable gap 412. When the port 406 is an inlet port, the slope 410 assists in pushing bubbles out of the staining surface area 404. Moreover, when the port 406 is an outlet port, the slope 410 assists in keeping liquid surrounding and flowing into the port 406 and not letting the port 406 be exposed to air (e.g.. when the port 406 is exposed to air, the air stops liquid from getting sucked into the port 406). In further embodiments, the port 406 may function as both an inlet and an outlet. Furthermore, the design of the port 406 is not limited to what is shown in the figures, and embodiments described herein include more complex shapes than a simple circle shape. For example, a slit shaped port directs the flowing staining liquid 420 across a larger distance. In embodiments, the port 406 may include a series of apertures arranged linearly, connected to the duct 409 by a manifold. A port 406 that is slit shaped or includes a series of apertures may extend across the staining surface 404 and may extend at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the width of the staining surface 404.

[0058] In embodiments, e.g., as shown in FIGS. 4A-4J, the port 406 may be approximately centered on staining surface 404. As shown in FIG. 4G-4J, the slope 410 may form a taper or volcano-like structure on the staining surface 404. In embodiments, the raised structure 410 may be configured to extend away from the port 406 a sufficient distance so as to prevent air from entering the port 406 when staining liquid is drained through the port. A sufficient distance may include distances greater than 2 mm, greater than 3 mm, greater than 4 mm, and/or greater than 5 mm. In an embodiment, the taper may be a uniform taper from the port 406 to the edges of the staining surface 404. Thus, the slope of the taper may be shallower in the long direction of the staining surface and steeper in the short direction of the staining surface 404. [0059] In FIGS. 4A and 4B, the staining plate 301 may be disposed in proximity to the staining surface 404 of the staining cartridge 403 in accordance with the operation 1004 of method 1000 to establish an overlap 411. Together, the overlap 411 and the gap 412 establish or define the staining volume 499. In embodiments, the overlap 411 may span the entirety of the staining surface 404 before any staining liquid is deposited. As shown in FIGS. 4B-4F, the staining liquid 420 is disposed on staining surface 404 in accordance with operation 1002 of the method 1000 by the port 406. According to embodiments herein, the port 406 may operate as both an inlet and an outlet such that the staining liquid 420 (and subsequent liquids in a staining process) is pumped in and out from the port 406. FIGS. 4G-4J illustrate the same movement of the staining liquid 420 shown in FIGS. 4B-4F, but as a cross-section view and from a different angle to better illustrate the slope 410 and the duct 409 in relation to the staining surface 404, the port 406 and the staining plate 401. As described herein, in accordance with operation 1006 of method 1000, the ledges 408 maintain the gap 412 between the staining plate 401 and the staining surface 404. Furthermore, in accordance with embodiments, the staining surface 404 may not be parallel with the ledges 408 (and therefore the staining plate 301). Instead, the staining surface 404 may taper due to the slope 410 so that the edges of the staining surface are farther from the staining plate 301 than center point (e.g., near the port 406). As such, the gap 412 is maintained but gradually decreases as the staining surface 404 tapers. The gap 412 is maintained and does not disappear. The size of the gap 412, however, changes in accordance w ith the taper of the slope 410 of the staining surface 404. In embodiments, the gap 412 may be less than 350 microns, less than 300 microns, less than 250 microns, less than 200 microns, less than 150 microns, less than 100 microns, or less than 50 microns at the edges of the slope 410. which may or may not coincide with the edges of the staining surface 404. In embodiments, the gap 412 at the location of the port 406 may be less than 300 microns, less than 250 microns, less than 200 microns, less than 150 microns, less than 100 microns, less than 50 microns, or less than 25 microns. The structure of staining cartridge 403 may distribute the staining liquid 420 (e.g., in accordance with operation 1008 of method 1000) such that the staining liquid 420 is self-centering on the port 406 and prevents air from getting sucked into the port 406 when the staining liquid 420 is pumped out.

[0060] Embodiments described herein may further include multiple staining liquids. For example, according to embodiments shown in FIGS. 4A-4J, embodiments that include multiple staining liquids may include removing the staining liquid 420 from the staining surface 404 (e.g. , via the port 406, through the duct 409 and out the drain 405) and adding a second staining liquid (e.g. , another liquid stain, wash, fixative, etc.) to the gap 412 between the staining surface 404 and the staining plate 401 (e.g., as described above and shown according to the operations of method 1000). As described herein, adding and extracting fluids may occur quite quickly. For example, according to the embodiments shown in FIGS. 4A-4J, adding and extracting fluids may require one second or less, 2 seconds or less, 5 seconds or less, or 10 seconds or less in either direction. The ability to quickly introduce and remove fluids into the staining volume 499 may provide the advantage of more even staining.

[0061] FIGS. 5A-5V illustrate stages in a process of distributing staining liquid over a substrate. According to the embodiment shown. FIGS. 5A-5V illustrate the staining plate 301, a stain delivery’ device 502, and a staining cartridge 503. As described herein, the staining plate 301 receives a sample. Disposing a sample on staining plate 301 may include steps and techniques adapted for spreading, smearing, or otherwise distributing a deposited sample across the staining plate 301 in preparation for staining. As described herein, the stain delivery' device 502 may include one or a plurality⁷ of tube shaped structures (e.g., 502A-502D) for delivering a staining liquid. The stain delivery device 502 is not limited to the shape illustrated in FIGS. 5A-5V, and any suitable structure for disposing a staining liquid onto a staining surface (e.g, as described above with respect to operation 1002 of method 1000) is within the scope of the claimed invention.

[0062] The staining cartridge 503 may also include any or all of a pair of ledges 508 forming a ledge structure, a staining surface 504 (e.g., a surface that receives the disposed staining liquid), a port 506. a trough 507 and a drain 505. The ledges 508 may be similar in structure and function to the ledges 308 and the ledges 408. The staining surface 504 may be similar in structure and function to the staining surface 304 and 504. The port 506, trough 507, and drain 505 may be similar in structure and function to the ports 306/406, troughs 307/407, and drains 305/405, including the addition of an absorbent pad, respectively. Thus, the cartridge 503 may incorporate a staining surface 504 having a port 506 at one end surrounded by a raised structure 510 similar to that of cartridge 303, which may have a conical, tapered, or volcano-like surface. In another embodiment (not shown), the cartridge 503 may incorporate a staining surface 504 having a port 506 at one end surrounded by a raised structure 510 similar to that of cartridge 403. The cartridge 503 may, in some embodiments, include no trough 507 as described above with respect to FIGS. 3A-4J.

[0063] As shown in FIGS. 5A-5C. a first staining liquid may be deposited by the stain delivery device 502 to the staining surface 504. The staining plate 501 may be advanced relative to the staining surface 504 to establish an overlap 511 and distribute the first staining liquid within the gap 512. Together, the overlap 511 and the gap 512 establish or define the staining volume 599. As the staining plate 501 advances across the staining surface 504, the overlap 51 1 (and the staining volume 599) is increased. According to an embodiment, the staining plate 301, the stain delivery device 502 and the staining cartridge 503 may move relative to one another such that the staining plate 301 is disposed in proximity to the staining surface 504 of staining cartridge 503 in accordance with operation 1004 of method 1000, and the ledges 508 maintain the gap 512 between the staining plate 301 and the staining cartridge 503 in accordance with operation 1006 of method 1000. Similar to the gap 312 discussed above, the gap 512 may be of constant height and/or may have a gap profile defined by a constant height portion and a variable height portion associated with the raised structure 510. The stain delivery device 502 may deliver staining liquid to the meniscus edge of the staining volume 599 at a rate corresponding to the increase size of the staining volume 599 as the staining plate 501 is advanced. Moreover, according to embodiments, the stain deliver}' device

502 may remain stationary as the staining cartridge 503 is mobile (e.g., the staining cartridge

503 moves beneath the stationary stain delivery device 502). According to other embodiments, the staining cartridge 503 may be stationary and the stain delivery device 502 may be mobile (e.g., the stain deliver}⁷ device 502 moves above the stationary staining cartridge 303).

[0064] Following distribution of the first staining liquid, the staining cartridge 503 may be operated in various ways. As shown in FIGS. 5D-5F, additional staining liquids (e.g., liquid stains, fixatives, washes, etc.) may be distributed to the open end (e.g.. meniscus edge) of the gap 512 at the end of the staining plate 301. Because the meniscus edge is open to an unconstrained environment, any accumulated bubbles may be released to the unconstrained environment. The additional staining liquids may be distributed as the port 506 is operated to remove the previous staining liquid. If the old staining liquid is removed at the same rate that the new staining liquid is added, the new staining liquid will flow smoothly through the gap 512 without ever generating bubbles due to a break in the liquid. In this manner, any number of staining liquids may be sequentially added and removed. This distribution represents an example of distributing each additional staining liquid according to operations 1002, 1004, 1006, and 1008.

[0065] FIGS. 5G-5J illustrate an alternative technique for delivering additional staining liquids. As shown in FIG. 5G, the first staining liquid may be removed via the port 506. To assist in removal, the staining plate 301 may be drawn or slid back to a starting position as shown in FIG. 5H. Next, as shown in FIG. 51 to FIG. 5J, a second staining liquid may be distributed by the stain delivery' device 502 while the staining plate 301 is advanced across the staining surface 504. In this manner, any number of staining liquids may be sequentially added and removed without the liquids touching each other. This distribution represents an example of distributing each additional staining liquid according to operations 1002, 1004, 1006, and 1008.

[0066] FIGS. 5K-5N illustrate the steps of FIGS. 5A-5C from a cross-section and side view to further illustrate the distribution of a staining liquid across the staining plate.

[0067] FIGS. 6A-6H illustrate stages in a process of distributing staining liquid over a substrate. According to the embodiment shown, FIGS. 6A-6H illustrate a staining plate 301 and a staining cartridge 603. As described herein, the staining plate 301 receives a sample. Disposing a sample on staining plate 301 may include steps and techniques adapted for spreading, smearing, or otherwise distributing a deposited sample across the staining plate 301 in preparation for staining. The staining cartridge 603 may further include a plurality' of staining surfaces 604A/B/C/D. The plurality' of staining surfaces 604A/B/C/D may be a series of series of structures raised above the trough 607. Each staining surface 604A/B/C/D provides a surface that is isolated from each other staining surface 604A/B/C/D. The plurality of staining surfaces 604A/B/C/D may be square, rectangular, oval, or any other suitable shape and are not limited to the number or shapes illustrated in FIGS. 6A-6N. For example, the plurality' of staining surfaces 604A/B/C/D could be larger or smaller, greater or lesser in number, configured differently and/or shaped differently. As described herein, the plurality of staining surfaces 604A/B/C/D encompass any suitable structure for receiving a staining liquid onto a staining surface (e.g., as described above with respect to operation 1002 of method 1000)..

[0068] The staining cartridge 503 may also include a pair of ledges 508 forming a ledge structure and a trough 507. The ledges 508 may be similar in structure and function to the ledges 308/408/508. The trough 507 may be similar in structure and function to the troughs 307/407/507. Further, the

[0069] As shown in FIGS. 6A-6H, staining liquid may be placed on each one of the exposed staining surfaces 604A/B/C/D in accordance with operation 1002 of method 1000. FIGS. 6A- 6E show perspective views of a staining cartridge 603, while FIGS. 6F-6G show alternative cross-sectional views. According to the embodiments shown in FIGS.6A-6H, a plurality of different staining liquids 620A/B/C/D may be deposited on the plurality of staining surfaces 604A/B/C/D. As shown, staining plate 301 (e.g., with a sample, not shown) is swiped over the plurality of staining surfaces 604A/B/C/D and the staining liquids 620A/B/C/D deposited thereon stains, spreads, or smears across the staining plate 301 with the liquids being deposited on staining surfaces 604/A/B/C/D as examples accordance with operation 1004 and 1008 of method 1000. According to embodiments described, the gap 612 between the staining plate 301 and the plurality of staining surfaces 604A/B/C/D is maintained and is less than 350 microns (or less than 300 microns, or less than 250 microns, or less than 200 microns, or less than 150 microns, or less than 100 microns, or less than 50 microns, or less than 25 microns) in accordance with operation 1006 of method 1000.

[0070] FIGS. 6A-6H illustrate the application of multiple liquids onto the staining plate 301 (e.g., multiple staining liquids). According to embodiments, the staining plate 301 moves across the staining cartridge 603 (e.g., on the ledges 608) and comes in contact or otherwise interacts with a staining liquid deposited on one of the plurality’ of staining surfaces 604A/B/C/D as an overlap 611 between the staining plate and the one of the plurality of the staining surfaces 604A/B/C/D is established. The multiple overlaps 611 and gaps 612 each establish a respective associated staining volume 699. For example, as shown in FIGS. 6C, staining plate 301 passes over the staining surface 604A and establishes an overlap 611 therewith, and when a stain or other liquid (e.g., the staining liquid 620 A) is pre-deposited on staining surface 604A, that stain or other liquid is smeared, spread, or otherwise distributed across the staining plate 301. As further shown in FIG. 6C, the staining plate 301 next passes over staining surface 604B, establishes an overlap 611 therewith, and when a stain or other liquid (e.g, the staining liquid 620B) is pre-deposited on staining surface 604B, that stain or other liquid is smeared, spread, or otherwise distributed across the staining plate 301 and replaces the stain or other liquid smeared on the staining plate 301 from on the staining surface 604A (e.g., the staining liquid 620A). The staining plate 301 next passes over staining surface 604C, establishes an overlap 611 therewith, and when a stain or other liquid (e.g., the staining liquid 620C) is pre-deposited on staining surface 604C, that stain or other liquid is smeared, spread, or otherwise distributed across the staining plate 301 and replaces the stains or other liquids smeared on the staining plate 301 from on the staining surfaces 604A and 604B (e.g., the staining liquids 620A and 620B). As shown in FIG. 6D , the staining plate 301 finally passes over staining surface 604D, establishes an overlap 611 therewith, and when a stain or other liquid (e.g.. the staining liquid 620D) is pre-deposited on staining surface 604D. that stain or other liquid is smeared, spread, or otherwise distributed across the staining plate 301 and replaces the stain or other liquid smeared on the staining plate 301 from on the staining surfaces 604A, 604B and 604C (e.g., the staining liquids 620A, 620B and 620C). Thus, different portions of the substrate 601 may be exposed to different staining liquids at any given time. Further, in embodiments, the staining surfaces 604 A, 604B and 604C may be sized differently, which may be permit adjustments to the amount of time that any portion of the substrate 601 (and a sample thereon) are exposed to a given staining liquid if the substrate 601 is advanced at a constant rate. In other embodiments, the rate of advance of the substrate 601 may be variable, and may include one or more pauses, accelerations, reversals, etc.

[0071] As described above, the embodiment of FIGS. 6A-6N does not require any mechanism to pump or otherwise move liquids during movement of the staining plate 301. Rather, as shown, liquids are pre-deposited on one or more of the staining surfaces 604A/B/C/D and sequentially interact with the staining plate 301 as the staining plate 301 advances across and interacts with the staining surfaces 604A/B/C/D.

[0072] FIGS. 7A-7D illustrate stages in a process of distributing staining liquid over a substrate. According to the embodiment shown. FIGS. 7A-7D illustrate a staining cartridge 703 and a stain distribution device 702, one or more stain storage portions 709, one or more ports 706, a staining surface 704, and one or more inlets 716. The stain storage portions 709 are cavities within the staining cartridge 703 that are configured to store staining liquids. The inlets 716 are holes that provide access to the stain storage portions 709. The ports 706 are outlet holes that provide access to the staining surface 704 from the stain storage portions 709. In embodiments, one or more of the ports 706 and the inlets 716 may include removable coverings, such as strips of adhesive foil, strips of plugs, valve coverings, flexible flaps, etc. Moreover, as described herein, the stain distribution device 702 includes an air jet device 702A. Together, the stain distribution device 702, the air jet 702A, the one or more stain storage portions 709, the one or more ports 706 and one or more inlets 716 form an assembly that disposes and/or distributes a liquid as described above with respect to operation 1002 of method 1000. Elements of the stain distribution device 702 and related components are not limited to the shape illustrated in FIGS. 7A-7D, and any suitable structure for dispersing a staining liquid onto a staining surface (e.g., as described above with respect to operation 1002 and 1008 of method 1000) is within the scope of the claimed invention. For example, stain storage portions 709 are not limited to the linear tubular shape illustrated in FIGS. 7A-7D, and any suitable structure for controlling the distribution of a staining liquid onto the staining surface 604 (e.g., a tubular structure that curves or snakes within the staining cartridge 703) is within the scope of the claimed invention. Moreover, the features of the stain distribution device 702 and the staining cartridge 703 may be integrated with other embodiments described herein.

[0073] The staining cartridge 703 also includes a staining surface 704 (e.g., a surface that receives the disposed staining liquid) and a trough 707. While not shown in FIGS. 7A-7D, the staining cartridge 703 may include a pair of ledges forming a ledge structure. The ledges may be similar in structure and function to the ledges 308, 408, 508, 608, as described above. [0074] According an embodiment, e.g., as shown in FIGS. 7A-7D. a staining liquid (e.g., a fixative, wash, etc.) is pre-deposited in the one or more stain storage portions 709. The predeposited stain or other liquids are subsequently deposited to the staining surface 704 via a jet of high-pressure air from the air jet 702 A of the stain distribution device 702 in accordance with operation 1002 of method 1000. According to embodiments, deposition occurs when the stain distribution device 702 couples to the one or more inlets 716 and the air jet 702A and expels a measured amount of air pressure into the one or more inlets 716 such that a liquid pre-deposited within the one or more stain storage portions 709 is forcefully push out of the one or more ports 706. The distribution of stain or other liquids occurs with a surprising amount of control in dispensing the stain or other liquid. As described herein, a staining liquid is deposited via ajet of air pressure from air jet 702A that pushes or otherwise disperse liquid out of a stain storage portion 709 in the staining cartridge 703 without making physical contact with a sample smeared on the staining plate 301. In embodiments, the pressure of the air may be adjusted to alter the flow rate of the staining liquid out of the storage area. After deposition of the staining liquid onto the staining surface 704, the staining liquid may be distributed across a staining plate 301 in accordance with operations 1004. 1006, and 1008.

[0075] FIGS. 7A-7D illustrate the application of multiple liquids onto staining plate 301 (e.g., multiple staining liquids) with one stain distribution device 702. According to embodiments, sequential distribution occurs when the stain distribution device 702 couples in turn to each inlet 716 and the air jet 702 A and expels a measured amount of air pressure into the coupled inlet 716 such that a liquid pre-deposited within the respective stain storage portion 709 is forcefully push out of the respective port 706. The stain distribution device 702 is not limited to a tubular shape, as illustrated in FIGS. 7A-7D, and any suitable structure for dispersing a staining liquid onto a staining surface (e.g.. a plurality of stain distribution devices 702 to enable simultaneous distribution of liquid pre-deposited within the one or more stain storage portion 709) is within the scope of the claimed invention.

[0076] In further embodiments, the stain storage portion 709 may be configured to store a staining powder. The staining powder may be hydrated to create a staining liquid prior to use in a staining procedure.

[0077] FIG. 8 A and FIG. 8B illustrate a staining cartridge having a stain reservoir. The stain reservoir 811 of the staining cartridge 803 may be combined with any of the staining cartridges and/or features for automated staining disclosed herein. The staining cartridge 803 may include ledges 808. a trough 807. a port 806. a drain 805, a staining surface 804, and a raised structure 810. Each of these may be similar to those discussed above with respect to other embodiments.

[0078] In the staining cartridge 803, the staining surface 804 further includes the staining reservoir 811. The staining reservoir 811 is a portion of the staining surface 804 located opposite the port 806 from a larger main portion of the staining surface 804. The staining reservoir 811 is characterized by a surface that slopes or tapers downward (e.g., with respect to a plane defined by the main portion of the staining surface 804) as it extends away from the port 806. A first end of the staining reservoir 811 is disposed at a peak of the raised structure 810 where the port 806 is disposed. The staining reservoir 81 1 slopes down and away from the port 806 to the second end. The effect of the staining reservoir 811 geometry is such that, when a substrate 301 (not pictured) is arranged to overlap the staining surface 804 and establish a staining volume, the staining reservoir 811 provides an overflow buffer for the staining volume. When the staining volume is established, a portion of the staining volume corresponding to the staining reservoir 811 has gap that expands as it extends past the port 806.

[0079] In operation, the staining volume (including at least a portion of the staining reservoir) may be filled with staining liquid by disposing additional staining liquid to the staining volume. As additional staining liquids are added to the staining volume and previous staining liquids are removed, by methods discussed herein, the staining reservoir 81 1 may act as a buffer in case there are mismatches between inflow and outflow rates. Such mismatches may occur, for example, due to slight variations in the gap, due to bubbles in the fluid line, and/or due to potential mismatches in pumping rates. If the inflow rate is too high in comparison to the outflow, additional staining liquid may fill the staining reservoir 811 without escaping the staining volume altogether. If the outflow⁷ rate is too high in comparison to the inflow, the staining reservoir 811 may supply additional staining liquid to the port 806. In this way, staining liquid flow' into the port 806 is not disrupted (e g., by the introduction of air or other environmental gas), which would cause a break in the capillary action inside the staining volume and prevent the staining liquid from flowing properly.

[0080] In further embodiments, a camera or other optical device may be trained on the staining reservoir 811 to measure the amount of liquid in the staining reserv oir 811. Based on the amount of staining liquid in the staining reservoir 811 , the system may be configured to adjust the inflow and outflow rates of the staining liquid to maintain appropriate fill levels for the staining volume.

[0081] In further embodiments, a staining cartridge may include an inlet port at a first end and an outlet port at a second end. The stain deliver,' device may be provided via the inlet port. A first staining liquid may be deposited on the staining surface by the inlet port and distributed across the staining plate by relative motion, as described herein. A second staining liquid may be deposited on the staining surface by the inlet port while the second staining liquid is withdrawn via the outlet port, causing distribution of the second staining liquid across the staining plate.

[0082] Further embodiments may include various systems, devices, and methods to facilitate, improve, and/or optimize the staining methods discussed herein. Each of these embodiments may be used in combination with any of the methods and devices described herein.

[0083] As discussed above, air bubbles may be prevented from entering a staining volume by distributing the staining liquid to an area or space that is open to an unconstrained environment. Air bubbles, however, may present an additional problem. As discussed above, amounts of staining liquid that are delivered may be tightly controlled. Air bubble present in distribution lines may prevent accurate measurement of dispensed amounts as the air bubbles represent liquid volumes that are not delivered. In embodiments, the presence of bubbles in distribution lines and/or delivered to a staining surface may be monitored by camera or other imaging devices or optical sensors. For example, an imaging device may monitor a distribution line. An air bubble in a distribution line may be detected and its size estimated and an amount of dispensed volume may be adjusted accordingly. In another embodiment, an imaging device configured to monitor flow from a stain delivery device to a staining surface may detect a lack of liquid flow indicative of a bubble and the system may then adjust the incoming flow rate and volume (e.g., by increasing the rate or flow) and the outgoing flow rate and volume (e.g., by pausing or slowing) to ensure that they remain balanced. In further embodiments, stain delivery systems may include mechanical bubble traps to prevent the disruption of flow by bubbles.

[0084] In combination with any of the methods and techniques discussed herein, control of the inflow and outflow of staining liquids with respect to the staining volume may be performed. Removal of the first staining liquid and introduction of the second staining liquid may be performed by matching the volumetric flow rates of each staining liquid. Matching the volumetric flow rates may include pumping the first staining liquid out at substantially the same average volumetric flow rate as the second staining liquid is pumped in. Substantially the same average volumetric flow rate may include average flow rates that are within 5%, within 3%, and within 1% of each other over a given time period (e.g., 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, 0.5 seconds, etc.). Matching the volumetric flow rates may provide at least two advantages. First, by ensuring that the outflow of the first staining liquid is not higher than the inflow of the second staining liquid, capillary action within the staining volume is not broken. If outflow is too high, then air may be pulled into the staining volume, which may break capillary action at the location of the air pocket. Second, by ensuring that the inflow is not greater than the outflow, overflowing of the staining volume can be prevented. If inflow exceeds outflow by too much, the staining volume may overflow, which may cause a break in the meniscus edges of the staining volume.

[0085] In embodiments, the volumetric flow rates of the staining liquids may be matched by setting the inflow and outflow rates to be substantially the same, e.g.. by controlling pumps that drive the inflow and outflow. In further embodiments, the volumetric flow rates, both inflow and outflow, may be monitored, for example by a flow meter or by one or more cameras or other optical devices configured to monitor the staining volume and/or fluid lines through which fluid is added or removed from the staining volume. In embodiments, monitoring the volumetric flow rates may be performed by monitoring a combined flow rate, e.g., the combination of the input and output flow rates. For example, in embodiments wherein a camera or optical device monitors the staining volume, the camera may be configured to monitor the combined flow rate, e.g., by monitoring a state of the staining volume (e.g., overfill or underfill), the camera may be monitoring the combined input and output flow rates to the staining volume. Pumps that drive the inflow and outflow of the staining liquids may be controlled, e.g., by closed loop control, based on the information derived from monitoring the volumetric flow rates. Closed loop control may be performed by a processor associated with the integrated sample staining and processing system 100 and/or by suitable hardware. Closed loop control may be advantageous in such a system to account for minor variations in the process, resulting, for example, from slide misalignment, manufacturing variability, bubbles, etc.

[0086] In embodiments, staining liquids used for performing methods associated herewith may be provided in various manners. For example, as discussed above, staining cartridges consistent with embodiments hereof may be configured to contain staining liquids. In other examples, staining liquids may be provided in small disposable containers that include only enough staining liquid to stain a single substrate. In still further examples, staining liquid kits may be provided, packaging several different small staining liquid disposable containers together to perform a selected staining process.

[0087] In further embodiments, staining cartridges and systems associated herewith may include devices or systems to increase staining rates and reduce staining times. For example, one or more of a staining surface and a staining plate (substrate) may be vibrated to reduce staining times. The staining surface and/or staining plate may be vibrated by the integrated sample staining and processing system and or by another device as appropriate. In further embodiments, one or more of the staining surface and the staining plate may be heated to reduce staining times. For example, staining cartridges disclosed herein may include integrated heaters, e.g., resistive heaters or other devices. Staining plates disclosed herein may also be heated during staining operations.

[0088] The staining devices and methods described herein may contribute to sample processing methods that provide for increased accuracy and consistency of staining procedures, resulting in decreased analysis time, and increased throughput of sample processing. Such advantages may be particularly valuable when provided in an on-site environment for providing rapid sample evaluation and thus improving outcomes. The sample processing methods described herein are not limited to on-site processing, and may be employed to improve sample processing in any environment, including surgical tumor or tissue resections, remote or off-site pathology laboratories, clinical facilities, academic research institutions, and others.

[0089] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "includes" and/or "including," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0090] Further embodiments are described in the following clauses:

[0091] Clause 1. A method of distributing a staining liquid over a substrate, the method comprising: disposing a substrate in proximity to a staining surface to establish an overlap between the staining surface and the substrate; maintaining a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, the staining volume having one or more meniscus edges; disposing a portion of the staining liquid on the staining surface; and distributing the staining liquid on the substrate.

[0092] Clause 2. The method of clause 1. wherein disposing the portion of the staining liquid includes disposing the portion of the staining liquid at the meniscus edge such that the staining liquid is open to an unconstrained environment..

[0093] Clause 3. The method of clause 1, wherein distributing the staining liquid includes increasing the overlap by relative movement of the substrate and the staining surface.

[0094] Clause 4. The method of clause 3, wherein the relative movement is relative linear movement between the substrate and the staining surface.

[0095] Clause 5. The method of clause 3, wherein the portion of the staining liquid is disposed at the meniscus edge at a first volumetric rate corresponding to an increase in the staining volume caused by increasing the overlap.

[0096] Clause 6. The method of clause 5, wherein the staining volume is filled with the staining liquid without introducing bubbles to the staining volume. [0097] Clause 7. The method of clause 1, further comprising: depositing a second portion of a second staining liquid on the staining surface at the meniscus edge of the staining volume at a second volumetric rate; and removing the portion of the staining liquid from the staining volume at a third volumetric rate corresponding to the second volumetric rate.

[0098] Clause 8. The method of clause 7, wherein depositing the second portion and removing the portion are performed substantially simultaneously.

[0099] Clause 9. The method of clause 7, wherein the portion of the staining liquid is removed from the staining volume via a port, the port being defined by a raised structure on the staining surface, wherein a size of the gap is decreased towards the port.

[00100] Clause 10. The method of clause 9, wherein removal of the portion of the staining liquid via the port through the gap is performed without introducing air bubbles into the portion of the staining liquid as it is removed.

[00101] Clause 11. The method of claiml, wherein the staining surface includes a staining reservoir extending away from a port, wherein the gap expands over a length of the staining reservoir extending away from the port.

[00102] Clause 12. The method of clause 11, further comprising: depositing a second portion of a second staining liquid on the staining surface at the meniscus edge of the staining volume at a second volumetric rate; and removing the portion of the staining liquid from the staining volume at a third volumetric rate corresponding to the second volumetric rate, wherein the staining reservoir is configured to provide a buffer for mismatches between a second volumetric rate and a third volumetric rate.

[00103] Clause 13. The method of clause 9, wherein the port includes a plurality of apertures extending across at least 40% of a width of the substrate.

[00104] Clause 14. The method of clause 9, wherein the port includes a slit extending across at least 40% of a width of the substrate.

[00105] Clause 15. The method of clause 7, wherein removing the portion of the staining liquid from the staining volume at a third volumetric rate corresponding to the second volumetric rate includes: monitoring a combined flow rate including the second volumetric rate and the third volumetric rate; and adjusting at least one of the second volumetric rate and the third volumetric rate according to the monitoring.

[00106] Clause 16. The method of clause 5, wherein disposing the portion of the staining liquid at the meniscus edge at the first volumetric rate corresponding to an increase in the staining volume caused by increasing the overlap includes: monitoring the overlap; and adjusting the first volumetric flow rate according to the monitoring.

[00107] Clause 17. The method of clause 1, wherein disposing the portion of the staining liquid includes releasing the portion of the staining liquid from within a staining cartridge comprising the staining surface.

[00108] Clause 18. The method of clause 1, wherein the portion of the staining liquid is a predetermined or measured portion.

[00109] Clause 19. The method of clause 1, wherein the staining surface is a coverslip.

[00110] Clause 20. The method of clause 1, wherein the substrate is a slide.

[00111] Clause 21. The method of clause 1, wherein the gap is a capillary gap.

[001 12] Clause 22. The method of clause 1 , wherein the gap is substantially a same height across the overlap.

[00113] Clause 23. The method of clause 1, wherein the gap is defined by a gap profile of variable height.

[00114] Clause 24. The method of clause 1, wherein the gap is maintained by a ledge structure. [00115] Clause 25. The method of clause 1, wherein the gap is maintained by a robotic actuator. [00116] Clause 26. The method of clause 1, wherein distributing the staining liquid is performed by generating respective movement between the staining surface and the substrate that increases the overlap between the staining surface and the substrate, the method further comprising filling the staining volume with the portion of the staining liquid as the overlap is increased.

[00117] Clause 27. The method of clause 1, wherein the staining liquid is a first staining liquid, the method further comprising removing the portion of the first staining liquid from the gap and adding a portion of a second staining liquid.

[00118] Clause 28. The method of clause 27, wherein the removing and the adding are performed by pumping.

[00119] Clause 29. The method of clause 27, wherein the removing and the adding are performed simultaneously.

[00120] Clause 30. The method of clause 27, wherein the removing and the adding are performed sequentially.

[00121] Clause 31. The method of clause 27, wherein the removing and the adding is performed by respective movement between the staining surface and the substrate. [00122] Clause 32. The method of clause 27, wherein at least one of the removing and the adding are performed by a combination of pumping and respective movement between the staining surface and the substrate.

[00123] Clause 33. The method of clause 1, wherein the substrate includes one or more raised portions, the gap being defined between the staining surface and the one or more raised portions.

[00124] Clause 34. The method of clause 1, wherein the gap is a tapered gap that increases in height at edges of the overlap.

[00125] Clause 35. The method of clause 34, wherein the gap is tapered according to a raised structure on the staining surface.

[00126] Clause 36. The method of clause 35, wherein the raised structure has a volcano-like shape.

[00127] Clause 37. The method of clause 1, wherein the staining surface includes a port and distributing the staining liquid includes pumping staining liquid into the gap via the port.

[00128] Clause 38. The method of clause 37, wherein the pumping is performed with an air gap in a staining liquid line.

[00129] Clause 39. The method of clause 1, wherein at least one of the substrate and the staining surface include a hydrophilic surface.

[00130] Clause 40. The method of clause 1. wherein at least one of the substrate and the staining surface include a hydrophobic surface.

[00131] Clause 41. The method of clause 1, further comprising hydrating a staining powder to create the staining liquid.

[00132] Clause 42. The method of clause 1. wherein the staining liquid comprises at least one of a fixative, a color stain, and a water wash.

[00133] Clause 43. The method of clause 1, wherein distributing the staining liquid on the substrate further comprises distributing a plurality of staining liquids on the substrate.

[00134] Clause 44. The method of clause 42, wherein distributing a plurality of staining liquids on the substrate comprises distributing at least one fixative, at least two color stains and at least one water wash.

[00135] Clause 45. The method of clause 42, wherein distributing a plurality of staining liquids on the substrate comprises distributing a fixative, a first color stain, a second color stain and a water wash. [00136] Clause 46. The method of clause 42, wherein distributing a plurality of staining liquids on the substrate takes approximately 10 seconds to distribute each staining liquid.

[00137] Clause 47. The method of clause 1, wherein distributing the staining liquid on the substrate takes less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, or less than 2 seconds distribute the staining liquid.

[00138] Clause 48. The method of clause 1, wherein distributing the staining liquid on the substrate takes less than 1 second to distribute the staining liquid.

[00139] Clause 49. The method of clause 9, wherein the raised structure extends at least a 2 mm distance from the port.

[00140] Clause 50. The method of any of clauses 1-49, wherein at least one of disposing the portion of the staining liquid, disposing the staining surface, maintaining the gap, and distributing the staining liquid is performed by an automated system.

[00141] Clause 51. A device for staining a biological sample, comprising: a staining structure including a base, a staining surface disposed on the base, and at least one gap maintenance structure disposed on the base,; and a substrate configured to receive the biological sample; wherein: the substrate is configured to be disposed to establish an overlap between the staining surface and the substrate, the at least one gap maintenance structure is configured to maintain a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, and the staining volume is configured to receive a staining liquid and to have one or more meniscus edges when the staining liquid is received.

[00142] Clause 52. The device of clause 51, wherein the staining cartridge is configured such that the staining liquid is open to an unconstrained environment at the one or more meniscus edges.

[00143] Clause 53. The device of clause 52, wherein the staining cartridge is further configured to receive the staining liquid at the one or more meniscus edges.

[00144] Clause 54. The device of clause 51, wherein the staining cartridge is configured for distributing the staining liquid on the substrate by increasing the overlap by relative movement of the substrate and the staining surface.

[00145] Clause 55. The device of clause 54, further comprising an actuator configured for increasing the overlap by relative movement.

[00146] Clause 56. The device of clause 51, further comprising a port defined by a raised structure on the staining surface, wherein a size of the gap is decreased towards the port, the port being configured to introduce or remove the staining liquid from the staining surface. [00147] Clause 57. The device of clause 56, wherein the staining surface includes a staining reservoir extending away from a port, wherein the gap expands over a length of the staining reservoir extending away from the port.

[00148] Clause 58. The device of clause 57, wherein the port includes a plurality of apertures extending across at least 40% of a width of the substrate.

[00149] Clause 59. The device of clause 56, wherein the port includes a slit extending across at least 40% of a width of the substrate.

[00150] Clause 60. The device of clause 51, wherein the staining cartridge is configured to contain the staining liquid.

[00151] Clause 61. The device of clause 51. wherein the staining surface is a coverslip.

[00152] Clause 62. The device of clause 51 , wherein the substrate is a slide.

[00153] Clause 63. The device of clause 51, wherein the gap is a capillary gap.

[00154] Clause 64. The device of clause 51, wherein the gap is substantially a same height across the overlap.

[00155] Clause 65. The device of clause 51, wherein the gap is defined by a gap profile of variable height.

[00156] Clause 66. The device of clause 51, further comprising a stain delivery device.

[00157] Clause 67. The device of clause 51, further comprising a drain configured for removal of the staining liquid.

[00158] Clause 68. The device of clause 51, wherein the gap is a tapered gap that increases in height at edges of the overlap.

[00159] Clause 69. The device of clause 66, wherein the gap is tapered according to a raised structure on the staining surface.

[00160] Clause 70. The device of clause 67, wherein the raised structure has a volcano-like shape.

[00161] Clause 71. The device of clause 51, wherein at least one of the substrate and the staining surface include a hydrophilic surface.

[00162] Clause 72. The device of clause 51, wherein at least one of the substrate and the staining surface include a hydrophobic surface.

[00163] Clause 73. The device of clause 51, wherein the staining liquid comprises at least one of a fixative, a color stain, and a water wash. [00164] Clause 74. The device of clause 51, wherein the gap maintenance structure includes one or more ledges having a top at a height above the staining surface and configured to support the substrate above the staining surface.

[00165] Clause 75. The device of clause 51, wherein the gap maintenance structure includes one or more ledges having a top at a height below the staining surface and configured to support the substrate below the staining surface.

[00166] Clause 764. The device of clam 74, further comprising a trough disposed on the base and located between the staining surface and the one or more ledges.

[00167] Clause 77. The device of clause 51, further comprising at least one additional staining surface.

[00168] Clause 78. A device for staining a biological sample, comprising: a staining structure including a base, a staining surface disposed on the base, and at least one gap maintenance device; and a substrate configured to receive the biological sample; wherein: the substrate is configured to be disposed to establish an overlap between the staining surface and the substrate, the at least one gap maintenance device is configured to maintain a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, and the staining volume is configured to receive a staining liquid and to have one or more meniscus edges when the staining liquid is received.

[00169] Clause 79. The device of clause 78, wherein the at least one gap maintenance device includes a robotic actuator.

[00170] The embodiments described above are illustrative examples and it should not be construed that the present invention is limited to these particular embodiments. It should be understood that various embodiments disclosed herein may be combined in different combinations than the combinations specifically presented in the description and accompanying drawings. It should also be understood that, depending on the example, certain acts or events of any of the processes or methods described herein may be performed in a different sequence, may be added, merged, or left out altogether (e.g., all described acts or events may not be necessary to cany out the methods or processes). In addition, while certain features of embodiments hereof are described as being performed by a single module, device, or unit for purposes of clarity, it should be understood that the features and functions described herein may be performed by any combination of units or modules. Thus, various changes and modifications may be affected by one skilled in the art without departing from the spirit or scope of the invention as defined in the appended claims.

Claims

WE CLAIM:

1. A method of distributing a staining liquid over a substrate, the method comprising: disposing a substrate in proximity to a staining surface to establish an overlap between the staining surface and the substrate; maintaining a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, the staining volume having one or more meniscus edges; disposing a portion of the staining liquid on the staining surface; and distributing the staining liquid on the substrate.

2. The method of claim 1 , wherein disposing the portion of the staining liquid includes disposing the portion of the staining liquid at a meniscus edge of the one or more meniscus edges such that the staining liquid is open to an unconstrained environment..

3. The method of claim 1, wherein distributing the staining liquid includes increasing the overlap by relative movement of the substrate and the staining surface.

4. The method of claim 3, wherein the relative movement is relative linear movement between the substrate and the staining surface.

5. The method of claim 3, wherein the portion of the staining liquid is disposed at a meniscus edge of the one or more meniscus edges at a first volumetric rate corresponding to an increase in the staining volume caused by increasing the overlap.

6. The method of claim 5, wherein the staining volume is filled with the staining liquid without introducing bubbles to the staining volume.

7. The method of claim 1, further comprising: depositing a second portion of a second staining liquid on the staining surface at a meniscus edge of the one or more meniscus edges of the staining volume at a second volumetric rate; and removing the portion of the staining liquid from the staining volume at a third volumetric rate corresponding to the second volumetric rate.

8. The method of claim 7, wherein depositing the second portion and removing the portion are performed substantially simultaneously.

9. The method of claim 7, wherein the portion of the staining liquid is removed from the staining volume via a port, the port being defined by a raised structure on the staining surface, wherein a size of the gap is decreased tow ards the port.

10. The method of claim 9, wherein removal of the portion of the staining liquid via the port through the gap is performed without introducing air bubbles into the portion of the staining liquid as it is removed.

11. The method of claim 1, wherein the staining surface includes a staining reservoir extending aw ay from a port, wherein the gap expands over a length of the staining reservoir extending aw ay from the port.

12. The method of claim 11. further comprising: depositing a second portion of a second staining liquid on the staining surface at a meniscus edge of the one or more meniscus edges of the staining volume at a second volumetric rate; and removing the portion of the staining liquid from the staining volume at a third volumetric rate corresponding to the second volumetric rate, wherein the staining reservoir is configured to provide a buffer for mismatches between a second volumetric rate and a third volumetric rate.

13. The method of claim 9, wherein the port includes a plurality of apertures extending across at least 40% of a width of the substrate.

14. The method of claim 9, wherein the port includes a slit extending across at least

40% of a width of the substrate.

15. The method of claim 7, wherein removing the portion of the staining liquid from the staining volume at a third volumetric rate corresponding to the second volumetric rate includes: monitoring a combined flow rate including the second volumetric rate and the third volumetric rate; and adjusting at least one of the second volumetric rate and the third volumetric rate according to the monitoring.

16. The method of claim 5, wherein disposing the portion of the staining liquid at the meniscus edge at the first volumetric flow rate corresponding to an increase in the staining volume caused by increasing the overlap includes: monitoring the overlap; and adjusting the first volumetric flow rate according to the monitoring.

17. The method of claim 1, wherein disposing the portion of the staining liquid includes releasing the portion of the staining liquid from within a staining cartridge comprising the staining surface.

18. The method of claim 1, wherein the portion of the staining liquid is a predetermined or measured portion.

19. The method of claim 1, wherein the staining surface is a coverslip.

20. The method of claim 1, wherein the substrate is a slide.

21. The method of claim 1, wherein the gap is a capillary gap.

22. The method of claim 1, wherein the gap is substantially a same height across the overlap.

23. The method of claim 1, wherein the gap is defined by a gap profile of variable height.

24. The method of claim 1, wherein the gap is maintained by a ledge structure.

25. The method of claim 1, wherein the gap is maintained by a robotic actuator.

26. The method of claim 1, wherein distributing the staining liquid is performed by generating respective movement between the staining surface and the substrate that increases the overlap between the staining surface and the substrate, the method further comprising filling the staining volume with the portion of the staining liquid as the overlap is increased.

27. The method of claim 1 , wherein the staining liquid is a first staining liquid, the method further comprising removing the portion of the first staining liquid from the gap and adding a portion of a second staining liquid.

28. The method of claim 27, wherein the removing and the adding are performed by pumping.

29. The method of claim 27. wherein the removing and the adding are performed simultaneously.

30. The method of claim 27, wherein the removing and the adding are performed sequentially.

31. The method of claim 27, wherein the removing and the adding is performed by respective movement between the staining surface and the substrate.

32. The method of claim 27. wherein at least one of the removing and the adding are performed by a combination of pumping and respective movement between the staining surface and the substrate.

33. The method of claim 1, wherein the substrate includes one or more raised portions, the gap being defined between the staining surface and the one or more raised portions.

34. The method of claim 1, wherein the gap is a tapered gap that increases in height at edges of the overlap.

35. The method of claim 34. wherein the gap is tapered according to a raised structure on the staining surface.

36. The method of claim 35, wherein the raised structure has a volcano-like shape. .

37. The method of claim 1, wherein the staining surface includes a port and distributing the staining liquid includes pumping staining liquid into the gap via the port.

38. The method of claim 37, wherein the pumping is performed with an air gap in a staining liquid line.

39. The method of claim 1, wherein at least one of the substrate and the staining surface include a hydrophilic surface.

40. The method of claim 1, wherein at least one of the substrate and the staining surface include a hydrophobic surface.

41. The method of claim 1, further comprising hydrating a staining powder to create the staining liquid.

42. The method of claim 1, wherein the staining liquid comprises at least one of a fixative, a color stain, and a water wash.

43. The method of claim 1, wherein distributing the staining liquid on the substrate further comprises distributing a plurality of staining liquids on the substrate.

44. The method of claim 42, wherein distributing a plurality of staining liquids on the substrate comprises distributing at least one fixative, at least two color stains and at least one water wash.

45. The method of claim 42, wherein distributing a plurality of staining liquids on the substrate comprises distributing a fixative, a first color stain, a second color stain and a water wash.

46. The method of claim 42, wherein distributing a plurality of staining liquids on the substrate takes approximately 10 seconds to distribute each staining liquid.

47. The method of claim 1, wherein distributing the staining liquid on the substrate takes less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, or less than 2 seconds distribute the staining liquid.

48. The method of claim 1, wherein distributing the staining liquid on the substrate takes less than 1 second to distribute the staining liquid.

49. The method of claim 9, wherein the raised structure extends at least a 2 mm distance from the port.

50. The method of any of claims 1-49, wherein at least one of disposing the portion of the staining liquid, disposing the staining surface, maintaining the gap, and distributing the staining liquid is performed by an automated system.

51. A device for staining a biological sample, comprising: a staining structure including a base, a staining surface disposed on the base, and at least one gap maintenance structure disposed on the base; and a substrate configured to receive the biological sample; wherein: the substrate is configured to be disposed to establish an overlap between the staining surface and the substrate, the at least one gap maintenance structure is configured to maintain a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, and the staining volume is configured to receive a staining liquid and to have one or more meniscus edges when the staining liquid is received.

52. The device of claim 51, wherein the staining cartridge is configured such that the staining liquid is open to an unconstrained environment at the one or more meniscus edges.

53. The device of claim 52, wherein the staining cartridge is further configured to receive the staining liquid at the one or more meniscus edges.

54. The device of claim 51, wherein the staining cartridge is configured for distributing the staining liquid on the substrate by increasing the overlap by relative movement of the substrate and the staining surface.

55. The device of claim 54, further comprising an actuator configured for increasing the overlap by relative movement.

56. The device of claim 51, further comprising a port defined by a raised structure on the staining surface, wherein a size of the gap is decreased towards the port, the port being configured to introduce or remove the staining liquid from the staining surface.

57. The device of claim 56, wherein the staining surface includes a staining reservoir extending away from a port, wherein the gap expands over a length of the staining reservoir extending away from the port.

58. The device of claim 57, wherein the port includes a plurality of apertures extending across at least 40% of a width of the substrate.

59. The device of claim 56, wherein the port includes a slit extending across at least 40% of a width of the substrate.

60. The device of claim 51, wherein the staining cartridge is configured to contain the staining liquid.

61. The device of claim 51, wherein the staining surface is a coverslip.

62. The device of claim 51, wherein the substrate is a slide.

63. The device of claim 51, wherein the gap is a capillary gap.

64. The device of claim 51, wherein the gap is substantially a same height across the overlap.

65. The device of claim 51, wherein the gap is defined by a gap profile of variable height.

66. The device of claim 51, further comprising a stain delivery device.

67. The device of claim 51, further comprising a drain configured for removal of the staining liquid.

68. The device of claim 51 , wherein the gap is a tapered gap that increases in height at edges of the overlap.

69. The device of claim 66, wherein the gap is tapered according to a raised structure on the staining surface.

70. The device of claim 67, wherein the raised structure has a volcano-like shape.

71. The device of claim 51, wherein at least one of the substrate and the staining surface include a hydrophilic surface.

72. The device of claim 51, wherein at least one of the substrate and the staining surface include a hydrophobic surface.

73. The device of claim 51, wherein the staining liquid comprises at least one of a fixative, a color stain, and a water wash.

74. The device of claim 51, wherein the gap maintenance structure includes one or more ledges having a top at a height above the staining surface and configured to support the substrate above the staining surface.

75. The device of claim 51, wherein the gap maintenance structure includes one or more ledges having a top at a height below the staining surface and configured to support the substrate below the staining surface.

764. The device of clam 74. further comprising a trough disposed on the base and located between the staining surface and the one or more ledges.

77. The device of claim 51, further comprising at least one additional staining surface.

78. A device for staining a biological sample, comprising: a staining structure including a base, a staining surface disposed on the base, and at least one gap maintenance device; and a substrate configured to receive the biological sample; wherein: the substrate is configured to be disposed to establish an overlap between the staining surface and the substrate, the at least one gap maintenance device is configured to maintain a gap between the staining surface and the substrate to define a staining volume according to the overlap and the gap, and the staining volume is configured to receive a staining liquid and to have one or more meniscus edges when the staining liquid is received.

79. The device of claim 78, wherein the at least one gap maintenance device includes a robotic actuator.